Deuterostome phylogeny and the sister group of the chordates: evidence from molecules and morphology

Complete coding regions of the 18S rRNA gene of an enteropneust hemichordate and an echinoid and ophiuroid echinoderm were obtained and aligned with 18S rRNA gene sequences of all major chordate clades and four outgroups. Gene sequences were analyzed to test morphological character phylogenies and to assess the strength of the signal. Maximum- parsimony analysis of the sequences fails to support a monophyletic Chordata; the urochordates form the sister taxon to the hemichordates, and together this clade plus the echinoderms forms the sister taxon to the cephalochordates plus craniates. Decay, bootstrap, and tree-length distribution analyses suggest that the signal for inference of dueterostome phylogeny is weak in this molecule. Parsimony analysis of morphological plus molecular characters supports both monophyly of echinoderms plus enteropneust hemichordates and a sister group relationship of this clade to chordates. Evolutionary parsimony does not support chordate monophyly. Neighbor-joining, Fitch-Margoliash, and maximum-likelihood analyses support a chordate lineage that is the sister group to an echinoderm-plus-hemichordate lineage. The results illustrate both the limitations of the 18S rRNA molecule alone for high- level phylogeny inference and the importance of considering both molecular and morphological data in phylogeny reconstruction.      

The interrelationships of the echinoderm classes: morphological and molecular evidence

The relationship between the five echinoderm classes has perplexed phylogeneticists for some time. Although each of the crinoids (C), asteroids (A), ophiuroids (O), echinoids (E) and holothuroids (H) are morphologically distinct, evidence from larval and adult morphology, molecular biology, and stratigraphy have failed to provide a single consensus solution. We have reviewed all available morphological and molecular data, added new data and reanalysed independent data sets individually and in combination, in order to resolve echinoderm class relationships. In total, we present 21 larval and 50 adult morphological characters, partial 28S-like large subunit rRNA gene data for 39 taxa and complete 18S-like small subunit rRNA gene data for 37 taxa. For a 5 taxon problem there are 105 possible rooted tree topologies, and yet we were consistently presented with three competing hypotheses when data sets were analysed both individually and in combination. The total evidence solution favoured (outgroup(C(A(O(E, H))))) although the alternative tree topology, (outgroup(C(O(A(E, H))))) was only one step longer and (outgroup(C((A, O),(E, H)))) was two steps longer. Only these three trees are serious contenders and the distribution of morphological characters suggests we should discount the solution placing ophiuroids as sister group to an asteroid+echinoid + holothurian clade. Thus we are left with (outgroup(C(A(O(E, H))))) and (outgroup(C((A, O),(E, H)))) as the two most plausible phylogenetic hypotheses. Our data showed high levels of phylogenetic signal and these trees best fit the available data.     

Rooting molecular trees: problems and strategies

In constructing phylogenies from molecular data both the composition of the ingroup and the choice of outgroup can strongly affect the chances of obtaining the correct topology. This is because tree topology and uneven rates of molecular evolution affect the ability of tree-building algorithms to find the correct tree. Outgroups can be added in one of two ways, either to a single sister clade (preferably the closest) or through addition of single taxa from different clades. On theoretical and empirical grounds the former strategy is shown to be much more beneficial, both in terms of redressing balance and reducing stemminess. For parsimony to perform well some selection of sequence data, through omission or weighting, is also often necessary to reduce levels of homoplasy.     

Phylogenetic relationships of Steinernema Travassos, 1927 (Nematoda: Cephalobina: Steinernematidae) based on nuclear, mitochondrial and morphological data

Entomopathogenic nematodes of the genus Steinernema are lethal parasites of insects that are used as biological control agents of several lepidopteran, dipteran and coleopteran pests. Phylogenetic relationships among 25 Steinernema species were estimated using nucleotide sequences from three genes and 22 morphological characters. Parsimony analysis of 28S (LSU) sequences yielded a well-resolved phylogenetic hypothesis with reliable bootstrap support for 13 clades. Parsimony analysis of mitochondrial DNA sequences (12S rDNA and cox 1 genes) yielded phylogenetic trees with a lower consistency index than for LSU sequences, and with fewer reliably supported clades. Combined phylogenetic analysis of the 3-gene dataset by parsimony and Bayesian methods yielded well-resolved and highly similar trees. Bayesian posterior probabilities were high for most clades; bootstrap (parsimony) support was reliable for approximately half of the internal nodes. Parsimony analysis of the morphological dataset yielded a poorly resolved tree, whereas total evidence analysis (molecular plus morphological data) yielded a phylogenetic hypothesis consistent with, but less resolved than trees inferred from combined molecular data. Parsimony mapping of morphological characters on the 3-gene trees showed that most structural features of steinernematids are highly homoplastic. The distribution of nematode foraging strategies on these trees predicts that S. hermaphroditum, S. diaprepesi and S. longicaudum (US isolate) have cruise forager behaviours.     

Phylogeny of sea skaters, Halobates Eschscholtz (Hemiptera, Gerridae), based on mtDNA sequence and morphology

We examined phylogenetic relationships among halobatine water striders (Hemiptera, Gerridae) using molecular and morphological data. The molecular data set was 780 bp DNA sequence data from the 3' half of the mitochondria! gene encoding cytochrome oxidase subunit I from 19 species of sea skaters, Halobates , and one species from each of three related genera, Asclepios annandalei, Austrobates rivularis , and Eurymetra natalensis. The morphological data set was a slightly modified version of a previously published data set. Unweighted parsimony analyses of the molecular data set gave one tree with weak support for most branches. Maximum likelihood analysis of the same data set gave a tree with slightly different topology, but reveiled many of the clades found in parsimony analyses of the morphological data set. Parsimony analyses of the combined molecular + morphology data sets gave a better resolved and better supported tree than did analyses of any single data set. The phytogeny of Halobates presented here allows a more rigorous evaluation of several prior hypotheses about evolutionary processes in marine water striders. In particular, it supports the hypothesis of at least two separate transitions from coastal to oceanic environments.     

The phylogenetic relationships of the suborder Acanthuroidei (Teleostei: Perciformes) based on molecular and morphological evidence

Fragments of 12S and 16S mitochondrial DNA genes were sequenced for 14 acanthuroid taxa (representing all six families) and seven outgroup taxa. The combined data set contained 1399 bp after removal of all ambiguously aligned positions. Examination of site saturation indicated that loop regions of both genes are saturated for transitions, which led to a weighted parsimony analysis of the data set. The resulting tree topology generally agreed with previous morphological hypotheses, most notably placing the Luvaridae within the Acanthuroidei, but it also differed in several areas. The putative sister group of Acanthuroidei, Drepane, was recovered within the suborder, and the sister group of the family Acanthuridae, Zanclus, was likewise recovered within the family. Morphological characters were included to produce a combined data set of 1585 characters for 14 acanthuroid taxa and a single outgroup taxon. An analysis of the same 15 taxa was performed with only the DNA data for comparison. The total-evidence analysis supports the monophyly of the Acanthuridae. A parametric bootstrap suggests the possibility that the paraphyly of Acanthuridae indicated by the molecular analyses is the result of long-branch attraction. The disagreement between molecular and morphological data on the relationships of the basal acanthuroids and its putative sister taxon is unresolved.     

Phylogenetic relationships of Steinernema Travassos, 1927 (Nematoda: Cephalobina: Steinernematidae) based on nuclear, mitochondrial and morphological data

Entomopathogenic nematodes of the genus Steinernema are lethal parasites of insects that are used as biological control agents of several lepidopteran, dipteran and coleopteran pests. Phylogenetic relationships among 25 Steinernema species were estimated using nucleotide sequences from three genes and 22 morphological characters. Parsimony analysis of 28S (LSU) sequences yielded a well-resolved phylogenetic hypothesis with reliable bootstrap support for 13 clades. Parsimony analysis of mitochondrial DNA sequences (12S rDNA and cox 1 genes) yielded phylogenetic trees with a lower consistency index than for LSU sequences, and with fewer reliably supported clades. Combined phylogenetic analysis of the 3-gene dataset by parsimony and Bayesian methods yielded well-resolved and highly similar trees. Bayesian posterior probabilities were high for most clades; bootstrap (parsimony) support was reliable for approximately half of the internal nodes. Parsimony analysis of the morphological dataset yielded a poorly resolved tree, whereas total evidence analysis (molecular plus morphological data) yielded a phylogenetic hypothesis consistent with, but less resolved than trees inferred from combined molecular data. Parsimony mapping of morphological characters on the 3-gene trees showed that most structural features of steinernematids are highly homoplastic. The distribution of nematode foraging strategies on these trees predicts that S. hermaphroditum, S. diaprepesi and S. longicaudum (US isolate) have cruise forager behaviours.     

Phylogeny in Echinocereus (Cactaceae) based on combined morphological and molecular evidence: taxonomic implications

Echinocereus is a morphologically diverse genus that includes 64 species grouped into eight taxonomic sections based on morphological traits. In previous molecular phylogenetic analyses, the relationships amongst Echinocereus species were not entirely revealed and useful characters to recognize clades were not provided. The inclusion of several sources of evidence in a phylogenetic analysis is likely to produce more supported hypotheses. Therefore, we performed a combined phylogenetic analysis with a set of 44 morphological characters and six chloroplast DNA sequences. Topologies from parsimony and Bayesian analyses were mostly congruent. However, the relationships of E. poselgeri were not consistent between analyses. A second Bayesian analysis using a long-branch extraction test resulted in a topology with the morphological position of E. poselgeri congruent with that in parsimony analysis. Parsimony and Bayesian analyses corroborated the monophyly of Echinocereus, which included eight monophyletic groups. The combined phylogeny integrated into different clades those taxa that were not determined in previous analyses and changed the relationships of some recognized clades. The clades did not recover the recent infrageneric classification. In the present study, a new sectional classification for Echinocereus is proposed based on the eight recovered clades, which is supported by a combination of morphological and molecular characters. An identification key for sections in the genus is included.     

Phylogeny of the arachnid order Opiliones (Arthropoda) inferred from a combined approach of complete 18S and partial 28S ribosomal DNA sequences and morphology

The phylogenetic relationships among the main evolutionary lines of the arachnid order Opiliones were investigated by means of molecular (complete 18S rDNA and the D3 region of the 28S rDNA genes) and morphological data sets. Equally and differentially weighted parsimony analyses of independent and combined data sets provide evidence for the monophyly of the Opiliones. In all the analyses, the internal relationships of the group coincide in the monophyly of the following main groups: Cyphophthalmi, Eupnoi Palpatores, Dyspnoi Palpatores, and Laniatores. The Cyphophthalmi are monophyletic and sister to a clade that includes all the remaining opilionid taxa (=Phalangida). Within the Phalangida the most supported hypothesis suggests that Palpatores are paraphyletic, as follows: (Eupnoi (Dyspnoi + Laniatores)), but the alternative hypothesis (Laniatores (Eupnoi + Dyspnoi)) is more parsimonious in some molecular data analyses. Relationships within the four main clades are also addressed. Evolution of some morphological characters is discussed, and plesiomorphic states of these characters are evaluated using molecular data outgroup polarization. Finally, Martens' hypothesis of opilionid evolution is assessed in relation to our results.     

Molecular phylogenetics of the butterflyfishes (Chaetodontidae): taxonomy and biogeography of a global coral reef fish family

Marine butterflyfishes (10 genera, 114 species) are conspicuously beautiful and abundant animals found on coral reefs worldwide, and are well studied due to their ecological importance and commercial value. Several phylogenies based on morphological and molecular data exist, yet a well-supported molecular phylogeny at the species level for a wide range of taxa remains to be resolved. Here we present a molecular phylogeny of the butterflyfishes, including representatives of all genera (except Parachaetodon) and at least one representative of all commonly cited subgenera of Chaetodon (except Roa sensuBlum, 1988). Genetic data were collected for 71 ingroup and 13 outgroup taxa, using two nuclear and three mitochondrial genes that total 3332 nucleotides. Bayesian inference, parsimony, and maximum likelihood methods produced a well-supported phylogeny with strong support for a monophyletic Chaetodontidae. The Chaetodon subgenera Exornator and Chaetodon were found to be polyphyletic, and the genus Amphichaetodon was not the basal sister group to the rest of the family as had been previously proposed. Molecular phylogenetic analysis of data from 5 genes resolved some clades in agreement with previous phylogenetic studies, however the topology of relationships among major butterflyfish groups differed significantly from previous hypotheses. The analysis recovered a clade containing Amphichaetodon, Coradion, Chelmonops, Chelmon, Forcipiger, Hemitaurichthys, Johnrandallia, and Heniochus. Prognathodes was resolved as the sister to all Chaetodon, as in previous hypotheses, although the topology of subgeneric clades differed significantly from hypotheses based on morphology. We use the species-level phylogeny for the butterflyfishes to resolve long-standing questions regarding the use of subgenera in Chaetodon, to reconstruct molecular rates and estimated dates of diversification of major butterflyfish clades, and to examine global biogeographic patterns.     

Relationships among seed plants inferred from highly conserved genes: sorting conflicting phylogenetic signals among ancient lineages

Phylogenetic studies based on different types and treatment of data provide substantially conflicting hypotheses of relationships among seed plants. We conducted phylogenetic analyses of sequences of two highly conserved chloroplast genes, psaA and psbB, for a comprehensive taxonomic sample of seed plants and land plants. Parsimony analyses of two different codon position partitions resulted in well-supported, but significantly conflicting, phylogenetic trees. First and second codon positions place angiosperms and gymnosperms as sister clades and Gnetales as sister to Pinaceae. Third positions place Gnetales as sister to all other seed plants. Maximum likelihood trees for the two partitions are also in conflict. Relationships among the main seed plant clades according to first and second positions are similar to those found in parsimony analysis for the same data, but the third position maximum likelihood tree is substantially different from the corresponding parsimony tree, although it agrees partially with the first and second position trees in placing Gnetales as the sister group of Pinaceae. Our results document high rate heterogeneity among lineages, which, together with the greater average rate of substitution for third positions, may reduce phylogenetic signal due to long-branch attraction in parsimony reconstructions. Whereas resolution of relationships among major seed plant clades remains pending, this study provides increased support for relationships within major seed plant clades.     

IRREGULAR ECHINOIDS—AN INSUFFICIENTLY KNOWN GROUP

Some aspects of echinoid evolution from an endocyclic towards an exocyclic apical system, in the light of embryological and palaeontological data, are discussed. A primitive feature—imbrication of interamhulacral plates in Jurassic irregular echinoids, recently described—proves once more that this group is insufficiently known. Every revision of taxonomic or phylogenetic concepts concerning this group of echinoids should be preceded by careful morphological analysis of the material.     

Relationships among the major lineages of Dictyoptera: the effect of outgroup selection on dictyopteran tree topology

Abstract Dictyoptera, comprising Blattaria, Isoptera, and Mantodea, are diverse in appearance and life history, and are strongly supported as monophyletic. We downloaded COII, 16S, 18S, and 28S sequences of 39 dictyopteran species from GenBank. Ribosomal RNA sequences were aligned manually with reference to secondary structure. We included morphological data (maximum of 175 characters) for 12 of these taxa and for an additional 15 dictyopteran taxa (for which we had only morphological data). We had two datasets, a 59‐taxon dataset with five outgroup taxa, from Phasmatodea (2 taxa), Mantophasmatodea (1 taxon), Embioptera (1 taxon), and Grylloblattodea (1 taxon), and a 62‐taxon dataset with three additional outgroup taxa from Plecoptera (1 taxon), Dermaptera (1 taxon) and Orthoptera (1 taxon). We analysed the combined molecular?morphological dataset using the doublet and MK models in Mr Bayes , and using a parsimony heuristic search in paup . Within the monophyletic Mantodea, Mantoida is recovered as sister to the rest of Mantodea, followed by Chaeteessa; the monophyly of most of the more derived families as defined currently is not supported. We recovered novel phylogenetic hypotheses about the taxa within Blattodea (following Hennig, containing Isoptera). Unique to our study, one Bayesian analysis places Polyphagoidea as sister to all other Dictyoptera; other analyses and/or the addition of certain orthopteran sequences, however, place Polyphagoidea more deeply within Dictyoptera. Isoptera falls within the cockroaches, sister to the genus Cryptocercus. Separate parsimony analyses of independent gene fragments suggest that gene selection is an important factor in tree reconstruction. When we varied the ingroup taxa and/or outgroup taxa, the internal dictyopteran relationships differed in the position of several taxa of interest, including Cryptocercus, Polyphaga, Periplaneta and Supella. This provides further evidence that the choice of both outgroup and ingroup taxa greatly affects tree topology.     

Evolutionary relationships, interisland biogeography, and molecular evolution in the Hawaiian violets (Viola: Violaceae)

The endemic Hawaiian flora offers remarkable opportunities to study the patterns of plant morphological and molecular evolution. The Hawaiian violets are a monophyletic lineage of nine taxa distributed across six main islands of the Hawaiian archipelago. To describe the evolutionary relationships, biogeography, and molecular evolution rates of the Hawaiian violets, we conducted a phylogenetic study using nuclear rDNA internal transcribed spacer sequences from specimens of each species. Parsimony, maximum likelihood (ML), and Bayesian inference reconstructions of island colonization and radiation strongly suggest that the Hawaiian violets first colonized the Maui Nui Complex, quickly radiated to Kaua'i and O'ahu, and recently dispersed to Hawai'i. The lineage consists of "wet" and "dry" clades restricted to distinct precipitation regimes. The ML and Bayesian inference reconstructions of shifts in habitat, habit, and leaf shape indicate that ecologically analogous taxa have undergone parallel evolution in leaf morphology and habit. This parallel evolution correlates with shifts to specialized habitats. Relative rate tests showed that woody and herbaceous sister species possess equal molecular evolution rates. The incongruity of molecular evolution rates in taxa on younger islands suggests that these rates may not be determined by growth form (or lifespan) alone, but may be influenced by complex dispersal events.     

Phylogenetic relationships in Taxodiaceae and Cupressaceae sensu stricto based on matK gene, chlL gene, trnL-trnF IGS region, and trnL intron sequences

Nucleotide sequences from four chloroplast genes, the matK, chlL, intergenic spacer (IGS) region between trnL and trnF, and an intron of trnL, were determined from all species of Taxodiaceae and five species of Cupressaceae sensu stricto (s.s.). Phylogenetic trees were constructed using the maximum parsimony and the neighbor-joining methods with Cunninghamia as an outgroup. These analyses provided greater resolution of relationships among genera and higher bootstrap supports for clades compared to previous analyses. Results indicate that Taiwania diverged first, and then Athrotaxis diverged from the remaining genera. Metasequoia, Sequoia, and Sequoiadendron form a clade. Taxodium and Glyptostrobus form a clade, which is the sister to Cryptomeria. Cupressaceae s.s. are derived from within Taxodiaceae, being the most closely related to the Cryptomeria/Taxodium/Glyptostrobus clade. These relationships are consistent with previous morphological groupings and the analyses of molecular data. In addition, we found acceleration of evolutionary rates in Cupressaceae s.s. Possible causes for the acceleration are discussed.     

Reconstructing the evolutionary history of the Lorisidae using morphological, molecular, and geological data

Major aspects of lorisid phylogeny and systematics remain unresolved, despite several studies (involving morphology, histology, karyology, immunology, and DNA sequencing) aimed at elucidating them. Our study is the first to investigate the evolution of this enigmatic group using molecular and morphological data for all four well-established genera: Arctocebus, Loris, Nycticebus, and Perodicticus. Data sets consisting of 386 bp of 12S rRNA, 535 bp of 16S rRNA, and 36 craniodental characters were analyzed separately and in combination, using maximum parsimony and maximum likelihood. Outgroups, consisting of two galagid taxa (Otolemur and Galagoides) and a lemuroid (Microcebus), were also varied. The morphological data set yielded a paraphyletic lorisid clade with the robust Nycticebus and Perodicticus grouped as sister taxa, and the galagids allied with Arctocebus. All molecular analyses maximum parsimony (MP) or maximum likelihood (ML) which included Microcebus as an outgroup rendered a paraphyletic lorisid clade, with one exception: the 12S + 16S data set analyzed with ML. The position of the galagids in these paraphyletic topologies was inconsistent, however, and bootstrap values were low. Exclusion of Microcebus generated a monophyletic Lorisidae with Asian and African subclades; bootstrap values for all three clades in the total evidence tree were over 90%. We estimated mean genetic distances for lemuroids vs. lorisoids, lorisids vs. galagids, and Asian vs. African lorisids as a guide to relative divergence times. We present information regarding a temporary land bridge that linked the two now widely separated regions inhabited by lorisids that may explain their distribution. Finally, we make taxonomic recommendations based on our results.     

Molecular phylogeny of new world primates (Platyrrhini) based on beta2-microglobulin DNA sequences

Neotropical primates, traditionally grouped in the infraorder Platyrrhini, comprise 16 extant genera. Cladistic analyses based on morphological characteristics and molecular data resulted in topologic arrangements depicting disparate phylogenetic relationships, indicating that the evolution of gross morphological characteristics and molecular traits is not necessarily congruent. Here we present a phylogenetic arrangement for all neotropical primate genera obtained from DNA sequence analyses of the beta2-microglobulin gene. Parsimony, distance, and maximum likelihood analyses favored two families, Atelidae and Cebidae, each containing 8 genera. Atelids were resolved into atelines and pitheciines. The well-supported ateline clade branched into alouattine (Alouatta) and ateline (Ateles, Lagothrix, Brachyteles) clades. In turn, within the Ateline clade, Lagothrix and Brachyteles were well-supported sister groups. The pitheciines branched into well-supported callicebine (Callicebus) and pitheciine (Pithecia, Cacajao, Chiropotes) clades. In turn, within the pitheciine clade, Cacajao and Chiropotes were well-supported sister groups. The cebids branched into callitrichine (Saguinus, Leontopithecus, Callimico, Callithrix-Cebuella), cebine (Cebus, Saimiri), and aotine (Aotus) clades. While the callitrichine clade and the groupings of species and genera within this clade were all well supported, the cebine clade received only modest support, and the position of Aotus could not be clearly established. Cladistic analyses favored the proposition of 15 rather than 16 extant genera by including Cebuella pygmaea in the genus Callithrix as the sister group of the Callithrix argentata species group. These analyses also favored the sister grouping of Callimico with Callithrix and then of Leontopithecus with the Callithrix-Callimico clade.     

Molecular phylogenetic relationships among Lemnaceae and Araceae using the chloroplast trnL-trnF intergenic spacer

We test competing hypotheses of relationships among Aroids (Araceae) and duckweeds (Lemnaceae) using sequences of the trnL-trnF spacer region of the chloroplast genome. Included in the analysis were 22 aroid genera including Pistia and five genera of Lemnaceae including the recently segregated genus Landoltia. Aponogeton was used as an outgroup to root the tree. A data set of 522 aligned nucleotides yielded maximum parsimony and maximum likelihood trees similar to those previously derived from restriction site data. Pistia and the Lemnaceae are placed in two separate and well-supported clades, suggesting at least two independent origins of the floating aquatic growth form within the aroid clade. Within the Lemnaceae there is only partial support for the paradigm of sequential morphological reduction, given that Wolffia is sister to Wolffiella+Lemna. As in the results of the restriction site analysis, pantropical Pistia is placed with Colocasia and Typhonium of southeastern Asia, indicative of Old World affinities. Branch lengths leading to duckweed terminal taxa are much longer relative to other ingroup taxa (including Pistia), evidently as a result of higher rates of nucleotide substitutions and insertion/deletion events. Morphological reduction within the duckweeds roughly correlates with accelerated chloroplast genome evolution.     

Phylogenetic relationships and rates of allozyme evolution among the lineages of sceloporine sand lizards

Parsimony analysis of characters derived from an electrophoretic survey of allozyme variation in the sceloporine sand lizards indicates that Uma is outside of a clade formed by the rest of the sand lizards and that Cophosaurus and Holbrookia share a more recent common ancestor with one another than either does with Callisaurus. Previous electrophoretic studies used phenetic clustering based on genetic distance data to assess relationships among these taxa. The resulting dendrograms were used to argue that Holbrookia is the sister group of all other sand lizards and that Callisaurus and Cophosaurus are sister taxa. When reanalysed using parsimony methods, the data from these previous studies are found to support the conclusions of the present study, namely, that Uma rather than Holbrookia is the sister group of all other sand lizards and that Cophosaurus is the sister taxon of Holbrookia rather than of Callisaurus. Relative rate tests indicate that the incongruencies between branching diagrams derived from phenetic clustering of genetic distances versus those derived from parsimony analysis of electrophoretic characters are attributable to increased rates of protein evolution in the Holbrookia lineage.     

Systematics of Nothofagus (Nothofagaceae) based on rDNA spacer sequences (ITS): taxonomic congruence with morphology and plastid sequences

Phylogenetic relationships were examined within the southern beech family Nothofagaceae using 22 species representing the four currently recognized subgenera and related outgroups. Nuclear ribosomal DNA sequences encoding the 5.8s rRNA and two flanking internal transcribed spacers (ITS) provided 95 phylogenetically informative nucleotide sites from a single alignment of ~588 bases per species. Parsimony analysis of this variation produced two equally parsimonious trees supporting four monophyletic groups, which correspond to groups designated by pollen type. These topologies were compared to trees from reanalyses of previously reported rbcL sequences and a modified morphological data set. Results from parsimony analysis of the three data sets were highly congruent, with topological differences restricted to the placement of a few terminal taxa. Combined analysis of molecular and morphological data produced six equally parsimonious trees. The consensus of these trees suggests two basal clades within Nothofagus. Within the larger of the two clades, tropical Nothofagus (subgenus Brassospora) of New Guinea and New Caledonia are strongly supported as sister to cool-temperate species of South America (subgenus Nothofagus). Most of the morphological apomorphies of the cupule, fruit, and pollen of Nothofagus are distributed within this larger clade. An area cladogram based on the consensus of combined data supports three trans-Antarctic relationships, two within pollen groups and one between pollen groups. Fossil data support continuous ancestral distributions for all four pollen groups prior to continental drift; therefore, vicariance adequately explains two of these disjunctions. Extinction of trans-Antarctic sister taxa within formerly widespread pollen groups explains the third disjunction; this results in a biogeographic pattern indicative of phylogenetic relationship not vicariance. For the biogeographically informative vicariant clades, area relationships based on total evidence support the recently advanced hypothesis that New Zealand and Australia share a unique common ancestry. Contrary to previous thought, the distribution of extant Nothofagus is informative on the area relationships of the Southern Hemisphere, once precise phylogenetic relationships are placed in the context of fossil data.