Taxa In Phylogenetic Analyses Research Articles

The effect of character and outgroup choice on the phylogenetic position of the Jurassic dinosaur Chilesaurus diegosaurezi

The systematic position of the unusual Late Jurassic dinosaur Chilesaurus diegosaurezi remains an area of real uncertainty. Originally thought to be an unusual theropod, Chilesaurus has, since its first discovery, been suggested to either be a sauropodomorph dinosaur, an early diverging, ‘transitional’ ornithischian, or a more ‘derived’ neornithischian dinosaur, close to the heterodontosaurs. However, despite these recent fluctuations, the original placement within Theropoda remains the most prevalent phylogenetic hypothesis. This study looks to critically assess these various competing hypotheses regarding Chilesaurus and to analyse various aspects of the anatomical datasets that have placed it within Theropoda, Sauropodomorpha and Ornithischia. The choice of anatomical characters in past studies is considered and, crucially, so too is the choice of operational taxonomic units. By revising the datasets used in the initial analyses of Chilesaurus, one cause of this phylogenetic uncertainty has been clearly identified — outgroup choice. The total absence of members of Ornithischia from the predominantly saurischian-taxon-based datasets used in the original phylogenetic analyses of Chilesaurus is here shown to have had a substantial effect on the systematic position that was first recovered for this taxon. By scoring a selection of basal ornithischian taxa into the primarily saurischian datasets previously used, this study has recovered a different position for the Chilesaurus within Dinosauria, different internal topologies within Theropoda, and even a different arrangement of three major dinosaurian groups. The inclusion of an ornithischian outgroup in an otherwise predominantly saurischian focused dataset has produced results that again suggest that Chilesaurus could be an early diverging member of Ornithischia. What is more, this result was achieved without the addition of any classical ornithischian-like characters to the data. Whilst the results of this analysis do not by any means end the debate on the phylogenetic position of Chilesaurus, or on the fundamental relationships within Dinosauria, they do serve to highlight the current instability of such cryptic taxa in phylogenetic analyses, as well as the need for more comprehensive taxon sampling in such analyses generally. The results further highlight the potentially adverse effects of the omission of certain outgroup taxa on the overall topologies recovered within clades.

Skeletal Anatomy of Acaenasuchus Geoffreyi Long and Murry, 1995 (Archosauria: Pseudosuchia) and its Implications for the Origin of the Aetosaurian Carapace

ABSTRACT Acaenasuchus geoffreyi is a diminutive armored archosaur from the Upper Triassic Chinle Formation of northern Arizona, U.S.A., with uncertain evolutionary relationships and skeletal maturity. Known only from osteoderms, the taxon has been considered a valid taxon of aetosaur, juvenile specimens synonymous with the aetosaur Desmatosuchus spurensis, or a non-aetosaurian pseudosuchian archosaur. Here, we describe new fossils of Acaenasuchus geoffreyi that represent cranial, vertebral, and appendicular elements as well as previously unknown variations in the dorsal carapace and ventral shield. The skull bones are ornamented with the same anastomosing complex of ridges and grooves found on the paramedian and lateral osteoderms, and the appendicular skeleton resembles that of Revueltosaurus callenderi, Euscolosuchus olseni, aetosaurs, and other armored archosaurs such as erpetosuchids. Histology of osteoderms from the hypodigm of Acaenasuchus geoffreyi shows multiple growth lines, laminar tissue, and low vascularity, evidence that the individuals were close to skeletal maturity and not young juveniles. A revised phylogenetic analysis of early archosaurs recovers Acaenasuchus geoffreyi and Euscolosuchus olseni as sister taxa and members of a new clade that is the sister taxon of Aetosauria. This new phylogeny depicts a broader distribution of osteoderm character states previously thought to only occur in aetosaurs, demonstrating the danger of using only armor character states in aetosaur taxonomy and phylogeny. Acaenasuchus geoffreyi is also a good example of how new fossils can stabilize ‘wild card’ taxa in phylogenetic analyses and contributes to our understanding of the evolution of the aetosaur carapace.

Size matters: the effects of ontogenetic disparity on the phylogeny of Trematopidae (Amphibia: Temnospondyli)

AbstractTrematopids are a clade of terrestrial Permo-Carboniferous temnospondyl amphibians. The intrarelationships of this clade are poorly known. This is largely attributable to a substantial disparity in size between type specimens, which range from the small-bodied lectotype of Mattauschia laticeps (< 4 cm skull length) to the large-bodied holotype of Acheloma cumminsi (> 15 cm skull length). Inferred correlation of size disparity with ontogenetic disparity has led previous workers either to omit taxa in phylogenetic analyses or to forgo an analysis altogether. Here, I take a specimen-level approach and multiple subsampling permutations to explore the phylogeny of the Trematopidae as a case study for assessing the effects of ontogenetic disparity on phylogenetic reconstruction in temnospondyls. The various analyses provide evidence that ontogenetic disparity confounds the phylogenetic inference of trematopids but without a directional bias. Tree topologies of most permutations are poorly resolved and weakly supported, reflecting character conflict that results from the inability of the analyses to differentiate retained plesiomorphies from juvenile features. These findings urge caution in the interpretation of phylogenetic analyses for which ontogenetic disparity exists, but is unaccounted for, and provide a strong impetus for more directed exploration of the interplay of ontogeny and phylogeny across Temnospondyli.

Overlap Indices: Tools to quantify the amount of anatomical overlap among groups of incomplete terminal taxa in phylogenetic analyses

AbstractPhylogenetic analyses of morphological data are often characterized by missing data due to incomplete operational taxonomic units, as in fossils. This incomplete knowledge derives from various reasons, including—in the case of fossils—the numerous filters an organism has to pass through during taphonomy, fossilization, weathering and collecting. Whereas several methods have been proposed to address issues raised by the inclusion of incomplete terminal taxa, until recently no tool existed to easily quantify the amount of anatomical overlap within a particular clade. The Overlap Indices provide such values and might prove useful for comparative cladistics. We herein describe these new indices and their applications in detail and provide an example file for their calculation. A case study of diplodocid sauropod dinosaurs shows how the Overlap Indices will help to explore and quantify, which one of a number of conflicting tree topologies is supported by more anatomical traits, which skeletal regions are underrepresented in a particular phylogenetic matrix, and which taxon would improve character state score completeness.

Comments on detecting rogue taxa using RogueNaRok

Rogues are relatively unstable taxa in phylogenetic analyses that are of concern if they obfuscate relationships, or support for relationships, of interest among more stable taxa. RogueNaRok is a recently developed heuristic solution to the problem of identifying rogue taxa. We illustrate the performance of RogueNaRok with simple examples designed to clarify its behaviour. We argue that the optimality criterion currently used by RogueNaRok may sometimes be poorly suited to the task of detecting rogues and that the scores reported by RogueNaRok should not be interpreted as measures of taxon instability. We suggest how RogueNaRok might be enhanced and recommend that it not be relied upon exclusively for detecting rogues.

Phylogenetic Stability, Tree Shape, and Character Compatibility: A Case Study Using Early Tetrapods

Phylogenetic tree shape varies as the evolutionary processes affecting a clade change over time. In this study, we examined an empirical phylogeny of fossil tetrapods during several time intervals, and studied how temporal constraints manifested in patterns of tree imbalance and character change. The results indicate that the impact of temporal constraints on tree shape is minimal and highlights the stability through time of the reference tetrapod phylogeny. Unexpected values of imbalance for Mississippian and Pennsylvanian time slices strongly support the hypothesis that the Carboniferous was a period of explosive tetrapod radiation. Several significant diversification shifts take place in the Mississippian and underpin increased terrestrialization among the earliest limbed vertebrates. Character incompatibility is relatively high at the beginning of tetrapod history, but quickly decreases to a relatively stable lower level, relative to a null distribution based on constant rates of character change. This implies that basal tetrapods had high, but declining, rates of homoplasy early in their evolutionary history, although the origin of Lissamphibia is an exception to this trend. The time slice approach is a powerful method of phylogenetic analysis and a useful tool for assessing the impact of combining extinct and extant taxa in phylogenetic analyses of large and speciose clades.

Assessment of available anatomical characters for linking living mammals to fossil taxa in phylogenetic analyses.

Analyses of living and fossil taxa are crucial for understanding biodiversity through time. The total evidence method allows living and fossil taxa to be combined in phylogenies, using molecular data for living taxa and morphological data for living and fossil taxa. With this method, substantial overlap of coded anatomical characters among living and fossil taxa is vital for accurately inferring topology. However, although molecular data for living species are widely available, scientists generating morphological data mainly focus on fossils. Therefore, there are fewer coded anatomical characters in living taxa, even in well-studied groups such as mammals. We investigated the number of coded anatomical characters available in phylogenetic matrices for living mammals and how these were phylogenetically distributed across orders. Eleven of 28 mammalian orders have less than 25% species with available characters; this has implications for the accurate placement of fossils, although the issue is less pronounced at higher taxonomic levels. In most orders, species with available characters are randomly distributed across the phylogeny, which may reduce the impact of the problem. We suggest that increased morphological data collection efforts for living taxa are needed to produce accurate total evidence phylogenies.

A pragmatic approach to the species problem from a paleontological perspective

The ideal scenario for paleontologists would be for the species they designate to be equivalent to the species recognized for modern animals, in the sense that they were formed as a result of the same evolutionary processes. This would mean, for example, that we could be confident that in combining extant and extinct taxa in phylogenetic analyses we would be dealing with equivalent operational taxonomic units. Notwithstanding the many thousands of pages that have been spent arguing over species concepts, the only concept that has won widespread acceptance for the designation of modern species is Mayr's Biological Species Concept (BSC).(1) In fact, whenever we complete a cladistic analysis, we assume reproductive isolation of our terminal taxa because otherwise their similarities could be the product of interbreeding rather than common ancestry. Fundamentally, we all behave as though the BSC is true.

A new albuliform (Teleostei: Elopomorpha) from the Lower Cretaceous Santana Formation, Araripe Basin, northeastern Brazil

A new albuliform fish Bullichthys santanensis gen. nov. sp. nov. is described from the Lower Cretaceous Santana Formation of northeastern Brazil. The specimen is identified as a new plesion of Albuliformes, on the basis of an inflated otic bulla, a well developed ectopterygoid process, and a deep subtemporal fossa. Bullichthys does not belong to the Albulidae or Pterothrissidae because it lacks the broad parasphenoid, parasphenoid and endopterygoid complex with a crushing dentition (opposed by similar dentition on the basibranchial tooth plate), an ossified interorbital septum, a caudal scute, and an occipital condyle bordered by both exoccipitals and basioccipital bones. Bullichthys santanensis gen. nov. sp. nov. differs from all other albuliforms by the presence of a hypurapophysis and a parhypurapophysis. The discovery of B. santanensis increases albuliform diversity during the Early Cretaceous at the very beginning of albuliform history. It presents a suite of both plesiomorphic and derived characters. Such a character assemblage clearly demonstrates the necessity of including stem taxa in phylogenetic analysis based on morphology to resolve the controversial relationships of these basal teleostean fishes.

The Use and Validity of Composite Taxa in Phylogenetic Analysis

In phylogenetic analysis, one possible approach to minimize missing data in DNA supermatrices consists in sampling sequences from different species to obtain a complete sequence for all genes included in the study. We refer to those complete sequences as composite taxa because DNA sequences that are combined belong to different species. An alternative approach is to analyze incomplete supermatrices by coding unavailable DNA sequences as missing. The accuracy of phylogenetic trees estimated using matrices that include composite taxa has recently been questioned, and the best approach for analyzing incomplete supermatrices is highly debated. Through computer simulations, we compared the phylogenetic accuracy of the 2 competing approaches. We explored the effect of composite taxa when inferring higher level relationships, that is, relationships between monophyletic groups. DNA sequences were simulated on a 42-taxon model tree and incomplete supermatrices containing different percentages of missing data were generated. These incomplete supermatrices were analyzed either by coding the missing data with "?" or by reducing the amount of missing data through the combination of 2 or more taxa to generate composite taxa. Of 180 comparisons (18 simulation cases with 2 different inference methods and 5 levels of incompleteness), we observed significantly higher phylogenetic accuracies for composite matrices in 46 comparisons, whereas missing data matrices outperformed composites in 8 comparisons. In all other cases, the phylogenetic accuracy obtained with composite matrices was not significantly different from that of missing data matrices. This study demonstrates that composite taxa represent an interesting approach to minimize the amount of missing data in supermatrices and we suggest that it is the optimal approach to use in phylogenomic studies to reduce computing time.

Estimating ancestral distributions of lineages with uncertain sister groups: a statistical approach to Dispersal–Vicariance Analysis and a case using Aesculus L. (Sapindaceae) including fossils

Abstract We propose a simple statistical approach for using Dispersal–Vicariance Analysis (DIVA) software to infer biogeographic histories without fully bifurcating trees. In this approach, ancestral ranges are first optimized for a sample of Bayesian trees. The probability P of an ancestral range r at a node is then calculated as where Y is a node, and F(rY) is the frequency of range r among all the optimal solutions resulting from DIVA optimization at node Y, t is one of n topologies optimized, and Pt is the probability of topology t. Node Y is a hypothesized ancestor shared by a specific crown lineage and the sister of that lineage “x”, where x may vary due to phylogenetic uncertainty (polytomies and nodes with posterior probability <100%). Using this method, the ancestral distribution at Y can be estimated to provide inference of the geographic origins of the specific crown group of interest. This approach takes into account phylogenetic uncertainty as well as uncertainty from DIVA optimization. It is an extension of the previously described method called Bayes‐DIVA, which pairs Bayesian phylogenetic analysis with biogeographic analysis using DIVA. Further, we show that the probability P of an ancestral range at Y calculated using this method does not equate to pp*F(rY) on the Bayesian consensus tree when both variables are <100%, where pp is the posterior probability and F(rY) is the frequency of range r for the node containing the specific crown group. We tested our DIVA‐Bayes approach using Aesculus L., which has major lineages unresolved as a polytomy. We inferred the most probable geographic origins of the five traditional sections of Aesculus and of Aesculus californica Nutt. and examined range subdivisions at parental nodes of these lineages. Additionally, we used the DIVA‐Bayes data from Aesculus to quantify the effects on biogeographic inference of including two wildcard fossil taxa in phylogenetic analysis. Our analysis resolved the geographic ranges of the parental nodes of the lineages of Aesculus with moderate to high probabilities. The probabilities were greater than those estimated using the simple calculation of pp*F(ry) at a statistically significant level for two of the six lineages. We also found that adding fossil wildcard taxa in phylogenetic analysis generally increased P for ancestral ranges including the fossil's distribution area. The ΔP was more dramatic for ranges that include the area of a wildcard fossil with a distribution area underrepresented among extant taxa. This indicates the importance of including fossils in biogeographic analysis. Exmination of range subdivision at the parental nodes revealed potential range evolution (extinction and dispersal events) along the stems of A. californica and sect. Parryana.

Missing Data, Incomplete Taxa, and Phylogenetic Accuracy

The problem of missing data is often considered to be the most important obstacle in reconstructing the phylogeny of fossil taxa and in combining data from diverse characters and taxa for phylogenetic analysis. Empirical and theoretical studies show that including highly incomplete taxa can lead to multiple equally parsimonious trees, poorly resolved consensus trees, and decreased phylogenetic accuracy. However, the mechanisms that cause incomplete taxa to be problematic have remained unclear. It has been widely assumed that incomplete taxa are problematic because of the proportion or amount of missing data that they bear. In this study, I use simulations to show that the reduced accuracy associated with including incomplete taxa is caused by these taxa bearing too few complete characters rather than too many missing data cells. This seemingly subtle distinction has a number of important implications. First, the so-called missing data problem for incomplete taxa is, paradoxically, not directly related to their amount or proportion of missing data. Thus, the level of completeness alone should not guide the exclusion of taxa (contrary to common practice), and these results may explain why empirical studies have sometimes found little relationship between the completeness of a taxon and its impact on an analysis. These results also (1) suggest a more effective strategy for dealing with incomplete taxa, (2) call into question a justification of the controversial phylogenetic supertree approach, and (3) show the potential for the accurate phylogenetic placement of highly incomplete taxa, both when combining diverse data sets and when analyzing relationships of fossil taxa.

Missing data, incomplete taxa, and phylogenetic accuracy.

The problem of missing data is often considered to be the most important obstacle in reconstructing the phylogeny of fossil taxa and in combining data from diverse characters and taxa for phylogenetic analysis. Empirical and theoretical studies show that including highly incomplete taxa can lead to multiple equally parsimonious trees, poorly resolved consensus trees, and decreased phylogenetic accuracy. However, the mechanisms that cause incomplete taxa to be problematic have remained unclear. It has been widely assumed that incomplete taxa are problematic because of the proportion or amount of missing data that they bear. In this study, I use simulations to show that the reduced accuracy associated with including incomplete taxa is caused by these taxa bearing too few complete characters rather than too many missing data cells. This seemingly subtle distinction has a number of important implications. First, the so-called missing data problem for incomplete taxa is, paradoxically, not directly related to their amount or proportion of missing data. Thus, the level of completeness alone should not guide the exclusion of taxa (contrary to common practice), and these results may explain why empirical studies have sometimes found little relationship between the completeness of a taxon and its impact on an analysis. These results also (1) suggest a more effective strategy for dealing with incomplete taxa, (2) call into question a justification of the controversial phylogenetic supertree approach, and (3) show the potential for the accurate phylogenetic placement of highly incomplete taxa, both when combining diverse data sets and when analyzing relationships of fossil taxa.

Relationships within Cornales and circumscription of Cornaceae— matK and rbcL sequence data and effects of outgroups and long branches

Phylogenetic relationships in Cornales were assessed using sequences rbcL and matK. Various combinations of outgroups were assessed for their suitability and the effects of long branches and outgroups on tree topology were examined using RASA 2.4 prior to conducting phylogenetic analyses. RASA identified several potentially problematic taxa having long branches in individual data sets that may have obscured phylogenetic signal, but when data sets were combined RASA no longer detected long branch problems. t RASA provides a more conservative measurement for phylogenetic signal than the PTP and skewness tests. The separate matK and rbcL sequence data sets were measured as the chloroplast DNA containing phylogenetic signal by RASA, but PTP and skewness tests suggested the reverse. Nonetheless, the matK and rbcL sequence data sets suggested relationships within Cornales largely congruent with those suggested by the combined matK– rbcL sequence data set that contains significant phylogenetic signal as measured by t RASA, PTP, and skewness tests. Our analyses also showed that a taxon having a long branch on the tree may not be identified as a “long-branched” taxon by RASA. The long branches identified by RASA had little effect on the arrangement of other taxa in the tree, but the placements of the long-branched taxa themselves were often problematic. Removing the long-branched taxa from analyses generally increased bootstrap support, often substantially. Use of non-optimal outgroups (as identified by RASA) decreased phylogenetic resolution in parsimony analyses and suggested different relationships in maximum likelihood analyses, although usually weakly supported clades (less than 50% support) were impacted. Our results do not recommend using t RASA as a sole criterion to discard data or taxa in phylogenetic analyses, but t RASA and the taxon variance ratio obtained from RASA may be useful as a guide for improved phylogenetic analyses. Results of parsimony and ML analyses of the sequence data using optimal outgroups suggested by RASA revealed four major clades within Cornales: (1) Curtisia–Grubbia, (2) Cornus–Alangium, (3) Nyssa–Camptotheca–Davidia–Mastixia–Diplopanax, and (4) Hydrangeaceae–Loasaceae, with clades (2) and (3) forming a monophyletic group sister to clade (4) and clade (1) sister to the remainder of Cornales. However, there was not strong bootstrap support for relationships among the major clades. The placement of Hydrostachys could not be reliably determined, although most analyses place the genus within Hydrangeaceae; ML analyses, for example, placed the genus as the sister of Hydrangeeae. Our results supported a Cornales including the systematically problematic Hydrostachys, a Cornaceae consisting of Cornus and Alangium, a Nyssaceae consisting of Nyssa and Camptotheca, a monogeneric Davidiaceae, a Mastixiaceae consisting of Mastixia and Diplopanax, and an expanded Grubbiaceae consisting of Grubbia and Curtisia, and two larger families, Hydrangeaceae and Loasaceae.

Phylogenetic biogeography with and without the fossil record: gauging the effects of extinction and paleontological incompleteness

Biogeography played an important role in early developments in evolutionary theory and continues to play an important role in evolutionary studies and paleogeographic reconstructions. The development of a phylogenetic approach to biogeographic analysis has been important; however, fossil taxa have not always played a role in phylogenetic biogeographic studies and their role has been criticized by some phylogenetic biogeographers. Here, simulation studies are used to show that phylogenetic biogeographic studies on extant organisms that do not include fossil taxa can often be artificially incongruent and inaccurate. This is because area cladograms for extant taxa alone may differ from those that also include extinct taxa, implying different patterns of biogeographic relationship between areas, and area cladograms are the fundamental data of phylogenetic biogeographic analysis. This finding is analogous to what is known about how including fossil taxa in phylogenetic analyses along with extant taxa can improve resolution and accuracy. The incompleteness of the fossil record can also lead to artificial incongruence in phylogenetic paleobiogeographic studies because not all taxa that have ever lived are preserved in the fossil record, and thus those missing taxa cannot be incorporated into area cladograms. Therefore, area cladograms based on all preserved fossil species may also differ fundamentally from the true area cladograms which would be available if all taxa were preserved in the fossil record. Again, this can lead to artificial incongruence and inaccurate results. Simulations are used to show that under average preservational regimes for skeletonized marine invertebrates in the fossil record, and with average extinction probabilities, phylogenetic biogeographic studies of the extant biota are typically more likely to be artificially incongruent than those relying on fossil biotas. Further, in phylogenetic and biogeographic studies extant groups that have persisted for a very long time, have high extinction rates, and low diversity should be avoided. Phylogenetic biogeographic studies of fossil taxa should avoid groups with low diversity and a poor fossil record; these studies should also avoid time periods or regions with a poor fossil record.

SYSTEMATICS OF ULVA AND ENTEROMORPHA (ULVALES), WITH EMPHASIS ON SPECIES FROM THE NORTHEAST PACIFIC

Hayden, H. S. & Waaland, J. R.Department of Botany, University of Washington, Seattle, WA 98195Ulva L. and Enteromorpha Link are two of the most common macroalgal genera world wide and are well known for their role in “green tides” and marine biofouling. Traditionally, these genera have been distinguished from one another by their different morphologies: Ulva species have distromatic blades, and Enteromorpha species form monostromatic tubes. Despite the distinct morphological difference between these two genera, morphological simplicity and plasticity of algae within each of them have confounded systematic and taxonomic efforts. Recent molecular studies of primarily European taxa indicate that Ulva and Enteromorpha are not reciprocally monophyletic yet within the Ulva/Enteromorpha assemblage several species can be identified. A study of Pacific Ulva and Enteromorpha was undertaken to reconstruct phylogeny and test species hypotheses based on morphology, development and hybridization experiments. Multiple samples of the eleven currently recognized Ulva and Enteromorpha species along the Pacific coast of North America were collected from Alaska to southern California. Collections were identified using existing morphological descriptions, and when possible, growth in culture. Sequences for the internal transcribed spacers of nuclear ribosomal DNA and the chloroplast‐encoded gene for the large subunit of RUBISCO were determined for these samples and additional ones from Australia, Chile, Hawaii and Japan. Sequences for Pacific collections were combined with those for European taxa in phylogenetic analyses using the maximum parsimony criterion. The results provide improved understanding of species diversity and biogeography in Ulva and Enteromorpha and additional evidence for the polyphyly of these genera.

Convergent evolution and character correlation in burrowing reptiles: towards a resolution of squamate relationships

The affinities of three problematic groups of elongate, burrowing reptiles (amphisbaenians, dibamids and snakes) are reassessed through a phylogenetic analysis of all the major groups of squamates, including the important fossil taxaSineoamphisbaena, mosasauroids andPachyrhachis; 230 phylogenetically informative osteological characters were evaluated in 22 taxa. Snakes (includingPachyrhachis) are anguimorphs, being related firstly to large marine mosasauroids, and secondly to monitor lizards (varanids). Scincids and cordylids are not related to lacertiforms as previously thought, but to anguimorphs. Amphisbaenians and dibamids are closely related, andSineoamphisbaenais the sister group to this clade. The amphisbaenian–dibamid–Sineoamphisbaenaclade, in turn, is related to gekkotans and xantusiids. When the fossil taxa are ignored, snakes, amphisbaenians and dibamids form an apparently well-corroborated clade nested within anguimorphs. However, nearly all of the characters supporting this arrangement are correlated with head-first burrowing (miniaturization, cranial consolidation, body elongation, limb reduction), and invariably co-occur in other tetrapods with similar habits. These characters are potentially very misleading because of their sheer number and because they largely represent reductions or losses. It takes very drastic downweighting of these linked characters to alter tree topology: if fossils are excluded from the analysis, a (probably spurious) clade consisting of elongate, fossorial taxa almost always results. These results underscore the importance of including all relevant taxa in phylogenetic analyses. Inferring squamate phylogeny depends critically on the inclusion of certain (fossil) taxa with combinations of character states that demonstrate convergent evolution of the elongate, fossorial ecomorph in amphisbaenians and dibamids, and in snakes. In the all-taxon analysis, the position of snakes within anguimorphs is more strongly-corroborated than the association of amphisbaenians and dibamids with gekkotans. When the critical fossil taxa are deleted, snakes «attract» the amphisbaenian–dibamid clade on the basis of a suite of correlated characters. While snakes remain anchored in anguimorphs, the amphisbaenian–dibamid clade moves away from gekkotans to join them. Regardless of the varying positions of the three elongate burrowing taxa, the interrelationships between the remaining limbed squamates («lizards») are constant; thus, the heterodox affinities of scincids, cordylids, and xantusiids identified in this analysis appear to be robust. Finally, the position ofPachyrhachisas a basal snake rather than (as recently suggested) a derived snake is supported on both phylogenetic and evolutionary grounds.

Convergent evolution and character correlation in burrowing reptiles: towards a resolution of squamate relationships

The affinities of three problematic groups of elongate, burrowing reptiles (amphisbaenians, dibamids and snakes) are reassessed through a phylogenetic analysis of all the major groups of squamates, including the important fossil taxaSineoamphisbaena, mosasauroids andPachyrhachis; 230 phylogenetically informative osteological characters were evaluated in 22 taxa. Snakes (includingPachyrhachis) are anguimorphs, being related firstly to large marine mosasauroids, and secondly to monitor lizards (varanids). Scincids and cordylids are not related to lacertiforms as previously thought, but to anguimorphs. Amphisbaenians and dibamids are closely related, andSineoamphisbaenais the sister group to this clade. The amphisbaenian–dibamid–Sineoamphisbaenaclade, in turn, is related to gekkotans and xantusiids. When the fossil taxa are ignored, snakes, amphisbaenians and dibamids form an apparently well-corroborated clade nested within anguimorphs. However, nearly all of the characters supporting this arrangement are correlated with head-first burrowing (miniaturization, cranial consolidation, body elongation, limb reduction), and invariably co-occur in other tetrapods with similar habits. These characters are potentially very misleading because of their sheer number and because they largely represent reductions or losses. It takes very drastic downweighting of these linked characters to alter tree topology: if fossils are excluded from the analysis, a (probably spurious) clade consisting of elongate, fossorial taxa almost always results. These results underscore the importance of including all relevant taxa in phylogenetic analyses. Inferring squamate phylogeny depends critically on the inclusion of certain (fossil) taxa with combinations of character states that demonstrate convergent evolution of the elongate, fossorial ecomorph in amphisbaenians and dibamids, and in snakes. In the all-taxon analysis, the position of snakes within anguimorphs is more strongly-corroborated than the association of amphisbaenians and dibamids with gekkotans. When the critical fossil taxa are deleted, snakes «attract» the amphisbaenian–dibamid clade on the basis of a suite of correlated characters. While snakes remain anchored in anguimorphs, the amphisbaenian–dibamid clade moves away from gekkotans to join them. Regardless of the varying positions of the three elongate burrowing taxa, the interrelationships between the remaining limbed squamates («lizards») are constant; thus, the heterodox affinities of scincids, cordylids, and xantusiids identified in this analysis appear to be robust. Finally, the position ofPachyrhachisas a basal snake rather than (as recently suggested) a derived snake is supported on both phylogenetic and evolutionary grounds.

The use of PCR‐based typing methods to assess the diversity of Frankia nodule endophytes of the actinorhizal shrub Ceanothus

Our understanding of the actinorhizal symbiosis, in particular of the Frankia‐Ceanothus association, has been hampered by the failure to isolate infective strains in pure culture. Recently, the polymerase chain reaction (PCR) has been utilized to amplify regions of the Frankia genome, allowing analysis of the microsymbiont genome without first isolating the microbe in pure culture. Root nodules were collected from six Ceanothus spp. common to the coastal regions of the Santa Monica Mountains of southern California. Individual lobes were surface‐sterilized, total DNA was extracted and amplified using prokaryotic‐specific primers. To assess the genetic diversity of Frankia endophytes in the population studied, the BOX primer was used to generate genomic fingerprints of prokaryotic nodule inhabitants using rep‐PCR. Fingerprint patterns fell into twelve distinct groups indicating the occurrence of genetic diversity of Frankia in the nodules sampled. DNA extracts of individual lobes that gave distinct BOX‐PCR fingerprints were also amplified by PCR using primers directed against conserved regions of the 16S ribosomal RNA gene. The nucleotide sequences of the PCR products were determined and aligned with the corresponding region from other taxa for phylogenetic analysis. The sequences from Ceanothus nodules share a common ancestor to that of the Elaeagnus–infective strains.

How Many Taxa Must Be Sampled to Identify the Root Node of a Large Clade?

The importance of choice of taxa in phylogenetic analysis has been explored mainly with reference to its effect on the accuracy of tree estimation. Taxon sampling can also introduce other kinds of errors. Even if the sampled topology agrees with the true topology, it may not include the true root node of a clade, a node that is of interest for many reasons. Using a simple Yule model for the diversification process, the probability of identifying this node is derived under random sampling of taxa. For large clades, the minimum sample size needed to be 95% confident of identifying the root node is approximately 40 and is independent of the size of the clade. If rates of diversification differ in the two sister groups descended from the root node, the minimum sample size needed increases markedly. If these two sister groups are so different in diversity that a Yule model would be rejected by conventional diversification tests, then the necessary sample size is an order of magnitude greater than when diversification is homogeneous. (Diver- sification; phylogeny; branching; speciation; Yule model; taxon sampling.) The recent publication of a very large phylogenetic analysis of seed plants based on chloroplast rbcL data (Chase et al., 1993) has raised a number of interesting ques- tions about phylogenetic analyses of large clades. Among these questions are com- putational issues related to reconstructing optimal trees using heuristic algorithms (Rice et alv 1995) and the choice of taxon sampling scheme for groups that are either large or poorly understood phylogeneti- cally. The rbcL analysis included nearly 500 sequences, a remarkable and possibly record-setting number but one that sam- ples barely 0.2% of seed plant diversity. Other similarly large clades probably will remain sparsely sampled by systematists for the foreseeable future. How much sam- pling is enough in groups that are excep- tionally species rich?