Phylogeny of seed plants based on all three genomic compartments: extant gymnosperms are monophyletic and Gnetales' closest relatives are conifers

Efforts to resolve Darwin's "abominable mystery"-the origin of angiosperms-have led to the conclusion that Gnetales and various fossil groups are sister to angiosperms, forming the "anthophytes." Morphological homologies, however, are difficult to interpret, and molecular data have not provided clear resolution of relationships among major groups of seed plants. We introduce two sequence data sets from slowly evolving mitochondrial genes, cox1 and atpA, which unambiguously reject the anthophyte hypothesis, favoring instead a close relationship between Gnetales and conifers. Parsimony- and likelihood-based analyses of plastid rbcL and nuclear 18S rDNA alone and with cox1 and atpA also strongly support a gnetophyte-conifer grouping. Surprisingly, three of four genes (all but nuclear rDNA) and combined three-genome analyses also suggest or strongly support Gnetales as derived conifers, sister to Pinaceae. Analyses with outgroups screened to avoid long branches consistently identify all gymnosperms as a monophyletic sister group to angiosperms. Combined three- and four-gene rooted analyses resolve the branching order for the remaining major groups-cycads separate from other gymnosperms first, followed by Ginkgo and then (Gnetales + Pinaceae) sister to a monophyletic group with all other conifer families. The molecular phylogeny strongly conflicts with current interpretations of seed plant morphology, and implies that many similarities between gnetophytes and angiosperms, such as "flower-like" reproductive structures and double fertilization, were independently derived, whereas other characters could emerge as synapomorphies for an expanded conifer group including Gnetales. An initial angiosperm-gymnosperm split implies a long stem lineage preceding the explosive Mesozoic radiation of flowering plants and suggests that angiosperm origins and homologies should be sought among extinct seed plant groups.     

MADS-box genes reveal that gnetophytes are more closely related to conifers than to flowering plants

The evolutionary origin of the angiosperms (flowering plants sensu stricto) is still enigmatic. Answers to the question of angiosperm origins are intimately connected to the identification of their sister group among extinct and extant taxa. Most phylogenetic analyses based on morphological data agree that among the groups of extant seed plants, the gnetophytes are the sister group of the angiosperms. According to this view, angiosperms and gnetophytes are the only extant members of a clade called "anthophytes" to emphasize their shared possession of flower-like reproductive structures. However, most phylogeny reconstructions based on molecular data so far did not support an anthophyte clade, but also could not clarify the case because support for alternative groupings has been weak or controversial. We have isolated 13 different homologs of MADS-type floral homeotic genes from the gnetophyte Gnetum gnemon. Five of these genes fall into monophyletic gene clades also comprising putatively orthologous genes from flowering plants and conifers, among them orthologs of floral homeotic B and C function genes. Within these clades the Gnetum genes always form distinct subclades together with the respective conifer genes, to the exclusion of the angiosperm genes. This provides strong molecular evidence for a sister-group relationship between gnetophytes and conifers, which is in contradiction to widely accepted interpretations of morphological data for almost a century. Our phylogeny reconstructions and the outcome of expression studies suggest that complex features such as flower-like reproductive structures and double-fertilization arose independently in gnetophytes and angiosperms.     

The root of the angiosperms revisited

Most recent phylogenetic analyses of basal angiosperms have converged on the placement of Amborella as sister to all other extant angiosperms. However, certain recent studies suggest that Amborella and Nymphaeales (water lilies) form a clade sister to all remaining angiosperms or that Nymphaeales alone are the sister to the remaining angiosperms. We report here (i) maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses of 11 genes (>15,000 bp per taxon) for 16 taxa, (ii) maximum parsimony analysis for a subset of these genes for 104 taxa, and (iii) tests of alternative rootings with the nonparametric bootstrap and the likelihood ratio test with the parametric bootstrap. In addition, we use simulation analyses to examine the amount of bias that may be present in our methods of phylogeny estimation. Amborella continues to receive strong bootstrap support as the sister to all other extant angiosperms, and three of four tests reject alternative hypotheses of the angiosperm root. Although we cannot conclusively choose between Amborella vs. Amborella + Nymphaeales as sister to all other angiosperms, most analyses favor the former rooting.     

Horizontal gene transfer from flowering plants to Gnetum

Although horizontal gene transfer is well documented in microbial genomes, no case has been reported in higher plants. We discovered horizontal transfer of the mitochondrial nad1 intron 2 and adjacent exons b and c from an asterid to Gnetum (Gnetales, gymnosperms). Gnetum has two copies of intron 2, a group II intron, that differ in their exons, nucleotide composition, domain lengths, and structural characteristics. One of the copies, limited to an Asian clade of Gnetum, is almost identical to the homologous locus in angiosperms, and partial sequences of its exons b and c show characteristic substitutions unique to angiosperms. Analyses of 70 seed plant nad1 exons b and c and intron 2 sequences, including representatives of all angiosperm clades, support that this copy originated from a euasterid and was horizontally transferred to Gnetum. Molecular clock dating, using calibrations provided by gnetalean macrofossils, suggests an age of 5 to 2 million years for the Asian clade that received the horizontal transfer.     

Independent and combined analyses of sequences from all three genomic compartments converge on the root of flowering plant phylogeny

Plant phylogenetic estimates are most likely to be reliable when congruent evidence is obtained independently from the mitochondrial, plastid, and nuclear genomes with all methods of analysis. Here, results are presented from separate and combined genomic analyses of new and previously published data, including six and nine genes (8, 911 bp and 12,010 bp, respectively) for different subsets of taxa that suggest Amborella + Nymphaeales (water lilies) are the first-branching angiosperm lineage. Before and after tree-independent noise reduction, most individual genomic compartments and methods of analysis estimated the Amborella + Nymphaeales basal topology with high support. Previous phylogenetic estimates placing Amborella alone as the first extant angiosperm branch may have been misled because of a series of specific problems with paralogy, suboptimal outgroups, long-branch taxa, and method dependence. Ancestral character state reconstructions differ between the two topologies and affect inferences about the features of early angiosperms.     

On the origin of and phylogenetic relationships among living amphibians

The phylogenetic relationships among the three orders of modern amphibians (Caudata, Gymnophiona, and Anura) have been estimated based on both morphological and molecular evidence. Most morphological and paleontological studies of living and fossil amphibians support the hypothesis that salamanders and frogs are sister lineages (the Batrachia hypothesis) and that caecilians are more distantly related. Previous interpretations of molecular data based on nuclear and mitochondrial rRNA sequences suggested that salamanders and caecilians are sister groups to the exclusion of frogs. In an attempt to resolve this apparent conflict, the complete mitochondrial genomes of a salamander (Mertensiella luschani) and a caecilian (Typhlonectes natans) were determined (16,656 and 17,005 bp, respectively) and compared with previously published sequences from a frog (Xenopus laevis) and several other groups of vertebrates. Phylogenetic analyses of the mitochondrial data supported with high bootstrap values the monophyly of living amphibians with respect to other living groups of tetrapods, and a sister group relationship of salamanders and frogs. The lack of phylogenetically informative sites in the previous rRNA data sets (because of its shorter size and higher among-site rate variation) likely explains the discrepancy between our results and those based on previous molecular data. Strong support of the Batrachia hypothesis from both molecule- and morphology-based studies provides a robust phylogenetic framework that will be helpful to comparative studies among the three living orders of amphibians and will permit better understanding of the considerably divergent vertebral, brain, and digit developmental patterns found in frogs and salamanders.     

Phylogenetic relationships among species of Anopheles (Nyssorhynchus) (Diptera, Culicidae) based on nuclear and mitochondrial gene sequences

Phylogenetic relationships among 21 species of mosquitoes in subgenus Nyssorhynchus were inferred from the nuclear white and mitochondrial NADH dehydrogenase subunit 6 (ND6) genes. Bayesian phylogenetic methods found that none of the three Sections within Nyssorhynchus (Albimanus, Argyritarsis, Myzorhynchella) were supported in all analyses, although Myzorhynchella was found to be monophyletic at the combined genes. Within the Albimanus Section the monophyly of the Strodei Subgroup was strongly supported and within the Myzorhynchella Section Anopheles antunesi and An. lutzii formed a strongly supported monophyletic group. The epidemiologically significant Albitarsis Complex showed evidence of paraphyly (relative to An. lanei-Myzorhynchella) and discordance across gene trees, and the previously synonomized species of An. dunhami and An. goeldii were recovered as sister species. Finally, there was evidence of complexes in several species, including An. antunesi, An. deaneorum, and An. strodei.     

Distribution of living Cupressaceae reflects the breakup of Pangea

Most extant genus-level radiations in gymnosperms are of Oligocene age or younger, reflecting widespread extinction during climate cooling at the Oligocene/Miocene boundary [～23 million years ago (Ma)]. Recent biogeographic studies have revealed many instances of long-distance dispersal in gymnosperms as well as in angiosperms. Acting together, extinction and long-distance dispersal are likely to erase historical biogeographic signals. Notwithstanding this problem, we show that phylogenetic relationships in the gymnosperm family Cupressaceae (162 species, 32 genera) exhibit patterns expected from the Jurassic/Cretaceous breakup of Pangea. A phylogeny was generated for 122 representatives covering all genera, using up to 10,000 nucleotides of plastid, mitochondrial, and nuclear sequence per species. Relying on 16 fossil calibration points and three molecular dating methods, we show that Cupressaceae originated during the Triassic, when Pangea was intact. Vicariance between the two subfamilies, the Laurasian Cupressoideae and the Gondwanan Callitroideae, occurred around 153 Ma (124-183 Ma), when Gondwana and Laurasia were separating. Three further intercontinental disjunctions involving the Northern and Southern Hemisphere are coincidental with or immediately followed the breakup of Pangea.     

Novelties of the flowering plant pollen tube underlie diversification of a key life history stage

The origin and rapid diversification of flowering plants has puzzled evolutionary biologists, dating back to Charles Darwin. Since that time a number of key life history and morphological traits have been proposed as developmental correlates of the extraordinary diversity and ecological success of angiosperms. Here, I identify several innovations that were fundamental to the evolutionary lability of angiosperm reproduction, and hence to their diversification. In gymnosperms pollen reception must be near the egg largely because sperm swim or are transported by pollen tubes that grow at very slow rates (< approximately 20 microm/h). In contrast, pollen tube growth rates of taxa in ancient angiosperm lineages (Amborella, Nuphar, and Austrobaileya) range from approximately 80 to 600 microm/h. Comparative analyses point to accelerated pollen tube growth rate as a critical innovation that preceded the origin of the true closed carpel, long styles, multiseeded ovaries, and, in monocots and eudicots, much faster pollen tube growth rates. Ancient angiosperm pollen tubes all have callosic walls and callose plugs (in contrast, no gymnosperms have these features). The early association of the callose-walled growth pattern with accelerated pollen tube growth rate underlies a striking repeated pattern of faster and longer-distance pollen tube growth often within solid pathways in phylogenetically derived angiosperms. Pollen tube innovations are a key component of the spectacular diversification of carpel (flower and fruit) form and reproductive cycles in flowering plants.     

Chloroplast and nuclear gene sequences indicate late Pennsylvanian time for the last common ancestor of extant seed plants.

We have estimated the time for the last common ancestor of extant seed plants by using molecular clocks constructed from the sequences of the chloroplastic gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and the nuclear gene coding for the small subunit of rRNA (Rrn18). Phylogenetic analyses of nucleotide sequences indicated that the earliest divergence of extant seed plants is likely represented by a split between conifer-cycad and angiosperm lineages. Relative-rate tests were used to assess homogeneity of substitution rates among lineages, and annual angiosperms were found to evolve at a faster rate than other taxa for rbcL and, thus, these sequences were excluded from construction of molecular clocks. Five distinct molecular clocks were calibrated using substitution rates for the two genes and four divergence times based on fossil and published molecular clock estimates. The five estimated times for the last common ancestor of extant seed plants were in agreement with one another, with an average of 285 million years and a range of 275-290 million years. This implies a substantially more recent ancestor of all extant seed plants than suggested by some theories of plant evolution.     

On the evolutionary history of Ephedra: Cretaceous fossils and extant molecules

Gnetales comprise three unusual genera of seed plants, Ephedra, Gnetum, and Welwitschia. Their extraordinary morphological diversity suggests that they are survivors of an ancient, more diverse group. Gnetalean antiquity is also supported by fossil data. Dispersed "ephedroid" (polyplicate) pollen first appeared in the Permian >250 million years ago (Myr), and a few megafossils document the presence of gnetalean features in the early Cretaceous. The Cretaceous welwitschioid seedling Cratonia cotyledon dates the split between Gnetum and Welwitschia to before 110 Myr. Ages and character evolution of modern diversity are, however, controversial, and, based on molecular data, it has recently been suggested that Ephedra is very young, only 8-32 Myr. Here, we present data on the evolutionary history of Ephedra. Fossil seeds from Buarcos, Portugal, unequivocally link one type of Cretaceous polyplicate pollen to Ephedra and document that plants with unique characters, including the peculiar naked male gametophyte, were established already in the Early Cretaceous. Clades in our molecular phylogeny of extant species correspond to geographical regions, with African species in a basal grade/clade. The study demonstrates extremely low divergence in both molecular and morphological characters in Ephedra. Features observed in the fossils are present in all major extant clades, showing that modern species have retained unique reproductive characters for >110 million years. A recent origin of modern species of Ephedra would imply that the Cretaceous Ephedra fossils discussed here were members of widespread, now extinct sister lineage(s), and that no morphological innovations characterized the second diversification.     

Independent adaptation to riverine habitats allowed survival of ancient cetacean lineages

The four species of "river dolphins" are associated with six separate great river systems on three subcontinents and have been grouped for more than a century into a single taxon based on their similar appearance. However, several morphologists recently questioned the monophyly of that group. By using phylogenetic analyses of nucleotide sequences from three mitochondrial and two nuclear genes, we demonstrate with statistical significance that extant river dolphins are not monophyletic and suggest that they are relict species whose adaptation to riverine habitats incidentally insured their survival against major environmental changes in the marine ecosystem or the emergence of Delphinidae.     

Date of the monocot-dicot divergence estimated from chloroplast DNA sequence data.

The divergence between monocots and dicots represents a major event in higher plant evolution, yet the date of its occurrence remains unknown because of the scarcity of relevant fossils. We have estimated this date by reconstructing phylogenetic trees from chloroplast DNA sequences, using two independent approaches: the rate of synonymous nucleotide substitution was calibrated from the divergence of maize, wheat, and rice, whereas the rate of nonsynonymous substitution was calibrated from the divergence of angiosperms and bryophytes. Both methods lead to an estimate of the monocot-dicot divergence at 200 million years (Myr) ago (with an uncertainty of about 40 Myr). This estimate is also supported by analyses of the nuclear genes encoding large and small subunit ribosomal RNAs. These results imply that the angiosperm lineage emerged in Jurassic-Triassic time, which considerably predates its appearance in the fossil record (approximately 120 Myr ago). We estimate the divergence between cycads and angiosperms to be approximately 340 Myr, which can be taken as an upper bound for the age of angiosperms.     

Multiple molecular evidences for a living mammalian fossil

Laonastes aenigmamus is an enigmatic rodent first described in 2005. Molecular and morphological data suggested that it is the sole representative of a new mammalian family, the Laonastidae, and a member of the Hystricognathi. However, the validity of this family is controversial because fossil-based phylogenetic analyses suggest that Laonastes is a surviving member of the Diatomyidae, a family considered to have been extinct for 11 million years. According to these data, Laonastes and Diatomyidae are the sister clade of extant Ctenodactylidae (i.e., gundies) and do not belong to the Hystricognathi. To solve the phylogenetic position of Laonastes, we conducted a large-scale molecular phylogeny of rodents. The analysis includes representatives of all major rodent taxonomic groups and was based on 5.5 kb of sequence data from four nuclear and two mitochondrial genes. To further validate the obtained results, a short interspersed element insertion analysis including 11 informative loci was also performed. Our molecular data based on sequence and short interspersed element analyses unambiguously placed Laonastes as a sister clade of gundies. All alternative hypotheses were significantly rejected based on Shimodaira-Hasegawa tests, supporting the idea that Laonastes does not belong to the Hystricognathi. Molecular dating analysis also supports an ancient divergence, approximately 44 Mya ago, between Ctenodactylidae and Laonastes. These combined analyses support the hypothesis that Laonastes is indeed a living fossil. Protection of this surviving species would conserve an ancient mammalian family.     

Molecular evolutionary history of ancient aquatic angiosperms.

Aquatic plants are notoriously difficult to study systematically due to convergent evolution and reductionary processes that result in confusing arrays of morphological features. Plant systematists have frequently focused their attention on the "water lilies," putative descendants of the most archaic angiosperms. Classification of these 10 plant genera varies from recognition of one to three orders containing three to six families. We have used DNA sequence analysis as a means of overcoming many problems inherent in morphologically based studies of the group. Phylogenetic analyses of sequence data obtained from a 1.2-kilobase portion of the chloroplast gene rbcL provide compelling evidence for the recognition of three distinct lineages of "water lily" plants. Molecular phylogenies including woody Magnoliidae sequences and sequences of these aquatic plants depict Ceratophyllum as an early diverging genus. Our results support hypotheses that most taxonomic concepts of the order Nymphaeales reflect polyphyletic groups and that the unusual genus Ceratophyllum represents descendants of some of the earliest angiosperms.     

The deepest divergences in land plants inferred from phylogenomic evidence

Phylogenetic relationships among the four major lineages of land plants (liverworts, mosses, hornworts, and vascular plants) remain vigorously contested; their resolution is essential to our understanding of the origin and early evolution of land plants. We analyzed three different complementary data sets: a multigene supermatrix, a genomic structural character matrix, and a chloroplast genome sequence matrix, using maximum likelihood, maximum parsimony, and compatibility methods. Analyses of all three data sets strongly supported liverworts as the sister to all other land plants, and analyses of the multigene and chloroplast genome matrices provided moderate to strong support for hornworts as the sister to vascular plants. These results highlight the important roles of liverworts and hornworts in two major events of plant evolution: the water-to-land transition and the change from a haploid gametophyte generation-dominant life cycle in bryophytes to a diploid sporophyte generation-dominant life cycle in vascular plants. This study also demonstrates the importance of using a multifaceted approach to resolve difficult nodes in the tree of life. In particular, it is shown here that densely sampled taxon trees built with multiple genes provide an indispensable test of taxon-sparse trees inferred from genome sequences.     

Anthropoid humeri from the late Eocene of Egypt

A number of recent studies have, by necessity, placed a great deal of emphasis on the dental evidence for Paleogene anthropoid interrelationships, but cladistic analyses of these data have led to the erection of phylogenetic hypotheses that appear to be at odds with biogeographic and stratigraphic considerations. Additional morphological data from the cranium and postcranium of certain poorly understood Paleogene primates are clearly needed to help test whether such hypotheses are tenable. Here we describe humeri attributable to Proteopithecus sylviae and Catopithecus browni, two anthropoids from late Eocene sediments of the Fayum Depression in Egypt. Qualitative and morphometric analyses of these elements indicate that humeri of the oligopithecine Catopithecus are more similar to early Oligocene propliopithecines than they are to any other Paleogene anthropoid taxon, and that Proteopithecus exhibits humeral similarities to parapithecids that may be symplesiomorphies of extant (or "crown") Anthropoidea. The humeral morphology of Catopithecus is consistent with certain narrowly distributed dental apomorphies-such as the loss of the upper and lower second premolar and the development of a honing blade for the upper canine on the lower third premolar-which suggest that oligopithecines constitute the sister group of a clade containing propliopithecines and Miocene-Recent catarrhines and are not most closely related to Proteopithecus as has recently been proposed.     

Chloroplast and mitochondrial DNA are paternally inherited in Sequoia sempervirens D. Don Endl

Restriction fragment length polymorphisms in controlled crosses were used to infer the mode of inheritance of chloroplast DNA and mitochondrial DNA in coast redwood (Sequoia sempervirens D. Don Endl.). Chloroplast DNA was paternally inherited, as is true for all other conifers studied thus far. Surprisingly, a restriction fragment length polymorphism detected by a mitochondrial probe was paternally inherited as well. This polymorphism could not be detected in hybridizations with chloroplast probes covering the entire chloroplast genome, thus providing evidence that the mitochondrial probe had not hybridized to chloroplast DNA on the blot. We conclude that mitochondrial DNA is paternally inherited in coast redwood. To our knowledge, paternal inheritance of mitochondrial DNA in sexual crosses of a multicellular eukaryotic organism has not been previously reported.     

Massive horizontal transfer of mitochondrial genes from diverse land plant donors to the basal angiosperm Amborella

Several plants are known to have acquired a single mitochondrial gene by horizontal gene transfer (HGT), but whether these or any other plants have acquired many foreign genes is entirely unclear. To address this question, we focused on Amborella trichopoda, because it was already known to possess one horizontally acquired gene and because it was found in preliminary analyses to contain several more. We comprehensively sequenced the mitochondrial protein gene set of Amborella, sequenced a variable number of mitochondrial genes from 28 other diverse land plants, and conducted phylogenetic analyses of these sequences plus those already available, including the five sequenced mitochondrial genomes of angiosperms. Results indicate that Amborella has acquired one or more copies of 20 of its 31 known mitochondrial protein genes from other land plants, for a total of 26 foreign genes, whereas no evidence for HGT was found in the five sequenced genomes. Most of the Amborella transfers are from other angiosperms (especially eudicots), whereas others are from nonangiosperms, including six striking cases of transfer from (at least three different) moss donors. Most of the transferred genes are intact, consistent with functionality and/or recency of transfer. Amborella mtDNA has sustained proportionately more HGT than any other eukaryotic, or perhaps even prokaryotic, genome yet examined.     

Phylogeny and evolution of antlered deer determined from mitochondrial DNA sequences.

Mitochondrial DNA sequences of both ribosomal RNA genes and three adjacent transfer RNA genes were obtained for the three extant subfamilies of antlered deer (Cervinae, Muntiacinae, and Odocoileinae) as well as for their antlerless sister group Hydropotinae (family Cervidae). Phylogenetic analysis of these sequences (each nearly 2.7 kilobase pairs in length) supports a cervine/muntiacine clade to the exclusion of odocoileines. These results are statistically significant, stable, and congruent with some independent data. Our mitochondrial DNA sequences, when coupled with other information, indicate that the earliest fossil antlered deer are not closely related to living muntiacines or any other contemporary subfamily. From this information, we hypothesize an Old World, Late Miocene origin of Odocoileinae.