Molecular phylogenetics of the Pectinidae (Mollusca: Bivalvia) and effect of increased taxon sampling and outgroup selection on tree topology

Evolutionary relationships of the Pectinidae were examined using two mitochondrial genes (12S rRNA, 16S rRNA) and one nuclear gene (Histone H3) for 46 species. Outgroup taxa from Propeamussidae, Spondylidae and Limidae were also sequenced to examine the impact of outgroup choice on pectinid topologies. Our phylogenetic analyses resolved the Pectinidae as monophyletic, but many of the subfamilies and tribes within the family do not form monophyletic clades. The paraphyletic Aequipectinini group is the most basal member of the Pectinidae, with the Chlamydinae and Palliolinae representing the most recently derived pectinid groups. These results are in contrast with the current morphological hypothesis of Pectinidae evolution, which suggests the Chlamydinae and Pallioline are basal groups within the Pectinidae. Ingroup topology was found to be sensitive to outgroup choice and increasing taxon sampling within the Pectinidae resulted in more robust phylogenies.     

Outgroup sampling in phylogenetics: Severity of test and successive outgroup expansion

Outgroup sampling is a fundamental step in the design of phylogenetic analyses, independent of optimality criterion, taxonomic group, or source of evidence. Studies have demonstrated the efficient analysis of many thousands of terminals, all of which could be included in any empirical investigation, yet outgroup samples typically include only a small number of terminals. Most discussion of outgroup sampling centers on employing “correct” or “appropriate” outgroup terminals to increase “accuracy” or “reliability” by preventing “errors” such as long branch attraction and “incorrect” ingroup rooting. As an alternative, I develop a theory of outgroup sampling grounded in the logic of scientific discovery, whereby the objective is to test nested hypotheses of ingroup topology and character‐state transformation as severely as possible by incorporating outgroup terminals in unconstrained, simultaneous analysis, using background knowledge to select the terminals that have the greatest chance of refuting those hypotheses. This framework provides a logical basis for selecting outgroup taxa but does not provide grounds for limiting the outgroup sample, given that, ceteris paribus, testability and explanatory power increase with the inclusion of additional terminals. Therefore, I propose the ancillary procedure of successively expanding the outgroup sample until ingroup hypotheses become stable (insensitive) to increased sampling, with each expansion guided by the scientific objectives of outgroup sampling. This is a heuristic procedure that does not prevent more outgroup terminals from being sampled or guarantee that ingroup hypotheses will remain insensitive to further outgroup expansion, and it has no bearing on the objective support of a given hypothesis. Nevertheless, it provides an objective, empirical basis for limiting outgroup sampling in a given research cycle. I illustrate this procedure by examining the effect of successive outgroup expansion on the relationships among the poison frog genera Adelphobates, Dendrobates, and Oophaga.     

Foundational issues concerning taxa and taxon names

In a series of articles, Rieppel (2005, Biol. Philos. 20:465-487; 2006a, Cladistics 22:186-197; 2006b, Systematist 26:5-9), Keller et al. (2003, Bot. Rev. 69:93-110), and Nixon and Carpenter (2000, Cladistics 16:298-318) criticize the philosophical foundations of the PhyloCode. They argue that species and higher taxa are not individuals, and they reject the view that taxon names are rigid designators. Furthermore, they charge supporters of the individuality thesis and rigid designator theory with assuming essentialism, committing logical inconsistencies, and offering proposals that render taxonomy untestable. These charges are unsound. Such charges turn on confusions over rigid designator theory and the distinction between kinds and individuals. In addition, Rieppel's, Keller et al.'s, and Nixon and Carpenter's proposed alternatives are no better and have their own problems. The individuality thesis and rigid designator theory should not be quickly abandoned.     

Assessing the value of DNA barcodes for molecular phylogenetics: effect of increased taxon sampling in lepidoptera

Background

A common perception is that DNA barcode datamatrices have limited phylogenetic signal due to the small number of characters available per taxon. However, another school of thought suggests that the massively increased taxon sampling afforded through the use of DNA barcodes may considerably increase the phylogenetic signal present in a datamatrix. Here I test this hypothesis using a large dataset of macrolepidopteran DNA barcodes.

Methodology/Principal Findings

Taxon sampling was systematically increased in datamatrices containing macrolepidopteran DNA barcodes. Sixteen family groups were designated as concordance groups and two quantitative measures; the taxon consistency index and the taxon retention index, were used to assess any changes in phylogenetic signal as a result of the increase in taxon sampling. DNA barcodes alone, even with maximal taxon sampling (500 species per family), were not sufficient to reconstruct monophyly of families and increased taxon sampling generally increased the number of clades formed per family. However, the scores indicated a similar level of taxon retention (species from a family clustering together) in the cladograms as the number of species included in the datamatrix was increased, suggesting substantial phylogenetic signal below the ‘family’ branch.

Conclusions/Significance

The development of supermatrix, supertree or constrained tree approaches could enable the exploitation of the massive taxon sampling afforded through DNA barcodes for phylogenetics, connecting the twigs resolved by barcodes to the deep branches resolved through phylogenomics.     

Detection of selection utilizing molecular phylogenetics: a possible approach

The neutral theory of molecular evolution (Kimura 1985) is the basis for most current statistical tests for detecting selection, mainly using polymorphism data within species, divergence data between species, and/or genomic structures like linkage disequilibrium (Wang et al. 2006). In most cases informative tests can only be constructed with ample variations within these parameters and many common tests are difficult to formulate when identity-by-descent is not clear, for example in gene families or repetitive elements. With the current progress being made toward whole-genome sequencing and re-sequencing efforts, as well as protein sequencing via tandem mass spectrometry where genomic sequencing is lacking, we felt it was necessary to re-visit possible methods for rapid screening and detection of evolutionary outliers. These outliers might be of interest for other research, such as candidate gene association studies or genome annotations, drug- and disease-target searches, and functional studies. We focused on methods that would work on both protein and nucleotide data, could be used on large gene or protein domain families, and could be generated quickly in order for “first pass” annotation of large scale data. For these reasons, we chose properties of trees generated routinely in molecular phylogenetic studies; genetic distance, tree shape and balance, and internal node statistics (Heard 1992). Our current research looking at protein domain family data and phylogenetic trees from PFAM (Finn et al. 2008) suggests this approach towards detecting evolutionary outliers is feasible, but additional work will be necessary to determine the parameters that suggest either positive or negative selection is occurring in specific gene families. This is particularly true when other factors such as rapid duplication and deletion of genes containing these domains is taking place, and we suggest phylogenetic statistics may be useful in combination with existing methodologies for detailed studies of gene family data.     

New insights on systematics and phylogenetics of Mediterranean Blaps species (Coleoptera: Tenebrionidae: Blaptini), assessed through morphology and dense taxon sampling

The genus Blaps is an emblematic group of large flightless beetles adapted to semi‐arid and arid environments. About 230 species have been described in the subgenus Blaps, which is the largest of the four extant subgenera. Within this subgenus, one large group of morphologically homogeneous species is of particular interest, and comprises the species formerly assigned to Seidlitz's division one. Strikingly, almost all species in this group are endemic to the Mediterranean basin, whereas the remaining Blaps species are mostly distributed in Asia. Here we present the first phylogenetic hypothesis for the Mediterranean Blaps and several taxonomic clarifications for the North African species belonging to this group. Based on careful examination of almost all type material and thousands of specimens for all known species and subspecies (initially 65 taxa), seven lectotypes are designated and six new synonymies are established: Blaps heydeni Allard, 1880 = Blaps heydenissp. taciturna Peyerimhoff syn.n. , Blaps emondi Solier = Blaps nitidula Solier syn.n. , Blaps tripolitanica Karsch = Blaps vialattei Peyerimhoff syn.n. , Blaps bifurcata strauchi Reiche = Blaps bifurcata ssp. gridellii Pierre syn.n. , Blaps cognata Solier = Blaps schweinfurhtii Seidlitz syn.n. and Blaps judaeorum Miller = Blaps pharao Seidlitz syn.n. Among the examined material, five species are also new for science and are assigned to extant species group. To infer species relationships, a morphological character matrix was assembled and analysed using both parsimony and probabilistic methods. All analyses yielded a similar and repeatable phylogenetic pattern, which allows us to discuss the systematics of the Mediterranean endemic species, the limit of extant species groups and to clarify the taxonomic status of several ambiguous taxa. We also reveal the existence of a large clade that encompasses the eastern Mediterranean species that are also mostly associated with arid regions. This clade is embedded within a second group that corresponds to western Mediterranean species, which are generally found in semi‐arid regions. Finally, we discuss the relevance of species groups in relation to their geographic distribution. This work represents a first step for further studies in taxonomy, systematics and historical biogeography of Blaps species.     

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.     

Taxonomy and phylogenetics: Some considerations

The relationships between taxonomy and phylogenetics are discussed. It is suggested that they are distinct biological subdisciplines and some consequences of this position are set out.     

Molecular phylogenetics: principles and practice

Phylogenies are important for addressing various biological questions such as relationships among species or genes, the origin and spread of viral infection and the demographic changes and migration patterns of species. The advancement of sequencing technologies has taken phylogenetic analysis to a new height. Phylogenies have permeated nearly every branch of biology, and the plethora of phylogenetic methods and software packages that are now available may seem daunting to an experimental biologist. Here, we review the major methods of phylogenetic analysis, including parsimony, distance, likelihood and Bayesian methods. We discuss their strengths and weaknesses and provide guidance for their use.     

Model selection and model averaging in phylogenetics: advantages of akaike information criterion and bayesian approaches over likelihood ratio tests

Model selection is a topic of special relevance in molecular phylogenetics that affects many, if not all, stages of phylogenetic inference. Here we discuss some fundamental concepts and techniques of model selection in the context of phylogenetics. We start by reviewing different aspects of the selection of substitution models in phylogenetics from a theoretical, philosophical and practical point of view, and summarize this comparison in table format. We argue that the most commonly implemented model selection approach, the hierarchical likelihood ratio test, is not the optimal strategy for model selection in phylogenetics, and that approaches like the Akaike Information Criterion (AIC) and Bayesian methods offer important advantages. In particular, the latter two methods are able to simultaneously compare multiple nested or nonnested models, assess model selection uncertainty, and allow for the estimation of phylogenies and model parameters using all available models (model-averaged inference or multimodel inference). We also describe how the relative importance of the different parameters included in substitution models can be depicted. To illustrate some of these points, we have applied AIC-based model averaging to 37 mitochondrial DNA sequences from the subgenus Ohomopterus(genus Carabus) ground beetles described by Sota and Vogler (2001).     

Legume comparative genomics: progress in phylogenetics and phylogenomics

The legumes are the focus of numerous rapidly expanding genomic projects, all of which involve members of one part of the Leguminosae, the subfamily Papilionoideae. This subfamily is monophyletic, and recent studies concur on a series of clades within it that are well supported and have received informal names. These include the Cladrastis clade, the genistoids (including Lupinus), the mirbelioids, the dalbergioids (including Arachis), the millettioids (including Glycine and Phaseolus), and the hologalegina (galegoid) legumes, which comprise the robinioids (including Lotus) and the inverted repeat loss (IRL) clade (including Medicago and Pisum). The canavanine-accumulating legumes appear to fall into a single clade, consistent with the idea that the production of this toxic amino acid evolved only once. Recent advances in analytical techniques for dating phylogenies support an 'early explosion hypothesis', suggesting that much of the morphological diversity of the legume family evolved rapidly around 50-60 million years ago. Within the papilionoids, the divergence between Glycine and Medicago is estimated to have taken place around 54 million years ago. There is strong evidence for a palaeoduplication event that affected both Glycine (a millettioid) and Medicago (from the IRL clade). As more genomic data are forthcoming for Arachis, it will be possible to test whether this event extends to the dalbergioids.     

EDIBLE: experimental design and information calculations in phylogenetics

SUMMARY: Although evolutionary inference from molecular sequences is a statistical problem, little attention has been paid to questions of experimental design. A computer program, EDIBLE, has been developed to perform likelihood calculations based on Markov process models of nucleotide substitution allied with phylogenetic trees, and from these to compute Fisher information measures under different experimental designs. These calculations can be used to answer questions of optimal experimental design in molecular phylogenetics. AVAILABILITY: Source code (ANSI C), executables and documentation for EDIBLE are available from http://ng-dec1.gen.cam. ac.uk/info/index.htmland 'downstream' Web pages. CONTACT: N.Goldman@gen.cam.ac.uk     

Species: the concept, category and taxon

The term species by itself is vague because it refers to the species concept, the species category and the species taxon, all of which are distinct although related to one another. The species concept is not primarily a part of systematics, but has always been an integral part of basic biological theory, It is based on evolutionary theory and applies only to sexually reproducing organisms. The species concept and the phyletic lineage concept are quite distinct although they are related to one another. The important aspect of the species concept is lack of gene flow between different species, and hence the defining criterion of the species is genetic isolation. The species concept is often considered as non‐dimensional, both in time and space. Species possess three different major properties, namely genetic isolation, reproductive isolation and ecological isolation; these properties evolve at different times and under the effect of different causes during the speciation process. Speciation requires an external isolating barrier during the initial allopatric phase in which genetic isolation evolves and must reach 100% efficiency. The subsequent sympatric phase of speciation occurs after the disappearance of the external isolating barrier when members of the two newly evolved species can interact with one another and exert mutual selective demands on one another. Much of the reproductive and ecological isolation evolves during this secondary sympatric phase. The species category is a rank in the taxonomic hierarchy and serves as the basis on which the diversity of organisms is described; it is not the same as the species concept. The species category applied to all organisms, sexually and asexually reproducing. The species taxon is the practical application of the species category in systematics with the recognition of species taxa requiring many arbitrary decisions. No single set of rules exist by which the species category can be applied to all organisms. Recognition of species taxa in asexually reproducing organisms is based on amount of variation and gaps in the variation of phenotypic features associated with ecological attributes of these organisms as compared with similar attributes in sympatric species taxa of sexually reproducing organisms. Species taxa are multidimensional in that they exist over space–time and often have fuzzy borders. Because recognition of species taxa, including those in sexually reproducing organisms, depends on many arbitrary decisions especially when dealing with broad geographical and temporal ranges, species taxa cannot be used as the foundation for developing and testing theoretical concepts in evolutionary theory which can only be done with the non‐dimensional species concept.     

Indicator selection in life cycle assessment to enable decision making: issues and solutions

Purpose

With an ever increasing list of indicators available, life cycle assessment (LCA) practitioners face the challenge of effectively communicating results to decision makers. Simplification of LCA is often limited to an arbitrary selection of indicators, use of single scores by using weighted values or single attribute indicators. These solutions are less attractive to decision makers, since value judgments are introduced or multi-indicator information is lost. Normalization could be a means to narrow the list of indicators by ranking indicators vs. a reference system. This paper shows three different normalization approaches that produce very different ranking of indicators. It is explained how normalization helps maintain a multi-indicator approach while keeping the most relevant indicators, allowing effective decision making.

Methods

The approaches are illustrated on a hand dishwashing case study, using ReCiPe as the impact assessment method and taking the European population (year 2000) as the reference situation. Indicators are ranked using midpoint normalization factors, and compared to the ranking from endpoint normalization broken down by midpoint contribution.

Results and discussion

Endpoint normalization shows Resources as the most relevant area of protection for this case, closely followed by Human Health and Ecosystem. Broken down by their key driving midpoints, fossil depletion, climate change and, to a lesser extent, particulate matter formation and metal depletion, are most relevant. Midpoint normalization, however, indicates Freshwater Eutrophication, Natural Land Transformation and Toxicity indicators (marine and freshwater ecotoxicity and human toxicity) are most relevant.

Conclusions

A three-step approach based on endpoint normalization is recommended to present only the most relevant indicators, allowing more effective decision making instead of communicating all LCA indicators. The selection process breaks out the normalized endpoint results into the most contributing midpoints (relevant indicators) and reports results with midpoint level units. Bias due to lack of data completeness is less of an issue in the endpoint normalization process (compared to midpoint normalization), while midpoint results are less subject to uncertainty (compared to endpoint results). Focusing on the relevant indicators and key contributing unit processes has proven to be effective for non-LCA expert decision makers to understand, use, and communicate complex LCA results.     

Character analysis in morphological phylogenetics: problems and solutions

Many aspects of morphological phylogenetics are controversial in the theoretical systematics literature and yet are often poorly explained and justified in empirical studies. In this paper, I argue that most morphological characters describe variation that is fundamentally quantitative, regardless of whether they are coded qualitatively or quantitatively by systematists. Given this view, three fundamental problems in morphological character analysis (definition, delimitation, and ordering of character states) may have a common solution: coding morphological characters as continuous quantitative traits. A new parsimony method (step-matrix gap-weighting, a modification of Thiele's approach) is proposed that allows quantitative traits to be analyzed as continuous variables. The problem of scaling or weighting quantitative characters relative to qualitative characters (and to each other) is reviewed, and three possible solutions are described. The new coding method is applied to data from hoplocercid lizards, and the results show the sensitivity of phylogenetic conclusions to different scaling methods. Although some authors reject the use of continuous, overlapping, quantitative characters in phylogenetic analysis, quantitative data from hoplocercid lizards that are coded using the new approach contain significant phylogenetic structure and exhibit levels of homoplasy similar to those seen in data that are coded qualitatively.     

Ethical issues associated with HIV phylogenetics in HIV transmission dynamics research: A review of the literature using the Emanuel Framework

The reduced costs of DNA sequencing and the use of such data for HIV‐1 clinical management and phylogenetic analysis have led to a massive increase of HIV‐1 sequences in the last few years. Phylogenetic analysis has shed light on the origin, spread and characteristics of HIV‐1 epidemics and outbreaks. Phylogenetic analysis is now also being used to advance our knowledge of the drivers of HIV‐1 transmission in order to design effective interventions. However, HIV phylogenetic analysis presents unique ethical challenges, which have not been fully explored. This review presents an analysis of what appear to be key ethical issues in HIV phylogenetics in the hope of stimulating further conceptual and empirical work in this rapidly emerging area. We structure the review using the Emanuel Framework, a systematic, holistic framework, which has been adapted for use in developing countries, which bear the brunt of the HIV‐1 pandemic.     

Technical issues in construction of nucleic acid vaccines

Delivery of antigen-encoding genes by nucleic acid vaccine vectors offers tremendous advantages in power and flexibility over conventional antigen delivery systems. Of the many forms of nucleic acid vaccine that can be constructed, circular DNA plasmids are the simplest. In this article, we consider the components that make up a generic DNA plasmid vaccine. We discuss some of the issues encountered when optimizing these vectors to exploit their potential for in vivo expression and presentation of antigens and thereby maximizing the immune response.     

Heterotachy and long-branch attraction in phylogenetics

Background  

Probabilistic methods have progressively supplanted the Maximum Parsimony (MP) method for inferring phylogenetic trees. One of the major reasons for this shift was that MP is much more sensitive to the Long Branch Attraction (LBA) artefact than is Maximum Likelihood (ML). However, recent work by Kolaczkowski and Thornton suggested, on the basis of simulations, that MP is less sensitive than ML to tree reconstruction artefacts generated by heterotachy, a phenomenon that corresponds to shifts in site-specific evolutionary rates over time. These results led these authors to recommend that the results of ML and MP analyses should be both reported and interpreted with the same caution. This specific conclusion revived the debate on the choice of the most accurate phylogenetic method for analysing real data in which various types of heterogeneities occur. However, variation of evolutionary rates across species was not explicitly incorporated in the original study of Kolaczkowski and Thornton, and in most of the subsequent heterotachous simulations published to date, where all terminal branch lengths were kept equal, an assumption that is biologically unrealistic.     

Model use in phylogenetics: nine key questions

Models of character evolution underpin all phylogeny estimations, thus model adequacy remains a crucial issue for phylogenetics and its many applications. Although progress has been made in selecting appropriate models for phylogeny estimation, there is still concern about their purpose and proper use. How do we interpret models in a phylogenetic context? What are their effects on phylogeny estimation? How can we improve confidence in the models that we choose? That the phylogenetics community is asking such questions denotes an important stage in the use of explicit models. Here, we examine these and other common questions and draw conclusions about how the community is using and choosing models, and where this process will take us next.