Higher taxonomic relationships among extant mammals based on morphology, with selected comparisons of results from molecular data.

Until a few decades ago, phylogenetic relationships among placental orders were ambiguous and usually depicted to radiate as an unresolved "bush." Resolution of this bush by various workers has been progressing slowly, but with promising results corroborated by nondental, dental, and molecular characters. In this study we continue to seek resolution. A total of 258 nondental and 2 dental characters was analyzed by PAUP and MacClade on 39 vertebrate taxa (3 reptiles, 1 nonmammalian therapsid, and 35 mammals; 20 of the mammals are extant and 15 are extinct) to study higher taxonomic relationships with emphasis on Placentalia (Eutheria). About two-thirds of the characters are osteological, the rest concern soft tissues, including myological but excluding molecular characters (most are our data, the rest are from the literature). Cladistic analysis included all 39 taxa (fossil taxa help to evaluate polarities of characters) and all characters were given equal weight. Extant Mammalia are divided into Prototheria and Theria, the latter into Marsupialia and Placentalia. Placentalia comprises Xenarthra and Epitheria. Within Epitheria, Lipotyphla and Preptotheria (emended) are sister-taxa. Preptotherian taxa group into: ungulate-related taxa and various nonungulates. The former include Carnivora, Pholidota, Tubulidentata, Artiodactyla, Cetacea, Perissodactyla, Hyracoidea, Proboscidea, and Sirenia. A possible association to embrace Lagomorpha, Rodentia, Macroscelidea, Scandentia, Primates, Chiroptera, and Dermoptera is suggested. Significant differences between our findings and those of recent investigators include the dissociation of Pholidota from Xenarthra and the plesiomorphous position of Lipotyphla within Epitheria. Congruence between morphological and molecular results is closer than previously reported.

Toward a phylogenetic classification of Primates based on DNA evidence complemented by fossil evidence.

A highly resolved primate cladogram based on DNA evidence is congruent with extant and fossil osteological evidence. A provisional primate classification based on this cladogram and the time scale provided by fossils and the model of local molecular clocks has all named taxa represent clades and assigns the same taxonomic rank to those clades of roughly equivalent age. Order Primates divides into Strepsirhini and Haplorhini. Strepsirhines divide into Lemuriformes and Loriformes, whereas haplorhines divide into Tarsiiformes and Anthropoidea. Within Anthropoidea when equivalent ranks are used for divisions within Platyrrhini and Catarrhini, Homininae divides into Hylobatini (common and siamang gibbon) and Hominini, and the latter divides into Pongina for Pongo (orangutans) and Hominina for Gorilla and Homo. Homo itself divides into the subgenera H. (Homo) for humans and H. (Pan) for chimpanzees and bonobos. The differences between this provisional age related phylogenetic classification and current primate taxonomies are discussed.

Understanding proboscidean evolution: a formidable task.

A new approach to proboscidean evolution depicts taxa in three major radiations. This approach highlights general proboscidean evolutionary trends and origins more than the specific relationships among them. Data from more than 55 million years of evolution help to interpret how the integration of primitive and derived characters was essential to proboscidean success. Only two, or perhaps three, species remain of approximately 164 that lived in the past. Extinct forms were extremely cosmopolitan, occupying a variety of habitats, from deserts to mountain tops, on all continents except Australia and Antarctica. Challenges for future investigators include a better understanding of structure and function of infrasonic call production and perception, brain features, and reproductive biology in extinct proboscideans based on inferences from living forms.

Proboscidean DNA from museum and fossil specimens: an assessment of ancient DNA extraction and amplification techniques.

Applications of reliable DNA extraction and amplification techniques to postmortem samples are critical to ancient DNA research. Commonly used methods for isolating DNA from ancient material were tested and compared using both soft tissue and bones from fossil and contemporary museum proboscideans. DNAs isolated using three principal methods served as templates in subsequent PCR amplifications, and the PCR products were directly sequenced. Authentication of the ancient origin of obtained nucleotide sequences was established by demonstrating reproducibility under a blind testing system and by phylogenetic analysis. Our results indicate that ancient samples may respond differently to extraction buffers or purification procedures, and no single method was universally successful. A CTAB buffer method, modified from plant DNA extraction protocols, was found to have the highest success rate. Nested PCR was shown to be a reliable approach to amplify ancient DNA templates that failed in primary amplification.

A blind testing design for authenticating ancient DNA sequences.

Reproducibility is a serious concern among researchers of ancient DNA. We designed a blind testing procedure to evaluate laboratory accuracy and authenticity of ancient DNA obtained from closely related extant and extinct species. Soft tissue and bones of fossil and contemporary museum proboscideans were collected and identified based on morphology by one researcher, and other researchers carried out DNA testing on the samples, which were assigned anonymous numbers. DNA extracted using three principal isolation methods served as template in PCR amplifications of a segment of the cytochrome b gene (mitochondrial genome), and the PCR product was directly sequenced and analyzed. The results show that such a blind testing design performed in one laboratory, when coupled with phylogenetic analysis, can nonarbitrarily test the consistency and reliability of ancient DNA results. Such reproducible results obtained from the blind testing can increase confidence in the authenticity of ancient sequences obtained from postmortem specimens and avoid bias in phylogenetic analysis. A blind testing design may be applicable as an alternative to confirm ancient DNA results in one laboratory when independent testing by two laboratories is not available.

Phylogenetic resolution within the Elephantidae using fossil DNA sequence from the American mastodon (Mammut americanum) as an outgroup.

DNA was extracted from the extinct American mastodon, the extinct woolly mammoth, and the modern Asian and African elephants to test the traditional morphologically based phylogeny within Elephantidae. Phylogenetic analyses of the aligned sequences of the mitochondrial gene cytochrome b support a monophyletic Asian elephant-woolly mammoth clade when the American mastodon is used as an outgroup. Previous molecular studies were unable to resolve the relationships of the woolly mammoth, Asian elephant, and African elephant because the sequences appear to have evolved at heterogeneous rates and inappropriate outgroups were used for analysis. The results demonstrate the usefulness of fossil molecular data from appropriate sister taxa for resolving phylogenies of highly derived or early radiating lineages.

Primate phylogeny: morphological vs. molecular results.

Our comparative study of morphological (our data on selected living primates) and molecular characters (from the literature) confirms that, overall, phylogenetic reconstructions of Primates, and consequently their classifications, are more similar than dissimilar. When data from fossil Primates are incorporated, there may be several possible relationships among living Primates; the difference between most of them hinges mainly on the position of Tarsius. In one hypothesis, tarsiers are closely related to lemurs and lorises, and thus Primates is divided into Prosimii [lorises, lemurs, and tarsiers] and Anthropoidea [Platyrrhini and Catarrhini, i.e., monkeys, apes, and humans]. Two additional alternatives are that Tarsius is a sister group to the clade embracing lorises + lemurs and Anthropoidea and that in which all three lineages (Tarsius, lorises + lemurs, and Anthropoidea) form a polychotomy. In another hypothesis, tarsiers are closely related to anthropoids, giving these two branches: Strepsirhini [lemurs, lorises] and Haplorhini [tarsiers and Anthropoidea (Platyrrhini, the New World monkeys, and Catarrhini, Old World monkeys and Hominoidea)]. The first three alternatives gain some support from the fossil record, and the fourth from morphology of the living Tarsius and molecular data. It is emphasized that the morphological characters employed in this study for Tarsius are based on the only surviving genus of once-diverse tarsiiform primates known from the Eocene, and, although considered a "living fossil," it cannot represent all of them. Furthermore, Tarsius embodies derived features of its own which may affect its systematic position, but not necessarily the position of Tarsiiformes. Although the early Tertiary adapoids might have more nearly resembled anthropoids in their biochemistry and placental developments, this hypothesis is not testable from fossils, and any inferred relationships here must be based on characters of skeletal anatomy. Alternatively, anthropoids may be derived from certain omomyids or from some as yet undiscovered Eocene African taxon. Close relationships among Homo, Pan, and Gorilla have been confirmed during recent decades; Pongo is the sister group to this trichotomy. With increasing molecular data, Homo and Pan appear to be closer to each other than to any other living hominid taxon. Gorilla is a sister group to the Homo-Pan clade and Pongo is a sister group to all of them. Morphologists have given limited evidence for such a dichotomous grouping. In this study, we support the Homo-Pan clade, although with characters not as strong as for other clades.

Mammalian phylogeny: comparison of morphological and molecular results.

In an attempt to resolve the "bushy" part at the root of the eutherian tree, 182 nondental morphological characters from 100 species (79 extant and 21 extinct; 98 mammalian and 2 nonmammalian) were analyzed using two maximum-parsimony tree-building algorithms. Parallel analyses of 2,258 pairwise immunodiffusion comparisons with chicken antisera on 101 mammalian species and of amino acid sequence data of alpha and beta hemoglobins and other published protein sequences were also carried out. The morphological and molecular phylogenies agree in depicting the infraclass Eutheria as consisting of five major clades (thus resolving part of the "bush"). Rates of evolution were also found to be similar in the two types of phylogenies.