Rudolf Meier

SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information

We present SequenceMatrix, software that is designed to facilitate the assembly and analysis of multi‐gene datasets. Genes are concatenated by dragging and dropping FASTA, NEXUS, or TNT files with aligned sequences into the program window. A multi‐gene dataset is concatenated and displayed in a spreadsheet; each sequence is represented by a cell that provides information on sequence length, number of indels, the number of ambiguous bases (“Ns”), and the availability of codon information. Alternatively, GenBank numbers for the sequences can be displayed and exported. Matrices with hundreds of genes and taxa can be concatenated within minutes and exported in TNT, NEXUS, or PHYLIP formats, preserving both character set and codon information for TNT and NEXUS files. SequenceMatrix also creates taxon sets listing taxa with a minimum number of characters or gene fragments, which helps assess preliminary datasets. Entire taxa, whole gene fragments, or individual sequences for a particular gene and species can be excluded from export. Data matrices can be re‐split into their component genes and the gene fragments can be exported as individual gene files. SequenceMatrix also includes two tools that help to identify sequences that may have been compromised through laboratory contamination or data management error. One tool lists identical or near‐identical sequences within genes, while the other compares the pairwise distance pattern of one gene against the pattern for all remaining genes combined. SequenceMatrix is Java‐based and compatible with the Microsoft Windows, Apple MacOS X and Linux operating systems. The software is freely available from http://code.google.com/p/sequencematrix/.

Episodic radiations in the fly tree of life

Flies are one of four superradiations of insects (along with beetles, wasps, and moths) that account for the majority of animal life on Earth. Diptera includes species known for their ubiquity (Musca domestica house fly), their role as pests (Anopheles gambiae malaria mosquito), and their value as model organisms across the biological sciences (Drosophila melanogaster). A resolved phylogeny for flies provides a framework for genomic, developmental, and evolutionary studies by facilitating comparisons across model organisms, yet recent research has suggested that fly relationships have been obscured by multiple episodes of rapid diversification. We provide a phylogenomic estimate of fly relationships based on molecules and morphology from 149 of 157 families, including 30 kb from 14 nuclear loci and complete mitochondrial genomes combined with 371 morphological characters. Multiple analyses show support for traditional groups (Brachycera, Cyclorrhapha, and Schizophora) and corroborate contentious findings, such as the anomalous Deuterophlebiidae as the sister group to all remaining Diptera. Our findings reveal that the closest relatives of the Drosophilidae are highly modified parasites (including the wingless Braulidae) of bees and other insects. Furthermore, we use micro-RNAs to resolve a node with implications for the evolution of embryonic development in Diptera. We demonstrate that flies experienced three episodes of rapid radiation—lower Diptera (220 Ma), lower Brachycera (180 Ma), and Schizophora (65 Ma)—and a number of life history transitions to hematophagy, phytophagy, and parasitism in the history of fly evolution over 260 million y.

Sepsid even-skipped Enhancers Are Functionally Conserved in Drosophila Despite Lack of Sequence Conservation

The gene expression pattern specified by an animal regulatory sequence is generally viewed as arising from the particular arrangement of transcription factor binding sites it contains. However, we demonstrate here that regulatory sequences whose binding sites have been almost completely rearranged can still produce identical outputs. We sequenced the even-skipped locus from six species of scavenger flies (Sepsidae) that are highly diverged from the model species Drosophila melanogaster, but share its basic patterns of developmental gene expression. Although there is little sequence similarity between the sepsid eve enhancers and their well-characterized D. melanogaster counterparts, the sepsid and Drosophila enhancers drive nearly identical expression patterns in transgenic D. melanogaster embryos. We conclude that the molecular machinery that connects regulatory sequences to the transcription apparatus is more flexible than previously appreciated. In exploring this diverse collection of sequences to identify the shared features that account for their similar functions, we found a small number of short (20–30 bp) sequences nearly perfectly conserved among the species. These highly conserved sequences are strongly enriched for pairs of overlapping or adjacent binding sites. Together, these observations suggest that the local arrangement of binding sites relative to each other is more important than their overall arrangement into larger units of cis-regulatory function.

The Use of Mean Instead of Smallest Interspecific Distances Exaggerates the Size of the “Barcoding Gap” and Leads to Misidentification

DNA barcoding is one of the best funded and most visible large-scale initiatives in systematic biology and has generated both much interest and controversy. DNA barcoding has also attracted significant support from foundations that had previously shown little interest in systematics. Yet, the project is controversial because many systematists feel that currently the conceptual foundation of DNA barcoding remains weak. This problem can only be alleviated through additional research that can lead to improved tools and concepts. Here, we scrutinize a key concept of DNA barcoding, the so-called barcoding gap (Meyer and Paulay, 2005), and use empirical data to document that it needs to be computed based on the smallest instead of the mean interspecific distances. In the literature on DNA barcoding, the “barcoding gap” (Meyer and Paulay, 2005) refers to the separation between mean intraand interspecific sequence variability for congeneric COI sequences. The barcoding gap is so essential to barcoding that a widely cited publication was dedicated to documenting these gaps across major metazoan taxa (Hebert et al., 2003b). It is also regularly mentioned in articles promoting barcoding to a broader audience (Check, 2005; Cognato and Caesar, 2006; Dasmahapatra and Mallet, 2006) and is one of the few metrics included in the Web-based identification system BOLD, “The Barcode of Life Data System,” which is a major identification tool for the DNA barcoding community (http://www.barcodinglife.org; Ratnasingham and Hebert, 2007). Large barcoding gaps are routinely used to predict DNA-barcoding success for the taxon under study (Hebert et al., 2003a, 2003b, 2004a, 2004b; Hogg and Hebert, 2004; Powers, 2004; Zehner et al., 2004; Armstrong and Ball, 2005; Ball et al., 2005; Barrett and Hebert, 2005; Lorenz et al., 2005; Saunders, 2005; Smith et al., 2005, 2006; Ward et al., 2005; Cywinska et al., 2006; Hajibabaei et al., 2006a, 2006b; Lefebure et al., 2006; Clare et al., 2007; Seifert et al., 2007). However, here we argue and document that barcoding gaps are currently incorrectly computed and that the values reported in the barcoding literature are misleading. The main problem is that the barcoding gap is generally quantified as the difference between intraspecific and mean interspecific, congeneric distances, whereas we will argue here that for species identification only the smallest interspecific distance should be used. Other authors have also pointed out that the use of smallest interspecific distances would be more appropriate (see Sperling, 2003; Moritz and Cicero, 2004; Vences et al., 2005a, 2005b; Cognato, 2006; Meier et al., 2006; Meyer and Paulay, 2005; Roe and Sperling, 2007), but currently we lack a comparative study that documents that the two measures yield different results. Here we provide evidence based on 43,137 COI sequences from 12,459 Metazoan species that barcoding gaps based on mean interspecific distances are artificially inflated and that only smallest interspecific distances correctly reflect that species identification gets more difficult as more species are sampled. Using DNA barcodes for species identification is analogous to identifying an unidentified specimen by comparing it to a reference collection of identified specimens. Initially one may compare an unidentified specimen to all identified material in the same genus, but ultimately the identification problem pares down to deciding whether a specimen belongs to one of a few, very similar, congeneric species. Determining an unidentified specimen to species is straightforward if the intraspecific variability is small—i.e., the unidentified specimen is a good match to a referenced species—and the differences between the best-matching species and the next best match is large—i.e., the specimen is a good match to only one of the referenced species. Analogously, the ease with which a query sequence can be identified to species is only dependent on how different it is from the most similar allospecific sequence, whereas its distinctness from a hypothetical “average” congeneric species does not matter (see Sperling, 2003; Moritz and Cicero, 2004; Vences et al., 2005a, 2005b; Cognato, 2006; Meier et al., 2006; Meyer and Paulay, 2005; Roe and Sperling, 2007). Yet, DNA barcoding publications and BOLD continue to report the mean instead of the smallest interspecific distances for congeneric species.

On the inappropriate use of Kimura-2-parameter (K2P) divergences in the DNA-barcoding literature

Here we present evidence, based on 10 datasets comprising 5283 sequences for 200 genera, that the use of the Kimura‐2‐parameter (K2P) model in DNA‐barcoding studies is poorly justified. We demonstrate that K2P is neither expected nor confirmed to be an appropriate model for closely related COI sequences. In addition, we show that the use of uncorrected distances yields higher or similar identification success rates for neighbour‐joining trees and distance‐based identification techniques. K2P also does not widen the barcoding gap for closely related sequences. We conclude that the spread of K2P through the barcoding literature is difficult to explain, and urge the use of evidence‐based approaches to DNA barcoding.

Proximate Causes of Rensch’s Rule: Does Sexual Size Dimorphism in Arthropods Result from Sex Differences in Development Time?

A prominent interspecific pattern of sexual size dimorphism (SSD) is Rensch’s rule, according to which male body size is more variable or evolutionarily divergent than female body size. Assuming equal growth rates of males and females, SSD would be entirely mediated, and Rensch’s rule proximately caused, by sexual differences in development times, or sexual bimaturism (SBM), with the larger sex developing for a proportionately longer time. Only a subset of the seven arthropod groups investigated in this study exhibits Rensch’s rule. Furthermore, we found only a weak positive relationship between SSD and SBM overall, suggesting that growth rate differences between the sexes are more important than development time differences in proximately mediating SSD in a wide but by no means comprehensive range of arthropod taxa. Except when protandry is of selective advantage (as in many butterflies, Hymenoptera, and spiders), male development time was equal to (in water striders and beetles) or even longer than (in drosophilid and sepsid flies) that of females. Because all taxa show female‐biased SSD, this implies faster growth of females in general, a pattern markedly different from that of primates and birds (analyzed here for comparison). We discuss three potential explanations for this pattern based on life‐history trade‐offs and sexual selection.

A phylogenetic analysis of the fungus-growing ants (Hymenoptera: Formicidae: Attini) based on morphological characters of the larvae

A phylogenetic hypothesis of the fungus‐growing ants (subfamily Myrmicinae, tribe Attini) is proposed, based on a cladistic analysis utilizing forty‐four morphological characters (109 states) of the prepupal worker larva. The fifty‐one attine species analysed include representatives of eleven of the twelve currently recognized attine genera, excluding only the monotypic workerless parasite Pseudoatta; the non‐attines include two outgroups (species of the basal myrmicine genera Myrmica and Pogonomyrmex), two myrmicine species presumed to be distantly related to the attines, and twelve species representing taxa that have been proposed by prior workers as possible sister groups of the Attini. There is strong character support for the monophyly of the Attini and for a sister‐group relationship of the Attini and the Neotropical Blepharidatta brasiliensis. The Attini are divided into two distinct lineages, an ‘apterostigmoid’ clade (containing Apterostigma and Mycocepurus) and an ‘attoid’ clade (containing all other attine genera except Myrmicocrypta). The attine genus Myrmicocrypta appears to be paraphyletic with respect to these two groups; the species M.buenzlii in particular retains many attine plesiomorphies.

Determining Species Boundaries in a World Full of Rarity: Singletons, Species Delimitation Methods

Singletons—species only known from a single specimen—and uniques—species that have only been collected once—are very common in biodiversity samples. Recent reviews suggest that in tropical arthropod samples, 30% of all species are represented by only one specimen (Bickel 1999; Novotny and Basset 2000; Coddington et al. 2009), with additional sampling helping little with eliminating rarity. Usually, such sampling only converts some of the singleton species to doubletons, with new singleton species being discovered in the process (Scharff et al. 2003; Coddington et al. 2009). Here, we first demonstrate that rare species are similarly common in specimen samples used for taxonomic research before we argue that the phenomenon of rarity has been insufficiently considered by the new quantitative techniques for species delimitation. Addressing this disconnect between theory and reality is pressing given that the last decade has seen a renewed interest in methods for species identification and delimitation (Sites and Marshall 2004; O’Meara 2010). Much of this interest has been fuelled by the availability of DNA sequences (Meier 2008). However, many newly proposed techniques implicitly or explicitly assume that all populations and species can be well sampled. But what is the value of these techniques if many species have only been collected once and/or are only known from one specimen? Here, we argue that all existing techniques need to be modified to accommodate the commonness of rarity and that all future techniques should be explicit about how rare species can be discovered and treated.

Morphological and molecular evidence converge upon a robust phylogeny of the megadiverse Holometabola

We present the largest morphological character set ever compiled for Holometabola. This was made possible through an optimized acquisition of data. Based on our analyses and recently published hypotheses based on molecular data, we discuss higher‐level phylogeny and evolutionary changes. We comment on the information content of different character systems and discuss the role of morphology in the age of phylogenomics. Microcomputer tomography in combination with other techniques proved highly efficient for acquiring and documenting morphological data. Detailed anatomical information (356 characters) is now available for 30 representatives of all holometabolan orders. A combination of traditional and novel techniques complemented each other and rapidly provided reliable data. In addition, our approach facilitates documenting the anatomy of model organisms. Our results show little congruence with studies based on rRNA, but confirm most clades retrieved in a recent study based on nuclear genes: Holometabola excluding Hymenoptera, Coleopterida (= Strepsiptera + Coleoptera), Neuropterida excl. Neuroptera, and Mecoptera. Mecopterida (= Antliophora + Amphiesmenoptera) was retrieved only in Bayesian analyses. All orders except Megaloptera are monophyletic. Problems in the analyses are caused by taxa with numerous autapomorphies and/or inapplicable character states due to the loss of major structures (such as wings). Different factors have contributed to the evolutionary success of various holometabolan lineages. It is likely that good flying performance, the ability to occupy different habitats as larvae and adults, parasitism, liquid feeding, and co‐evolution with flowering plants have played important roles. We argue that even in the “age of phylogenomics”, comparative morphology will still play a vital role. In addition, morphology is essential for reconstructing major evolutionary transformations at the phenotypic level, for testing evolutionary scenarios, and for placing fossil taxa.
© The Willi Hennig Society 2010.

Molecular phylogeny of the Calyptratae (Diptera: Cyclorrhapha) with an emphasis on the superfamily Oestroidea and the position of Mystacinobiidae and McAlpine's fly.

The dipteran clade Calyptratae is comprised of approximately 18 000 described species (12% of the known dipteran diversity) and includes well‐known taxa such as houseflies, tsetse flies, blowflies and botflies, which have a close association with humans. However, the phylogenetic relationships within this insect radiation are very poorly understood and controversial. Here we propose a higher‐level phylogenetic hypothesis for the Calyptratae based on an extensive DNA sequence dataset for 11 noncalyptrate outgroups and 247 calyptrate species representing all commonly accepted families in the Oestroidea and Hippoboscoidea, as well as those of the muscoid grade. DNA sequences for genes in the mitochondrial (12S, 16S, cytochrome c oxidase subunit I and cytochrome b) and nuclear genome [18S, 28S, the carbamoyl phosphate synthetase region of CAD (rudimentary), Elongation factor one alpha] were used to reconstruct the relationships. We discuss problems relating to the alignment and analysis of large datasets and emphasize the advantages of utilizing a guide tree‐based approach for the alignment of the DNA sequences and using the leaf stability index to identify ‘wildcard’ taxa whose excessive instability obscures the phylogenetic signal. Our analyses support the monophyly of the Calyptratae and demonstrate that the superfamily Oestroidea is nested within the muscoid grade. We confirm that the monotypic family Mystacinobiidae is an oestroid and further revise the composition of the Oestroidea by demonstrating that the previously unplaced and still undescribed ‘McAlpine’s fly’ is nested within this superfamily as a probable sister group to Mystacinobiidae. Within the Oestroidea we confirm with molecular data that the Calliphoridae are a paraphyletic grade of lineages. The families Sarcophagidae and Rhiniidae are monophyletic, but support for the monophyly of Tachinidae and Rhinophoridae depends on analytical technique (e.g. parsimony or maximum likelihood). The superfamilies Hippoboscoidea and Oestroidea are consistently found to be monophyletic, and the paraphyly of the muscoid grade is confirmed. In the overall relationships for the calyptrates, the Hippoboscoidea are sister group to the remaining Calyptratae, and the Fanniidae are sister group to the nonhippoboscoid calyptrates, whose relationships can be summarized as (Muscidae (Oestroidea (Scathophagidae, Anthomyiidae))).