Mitochondrial DNA size variation within individual crickets.

The mitochondrial DNA's of two closely related cricket species (genus Gryllus) share a size polymorphism as evidenced by analysis of restriction fragment patterns. Moreover, 12 of 100 field-collected crickets are heteroplasmic, that is these individuals have more than one size class of mitochondrial DNA. No heteroplasmy for restriction site variation is observed. Intraindividual variation in cricket mitochondrial DNA provides a useful marker for studying the transmission genetics of mitochondrial DNA. Available data on patterns of variation in mothers and offspring suggest that random segregation of mitochondrial DNA variants does not occur rapidly in cricket germ-cell lineages.

Collection and storage of invertebrate samples.

The validity of any comparative study is dependent on the reliability of the identification of the samples in the study. Not all researchers are experts in the field of identification of samples, nor do all researchers have quick and ready access to expert systematists who can accomplish the task of identification. The importance of verification of sample identity for comparative studies is vital. We describe several methods by which researchers can obtain and identify samples from the wild, and we suggest methods by which voucher samples can be obtained for future reference to these collected samples. We outline alternatives to collection of samples from the wild, such as purchase from stock centers and biological supply companies. Museum collections can also be extremely helpful in obtaining complete organismal samples for comparative studies.

Sources of ambiguity in nucleic acid sequence alignment.

The discussion of molecular sequence alignment is becoming more prominent in studies of molecular systematics and evolution. As the basis for initial homology statements, alignment is crucial to comparative molecular biology. Although fundamental, alignment is not a process which necessarily yields objective, precise results. Ambiguities can appear in alignment due to a number of factors. Three such sources of ambiguity are discussed here. These are ambiguity in the establishment of alignment parameters, pair-wise order and individual "path" variation. The first arises from the necessary but empirically untestable assumptions of gap costs and other factors which are required to align sequences objectively. The second is due to the possible existence of non-unique solutions to the same alignment parameters in heuristic and exact solutions. The third is a result of multiple optimal paths within single alignments, potentially generating huge numbers of equally costly but unique alignments. Some of the problems with and several possible solutions to the difficult situation of non-unique alignments are discussed.

Efficiency of parallel direct optimization.

Tremendous progress has been made at the level of sequential computation in phylogenetics. However, little attention has been paid to parallel computation. Parallel computing is particularly suited to phylogenetics because of the many ways large computational problems can be broken into parts that can be analyzed concurrently. In this paper, we investigate the scaling factors and efficiency of random addition and tree refinement strategies using the direct optimization software, POY, on a small (10 slave processors) and a large (256 slave processors) cluster of networked PCs running LINUX. These algorithms were tested on several data sets composed of DNA and morphology ranging from 40 to 500 taxa. Various algorithms in POY show fundamentally different properties within and between clusters. All algorithms are efficient on the small cluster for the 40-taxon data set. On the large cluster, multibuilding exhibits excellent parallel efficiency, whereas parallel building is inefficient. These results are independent of data set size. Branch swapping in parallel shows excellent speed-up for 16 slave processors on the large cluster. However, there is no appreciable speed-up for branch swapping with the further addition of slave processors (>16). This result is independent of data set size. Ratcheting in parallel is efficient with the addition of up to 32 processors in the large cluster. This result is independent of data set size.

Molecular evolution and phylogenetic utility of the polyubiquitin locus in mammals and higher vertebrates.

The product of ubiquitin genes is a small protein involved in intracellular sorting of other proteins. The locus consists of tandemly arrayed, uninterrupted copies of the gene. As several studies have noted, the Polyubiquitin locus is a model system for studying concerted evolution. While the protein is among the most conserved known, individual copies within an organism show variation in nucleotide sequence despite clear evidence of concerted evolution. When treated as individuals, repeats from a given locus form a monophyletic group. Furthermore adjacent copies often cluster, suggestive of the mechanism of concerted evolution. Due to this concerted evolution of repeats (and loci in organisms with multiple polyubiquitins), sequencing of heterogeneous PCR products consisting of all the repeats in a given organism may yield phylogenetic signal, as with other multicopy genes. We test this possibility through 22 original sequences using primers designed so that only tandem copies are amplified. Using these and previously published data, we further explore these phenomena in higher vertebrates and mammals in particular. We suggest that multiple locus duplications have occurred within mammals. Positional codon bias is strongly evident. We also find substitutional bias with regard to codon type. GC content of the locus appears to be generally high across vertebrates. Intraorganismal variation is tallied as an indication of frequency of change in codon position and transition/transversion ratios to further elucidate the tempo and mode of molecular evolution. Using these data, a weighting scheme for ubiquitin is also presented. Despite the gene's high GC content, transitional changes still appear more frequent. While the phylogenetic utility of ubiquitin does not appear great, its mechanistic insights seem far from exhausted.

Paired sequence difference in ribosomal RNAs: evolutionary and phylogenetic implications.

Ribosomal RNAs have secondary structures that are maintained by internal Watson-Crick pairing. Through analysis of chordate, arthropod, and plant 5S ribosomal RNA sequences, we show that Darwinian selection operates on these nucleotide sequences to maintain functionally important secondary structure. Insect phylogenies based on nucleotide positions involved in pairing and the production of secondary structure are incongruent with those constructed on the basis of positions that are not. Furthermore, phylogeny reconstruction using these nonpairing bases is concordant with other, morphological data.

On gaps.

Gaps result from the alignment of sequences of unequal length during primary homology assessment. Viewed as character states originating from particular biological events (mutation), gaps contain historical information suitable for phylogenetic analysis. The effect of gaps as a source of phylogenetic data is explored via sensitivity analysis and character congruence among different data partitions. Example data sets are provided to show that gaps contain important phylogenetic information not recovered by those methods that omit gaps in their calculations. However, gap cost schemes are arbitrary (although they must be explicit) and thus data exploration is a necessity of molecular analyses, while character congruence is necessary as an external criterion for hypothesis decision.

Elision: a method for accommodating multiple molecular sequence alignments with alignment-ambiguous sites.

The process of multiple sequence alignment provides homology statements for the phylogenetic analysis of molecular data. Unfortunately, multiple alignments are frequently nonunique. Two sources of these multiple alignments are analysis based on different sets of alignment parameter values (gap:change cost ratios) and nonunique equally costly alignments based on a single set of alignment parameters. By "eliding" these individual alignments into a single grand alignment, phylogeny that is weighted toward those positions that align more consistently can be reconstructed. Positions that show greater variation among alignments will be relatively downweighted. The technique results in a weighting procedure that is a posteriori and based on the evidence established from the original sequence alignments.

Arthropod phylogeny based on eight molecular loci and morphology.

The interrelationships of major clades within the Arthropoda remain one of the most contentious issues in systematics, which has traditionally been the domain of morphologists. A growing body of DNA sequences and other types of molecular data has revitalized study of arthropod phylogeny and has inspired new considerations of character evolution. Novel hypotheses such as a crustacean-hexapod affinity were based on analyses of single or few genes and limited taxon sampling, but have received recent support from mitochondrial gene order, and eye and brain ultrastructure and neurogenesis. Here we assess relationships within Arthropoda based on a synthesis of all well sampled molecular loci together with a comprehensive data set of morphological, developmental, ultrastructural and gene-order characters. The molecular data include sequences of three nuclear ribosomal genes, three nuclear protein-coding genes, and two mitochondrial genes (one protein coding, one ribosomal). We devised new optimization procedures and constructed a parallel computer cluster with 256 central processing units to analyse molecular data on a scale not previously possible. The optimal 'total evidence' cladogram supports the crustacean-hexapod clade, recognizes pycnogonids as sister to other euarthropods, and indicates monophyly of Myriapoda and Mandibulata.

Higher level relationships of the arctoid Carnivora based on sequence data and "total evidence".

The relationships of the lesser or red panda, Ailurus, have remained elusive even as any doubts about the identity of the giant panda as a bear have been erased. While usually classified as a member of the Procyonidae (raccoons), recent anatomical studies have suggested that the red panda may not fall in any of the arctoid carnivore families but instead may reflect an early offshoot of the lineage leading to ursids (bears) and pinnipeds (seals, sea lions, and walruses). Sequence data from the cytochrome b and 12S genes for multiple representatives of all relevant families support this hypothesis. Such a systematic position makes this threatened species particularly worthy of conservation. Sequence data alone, as well as a combined analysis of the sequence and anatomical data, strongly support a single origin of pinnipeds and their aquatic adaptations, lending some resolution to the general disagreement about familial relationships in this group. These molecular data also support canids as the basal members of this caniform clade, but are unresolved with respect to whether mustelids or procyonids constitute the sister group to the (ursid, pinniped, Ailurus) clade. There is support for the notion that skunks are a genetically divergent and possibly nonmustelid lineage.

The Strepsiptera problem: phylogeny of the holometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology.

Phylogenetic relationships among the holometabolous insect orders were inferred from cladistic analysis of nucleotide sequences of 18S ribosomal DNA (rDNA) (85 exemplars) and 28S rDNA (52 exemplars) and morphological characters. Exemplar outgroup taxa were Collembola (1 sequence), Archaeognatha (1), Ephemerida (1), Odonata (2), Plecoptera (2), Blattodea (1), Mantodea (1), Dermaptera (1), Orthoptera (1), Phasmatodea (1), Embioptera (1), Psocoptera (1), Phthiraptera (1), Hemiptera (4), and Thysanoptera (1). Exemplar ingroup taxa were Coleoptera: Archostemata (1), Adephaga (2), and Polyphaga (7); Megaloptera (1); Raphidioptera (1); Neuroptera (sensu stricto = Planipennia): Mantispoidea (2), Hemerobioidea (2), and Myrmeleontoidea (2); Hymenoptera: Symphyta (4) and Apocrita (19); Trichoptera: Hydropsychoidea (1) and Limnephiloidea (2); Lepidoptera: Ditrysia (3); Siphonaptera: Pulicoidea (1) and Ceratophylloidea (2); Mecoptera: Meropeidae (1), Boreidae (1), Panorpidae (1), and Bittacidae (2); Diptera: Nematocera (1), Brachycera (2), and Cyclorrhapha (1); and Strepsiptera: Corioxenidae (1), Myrmecolacidae (1), Elenchidae (1), and Stylopidae (3). We analyzed approximately 1 kilobase of 18S rDNA, starting 398 nucleotides downstream of the 5' end, and approximately 400 bp of 28S rDNA in expansion segment D3. Multiple alignment of the 18S and 28S sequences resulted in 1,116 nucleotide positions with 24 insert regions and 398 positions with 14 insert regions, respectively. All Strepsiptera and Neuroptera have large insert regions in 18S and 28S. The secondary structure of 18S insert 23 is composed of long stems that are GC rich in the basal Strepsiptera and AT rich in the more derived Strepsiptera. A matrix of 176 morphological characters was analyzed for holometabolous orders. Incongruence length difference tests indicate that the 28S + morphological data sets are incongruent but that 28S + 18S, 18S + morphology, and 28S + 18S + morphology fail to reject the hypothesis of congruence. Phylogenetic trees were generated by parsimony analysis, and clade robustness was evaluated by branch length, Bremer support, percentage of extra steps required to force paraphyly, and sensitivity analysis using the following parameters: gap weights, morphological character weights, methods of data set combination, removal of key taxa, and alignment region. The following are monophyletic under most or all combinations of parameter values: Holometabola, Polyphaga, Megaloptera + Raphidioptera, Neuroptera, Hymenoptera, Trichoptera, Lepidoptera, Amphiesmenoptera (Trichoptera + Lepidoptera), Siphonaptera, Siphonaptera + Mecoptera, Strepsiptera, Diptera, and Strepsiptera + Diptera (Halteria). Antliophora (Mecoptera + Diptera + Siphonaptera + Strepsiptera), Mecopterida (Antliophora + Amphiesmenoptera), and Hymenoptera + Mecopterida are supported in the majority of total evidence analyses. Mecoptera may be paraphyletic because Boreus is often placed as sister group to the fleas; hence, Siphonaptera may be subordinate within Mecoptera. The 18S sequences for Priacma (Coleoptera: Archostemata), Colpocaccus (Coleoptera: Adephaga), Agulla (Raphidioptera), and Corydalus (Megaloptera) are nearly identical, and Neuropterida are monophyletic only when those two beetle sequences are removed from the analysis. Coleoptera are therefore paraphyletic under almost all combinations of parameter values. Halteria and Amphiesmenoptera have high Bremer support values and long branch lengths. The data do not support placement of Strepsiptera outside of Holometabola nor as sister group to Coleoptera. We reject the notion that the monophyly of Halteria is due to long branch attraction because Strepsiptera and Diptera do not have the longest branches and there is phylogenetic congruence between molecules, across the entire parameter space, and between morphological and molecular data.

Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cycliophora, Plathelminthes, and Chaetognatha: a combined approach of 18S rDNA sequences and morphology.

Triploblastic relationships were examined in the light of molecular and morphological evidence. Representatives for all triploblastic "phyla" (except Loricifera) were represented by both sources of phylogenetic data. The 18S ribosomal (rDNA) sequence data for 145 terminal taxa and 276 morphological characters coded for 36 supraspecific taxa were combined in a total evidence regime to determine the most consistent picture of triploblastic relationships for these data. Only triploblastic taxa are used to avoid rooting with distant outgroups, which seems to happen because of the extreme distance that separates diploblastic from triploblastic taxa according to the 18S rDNA data. Multiple phylogenetic analyses performed with variable analysis parameters yield largely inconsistent results for certain groups such as Chaetognatha, Acoela, and Nemertodermatida. A normalized incongruence length metric is used to assay the relative merit of the multiple analyses. The combined analysis having the least character incongruence yields the following scheme of relationships of four main clades: (1) Deuterostomia [((Echinodermata + Enteropneusta) (Cephalochordata (Urochordata + Vertebrata)))]; (2) Ecdysozoa [(((Priapulida + Kinorhyncha) (Nematoda + Nematomorpha)) ((Onychophora + Tardigrada) Arthropoda))]; (3) Trochozoa [((Phoronida + Brachiopoda) (Entoprocta (Nemertea (Sipuncula (Mollusca (Pogonophora (Echiura + Annelida)))))))]; and (4) Platyzoa [((Gnathostomulida (Cycliophora + Syndermata)) (Gastrotricha + Plathelminthes))]. Chaetognatha, Nemertodermatida, and Bryozoa cannot be assigned to any one of these four groups. For the first time, a data analysis recognizes a clade of acoelomates, the Platyzoa (sensu Cavalier-Smith, Biol. Rev. 73:203-266, 1998). Other relationships that corroborate some morphological analyses are the existence of a clade that groups Gnathostomulida + Syndermata (= Gnathifera), which is expanded to include the enigmatic phylum Cycliophora, as sister group to Syndermata.