Data partitions and complex models in Bayesian analysis: the phylogeny of Gymnophthalmid lizards

Phylogenetic studies incorporating multiple loci, and multiple genomes, are becoming increasingly common. Coincident with this trend in genetic sampling, model-based likelihood techniques including Bayesian phylogenetic methods continue to gain popularity. Few studies, however, have examined model fit and sensitivity to such potentially heterogeneous data partitions within combined data analyses using empirical data. Here we investigate the relative model fit and sensitivity of Bayesian phylogenetic methods when alternative site-specific partitions of among-site rate variation (with and without autocorrelated rates) are considered. Our primary goal in choosing a best-fit model was to employ the simplest model that was a good fit to the data while optimizing topology and/or Bayesian posterior probabilities. Thus, we were not interested in complex models that did not practically affect our interpretation of the topology under study. We applied these alternative models to a four-gene data set including one protein-coding nuclear gene (c-mos), one protein-coding mitochondrial gene (ND4), and two mitochondrial rRNA genes (12S and 16S) for the diverse yet poorly known lizard family Gymnophthalmidae. Our results suggest that the best-fit model partitioned among-site rate variation separately among the c-mos, ND4, and 12S + 16S gene regions. We found this model yielded identical topologies to those from analyses based on the GTR+I+G model, but significantly changed posterior probability estimates of clade support. This partitioned model also produced more precise (less variable) estimates of posterior probabilities across generations of long Bayesian runs, compared to runs employing a GTR+I+G model estimated for the combined data. We use this three-way gamma partitioning in Bayesian analyses to reconstruct a robust phylogenetic hypothesis for the relationships of genera within the lizard family Gymnophthalmidae. We then reevaluate the higher-level taxonomic arrangement of the Gymnophthalmidae. Based on our findings, we discuss the utility of nontraditional parameters for modeling among-site rate variation and the implications and future directions for complex model building and testing.     

Horned lizard (Phrynosoma) phylogeny inferred from mitochondrial genes and morphological characters: understanding conflicts using multiple approaches

The genus Phrynosoma includes 13 species of North American lizards characterized by unique and highly derived morphologies and ecologies. Understanding interspecific relationships within this genus is essential for testing hypotheses about character evolution in this group. We analyzed mitochondrial ND4 and cytochrome b gene sequence data from all species of Phrynosoma in conjunction with a previously published dataset including 12S and 16S rRNA gene sequences and morphological characters. We used multiple phylogenetic methods and diagnostic tests for data combinability and taxonomic congruence to investigate the data in separate and combined analyses. Separate data partitions resulted in several well-supported lineages, but taxonomic congruence was lacking between topologies from separate and combined analyses. Partitioned Bremer support analyses also reveals conflict between data partitions in certain tree regions. When taxa associated with well-supported clades were removed from analyses, phylogenetic signal was lost. Combined, our results initially suggest conflict between data partitions, but further tests show the data are only appropriate for phylogenetic reconstruction of those parts of the topology that were well resolved. Nonetheless, our data analyses reveal five well-supported clades: (1) Phrynosoma ditmarsi and Phrynosoma hernandesi, (2) P. ditmarsi, P. hernandesi, and Phrynosoma douglasii, (3) P. ditmarsi, P. hernandesi, P. douglasii, and Phrynosoma orbiculare, (4) Phrynosoma mcallii and Phrynosoma platyrhinos, and (5) Phrynosoma braconnieri and Phrynosoma taurus.     

背瘤丽蚌F型线粒体基因组全序列分析

部分双壳贝类的线粒体遗传方式是特殊的双重单亲遗传方式:F型存在于雌性体细胞组织和性腺中,M型仅存在于雄性个体的性腺中。通过LA-PCR扩增、SHOT-GUN测序、软件拼接获得背瘤丽蚌(Lamprotula leai)F型线粒体基因组全序列。线粒体基因组全长为16530 bp,包括13个蛋白质编码基因,22个tRNA其中包括2个tRNA^Ser和2个tRNA^Leu,2个SrRNA及27个长度不等的非编码区,最长的两个非编码区分别为969 bp、228 bp。比较分析已登录到GenBank中的淡水蚌类F型线粒体结构特征,结果显示背瘤丽蚌F型A+T含量为60.28%,表现出A+T偏好性,淡水蚌类线粒体基因组长度的差异主要表现为非编码区长度的差异。此外,背瘤丽蚌mtDNA的COⅡ-12S rRNA区域基因排列存在差异,是ND3、tRNA^His、tRNA^Ala、tRNA^Ser1、tRNA^Ser2、tRNA^Glu、ND2、tRNA^Met 8个基因发生重排造成。F型线粒体序列构建的系统进化树中,淡水蚌类和海水双壳贝类分别聚为一支。研究结果为进一步研究淡水珍珠蚌的DUI线粒体遗传方式和种质资源保护奠定基础,为双壳贝类mtDNA基因重排提供依据。     

Complete mitochondrial genome of the boreal digging frog Kaloula borealis (Anura, Microhylidae)

We sequenced the complete mitochondrial genome from the boreal digging frog Kaloula borealis. The genome sequence was 17,173 bp in size, and the gene order and contents were identical to those of previously reported amphibian mitochondrial genomes. Of 13 protein-coding genes (PCGs), 5 genes (CO2, ATPase 6, CO3, ND3, and ND4) had incomplete stop codons. Also ND1 gene used GTG as a start codon, while CO1 and ND5 genes used AGG as a stop codon. The base composition of K. borealis mitogenome showed a strong anti-G bias (6.11%) on the 3rd position of PCGs.     

Congruence between nuclear and mitochondrial genes in Demospongiae: a new hypothesis for relationships within the G4 clade (Porifera: Demospongiae)

The current morphological classification of the Demospongiae G4 clade was tested using large subunit ribosomal RNA (LSU rRNA) sequences from 119 taxa. Fifty-three mitochondrial cytochrome oxidase 1 (CO1) barcoding sequences were also analysed to test whether the 28S phylogeny could be recovered using an independent gene. This is the largest and most comprehensive study of the Demospongiae G4 clade. The 28S and CO1 genetrees result in congruent clades but conflict with the current morphological classification. The results confirm the polyphyly of Halichondrida, Hadromerida, Dictyonellidae, Axinellidae and Poecilosclerida and show that several of the characters used in morphological classifications are homoplasious. Robust clades are clearly shown and a new hypothesis for relationships of taxa allocated to G4 is proposed.     

Phylogenetic relationships of silver crucian carp Carassius auratus gibelio, C. auratus cuvieri, crucian carp C. carassius, and common carp Cyprinus carpio as inferred from mitochondrial DNA variation

PCR-RFLP analysis of the ND3/ND4L/ND4 and 12S/16S rRNA regions and nucleotide sequence variation of the cytochrome b gene were used to study the mtDNA divergence in species of the family Cyprinidae, to examine the phylogenetic relationships of the species, and to identify their taxonomic status. The results indicated that an ancestral form diverged into silver crucian carp and crucian carp after its separation from the common carp lineage. The divergence of continental Carassius auratus gibelio and Japanese C. auratus cuvieri occurred more recently. Two well distinguishable mtDNA phylogroups, suggesting divergent evolution, were observed in continental C. auratus gibelio populations. The divergence was possibly related to the formation of two silver crucian carp groups with different types of reproduction, triploid gynogenetic and diploid gonochoric. At the same time, the results supported the high probability of current genetic exchange between the forms. In view of these findings and high morphological similarity of the two forms, they were not considered to be separate species.     

A molecular phylogenetic analysis of the Scarabaeinae (dung beetles)

The dung beetles (Scarabaeinae) include ca. 5000 species and exhibit a diverse array of morphologies and behaviors. This variation presumably reflects the adaptation to a diversity of food types and the different strategies used to avoid competition for vertebrate dung, which is the primary breeding environment for most species. The current classification gives great weight to the major behavioral types, separating the ball rollers and the tunnelers, but existing phylogenetic studies have been based on limited taxonomic or biogeographic sampling and have been contradictory. Here, we present a molecular phylogenetic analysis of 214 species of Scarabaeinae, representing all 12 traditionally recognized tribes and six biogeographical regions, using partial gene sequences from one nuclear (28S) and two mitochondrial (cox1, rrnL) genes. Length variation in 28S (588-621 bp) and rrnL (514-523 bp) was subjected to a thorough evaluation of alternative alignments, gap-coding methods, and tree searches using model-based (Bayesian and likelihood), maximum parsimony, and direct optimization analyses. The small-bodied, non-dung-feeding Sarophorus+Coptorhina were basal in all reconstructions. These were closely related to rolling Odontoloma+Dicranocara, suggesting an early acquisition of rolling behavior. Smaller tribes and most genera were monophyletic, while Canthonini and Dichotomiini each consisted of multiple paraphyletic lineages at hierarchical levels equivalent to the smaller tribes. Plasticity of rolling and tunneling was evidenced by a lack of monophyly (S-H test, p > 0.05) and several reversals within clades. The majority of previously unrecognized clades were geographical, including the well-supported Neotropical Phanaeini+Eucraniini, and a large Australian clade of rollers as well as tunneling Coptodactyla and Demarziella. Only three lineages, Gymnopleurini, Copris+Microcopris and Onthophagus, were widespread and therefore appear to be dispersive at a global scale. A reconstruction of biogeographical characters recovered 38-48 transitions between regions and an African origin for most lineages. Dispersal-vicariance analysis supported an African origin with links to all other regions and little back-migration. Our results provide a new synthesis of global-scale dung beetle evolution, demonstrating the great plasticity of behavioral and morphological traits and the importance of biogeographic distributions as the basis for a new classification.     

Decoupling of molecular and morphological evolution in deep lineages of a meiobenthic harpacticoid copepod

Molecular and biochemical genetic analyses have revealed that many marine invertebrate taxa, including some well-studied and presumably cosmopolitan species, are actually complexes of sibling species. When morphological differences are slight and estimated divergence times are old, data suggest either unusually high rates of sequence evolution or long-term morphological stasis. Here, five gene regions (mitochondrial cytochrome oxidase subunit I and large-subunit ribosomal 16S rDNA and nuclear ITS1, 5.8S rDNA, and ITS2) were analyzed in four geographic samples of the meiobenthic harpacticoid copepod Cletocamptus deitersi. Molecular sequences revealed four extremely differentiated molecular lineages with unalignable nuclear intergenic spacers and mitochondrial uncorrected divergences reaching 25% (cytochrome oxidase) and 36% (16S rDNA). These levels of divergence are greater than those reported previously for congeneric species in diverse invertebrate taxa, including crustaceans. The nominally intraspecific divergence matches or exceeds the corresponding divergence from a known congener (Cletocamptus helobius). A molecular clock applied to the cytochrome oxidase subunit I data suggests that these lineages split in the Miocene, consistent with the fossil record of a North American Cletocamptus from the same period. Morphological differences among the major lineages are subtle but congruent with the patterns of genetic differentiation. Our conclusion, based on concordant patterns of variation in two mitochondrial and three nuclear gene regions, as well as morphological observations, is that C. deitersi in North America is composed of at least four separate species by the genealogical concordance, phylogenetic, and morphological-species criteria. Alternative explanations for the deep phylogenetic nodes and apparent morphological stasis, including high rates of sequence evolution, balancing selection, and genetic signatures of historical events, are considered unlikely.     

Pattern of morphological diversification in the Leptocarabus ground beetles (Coleoptera: Carabidae) as deduced from mitochondrial ND5 gene and nuclear 28S rDNA sequences

Most of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene and a part of nuclear 28S ribosomal RNA gene were sequenced for 14 species of ground beetles belonging to the genus Leptocarabus. In both the ND5 and the 28S rDNA phylogenetic trees of Leptocarabus, three major lineages were recognized: (1) L. marcilhaci/L. yokoael/Leptocarabus sp. from China, (2) L. koreanus/L. truncaticollis/L. seishinensis/L. semiopacus/L. canaliculatus/L. kurilensis from the northern Eurasian continent including Korea and Hokkaido, Japan, and (3) all of the Japanese species except L. kurilensis. Clustering of the species in the trees is largely linked to their geographic distribution and does not correlate with morphological characters. The species belonging to different species groups are clustered in the same lineages, and those in the same species group are scattered among the different lineages. One of the possible interpretations of the present results would be that morphological transformations independently took place in the different lineages, sometimes with accompanying parallel morphological evolution, resulting in the occurrence of the morphological species belonging to the same species group (= type) in the different lineages.     

Complete mitochondrial genome of the rabbitfish Siganus fuscescens (Perciformes, Siganidae).

We determined the complete nucleotide sequence of the mitochondrial genome for the rabbitfish Siganus fuscescens (Perciformes, Siganidae). This mitochondrial genome, consisting of 16,491 base pairs (bp), included 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a noncoding control region similar those found in other vertebrates; the gene order was identical to that of typical vertebrates. Most of the genes of S. fuscescens were encoded on the H-strand, while the ND6 and eight tRNA (Gln, Ala, Asn, Cys, Tyr, Ser [UCN], Glu, and Pro) genes were encoded on the L-strand. The reading frames of ATPase 8 and 6 and those of ND4L and ND4 overlapped by ten and seven nucleotides, respectively. All mitochondrial protein-coding genes began with an ATG start codon, except for CO1, which started with GTG. Open reading frames of S. fuscescens ended with TAA (ND1, CO1, ATPase 8, ND4L, ND5 and ND6), and the remainder had incomplete stop codons, either TA (ATPase 6 and CO3) or T (ND2, CO2, ND3, ND4, and Cytb). The origin of L-strand replication in S. fuscescens was located in a cluster of five tRNA genes (WANCY) and was 34 nucleotides in length. A major noncoding region between the tRNA-Pro and tRNA-Phe genes (828 bp) was considered to be the control region (D-loop). Within this sequence, we identified a conserved sequence block characteristic of this region. The rabbitfish was grouped with Siganus canaliculatus in most parsimony analyses, which showed 100% bootstrap support for their divergence. These findings are useful for inferring phylogenetic relationships and identification within the suborder Acanthuroidei.     

Molecular evidence of natural hybridization between Fasciola hepatica and F. gigantica.

Nucleotide sequences of two regions, cytochrome c-oxidase subunit 1 (CO1) and NADH dehydrogenase subunit 1 (ND1) of the mitochondrial DNA and two regions, internal-transcribed spacer 2 (ITS2) and the D2 region in the 28S rDNA (28S) of the nuclear DNA were obtained from five Korean worms of the genus Fasciola in order to elucidate their taxonomic status. The CO1 and ND1 regions are all monomorphic in the Korean worms and similar to those of F. gigantica. On the other hand, the ITS2 and D2 regions were found to be polymorphic; that is, out of five worms, two possessed a F. gigantica-type sequence, one, a F. hepatica-type sequence and two possessed sequences of both types indicating an existence of different alleles at the loci. It should be noted that these variations of the ITS2 and D2 regions co-occur at the same individual worms. This was confirmed by sequencing five to six cloned PCR products for each worm. The present study strongly suggests interspecific cross-hybridization between the two species coexisting in Korea.     

New primers for amplifying and sequencing the mitochondrial ND4/ND5 gene region of the Cypriniformes (Actinopterygii: Ostariophysi)

We provide 15 new primers for amplifying and sequencing the mitochondrial ND4/ND5 gene region of the Cypriniformes in an attempt to resolve relationships of this diverse group of freshwater fishes with extensive taxonomic sampling. Sequences from this region have the following desirable characteristics for phylogenetic analyses, some of which are lacking from the more commonly used cyt b and 12S/16S rRNA genes: they are (1) easy to align, (2) relatively long (ca. 3.4 kb), and (3) contain more phylogenetically informative variation at 1st and 2nd codon positions. Moreover, the ND4/ND5 gene region is easy to amplify and sequence when employing the protocol suggested herein.     

Among-site rate variation and phylogenetic analysis of 12S rRNA in sigmodontine rodents

We analyze sequences from two mitochondrial genes, cytochrome b (cyt b) and 12S rRNA (12S), for a group of sigmodontine rodents among which phylogenetic relationships are well understood based on concordance of morphological, chromosomal, allozyme, and other DNA data sets. Because these two genes are physically linked on the nonrecombining mitochondrial genome, they necessarily share the same history. Phylogenetic analysis of the cyt b gene recovers the well-corroborated relationships, generally with strong support. None of the methods that we employed, including variously weighted parsimony, neighbor joining on both single-rate and gamma-corrected distances, and maximum likelihood, were able to recover these relationships for the 12S gene. Parsimony analyses of the 12S data resulted in a relatively strongly supported placement of Peromyscus eremicus that conflicts with that suggested by cyt b and all other data. There is extreme among-site rate variation in the 12S sequences and moderate levels in the cyt b sequences. This highly skewed distribution of rates in the 12S gene makes phylogenetic analyses of these sequences particularly susceptible to the misleading effects of nonindependence and other nonrandom noise, suggesting that phylogenetic analyses of data sets that contain a great deal of among-site rate variation be interpreted with caution.      

Genetic characterization of the mitochondrial DNA from Lepeophtheirus salmonis (Crustacea; Copepoda). A new gene organization revealed

The mitochondrial DNA (mtDNA) from the salmon louse, Lepeophtheirus salmonis, is 15445 bp. It includes the genes coding for cytochrome B (Cyt B), ATPase subunit 6 and 8 (A6 and A8), NADH dehydrogenase subunits 1-6 and 4L (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6), cytochrome c oxidase subunits I-III (COI, COII and COIII), two rRNA genes (12S rRNA and 16S rRNA) and 22 tRNAs. Two copies of tRNA-Lys are present in the mtDNA of L. salmonis, while tRNA-Cys was not identified. Both DNA strands contain coding regions in the salmon louse, in contrast to the other copepod characterized Tigriopus japonicus, but only a few genes overlap. In vertebrates, ND4 and ND4L are transcribed as one bicistronic mRNA, and are therefore localized together. The same organization is also found in crustaceans, with the exceptions of T. japonicus, Neocalanus cristatus and L. salmonis that deviate from this pattern. Another exception of the L. salmonis mtDNA is that A6 and A8 do not overlap, but are separated by several genes. The protein-coding genes have a bias towards AT-rich codons. The mitochondrial gene order in L. salmonis differs significantly from the copepods T. japonicus, Eucalanus bungii, N. cristatus and the other 13 crustaceans previously characterized. Furthermore, the mitochondrial rRNA genes are encoded on opposite strands in L. salmonis. This has not been found in any other arthropods, but has been reported in two starfish species. In a phylogenetic analysis, using an alignment of mitochondrial protein sequences, L. salmonis groups together with T. japonicus, being distant relatives to the other crustaceans.     

Structure and organization of Marchantia polymorpha chloroplast genome. IV. Inverted repeat and small single copy regions

We characterized the genes in the regions of large inverted repeats (IRA and IRB, 10,058 base-pairs each) and a small single copy (SSC 19,813 bp) of chloroplast DNA from Marchantia polymorpha. The inverted repeat (IR) regions contain genes for four ribosomal RNAs (16 S, 23 S, 4.5 S and 5 S rRNAs) and five transfer RNAs (valine tRNA(GAC), isoleucine tRNA(GAU), alanine tRNA(UGC), arginine tRNA(ACG) and asparagine tRNA(GUU)). The gene organization of the IR regions in the liverwort chloroplast genome is conserved, although the IR regions are smaller (10,058 base-pairs) than any reported in higher plant chloroplasts. The small single-copy region (19,813 base-pairs) encoded genes for 17 open reading frames, a leucine tRNA(UAG) and a proline tRNA(GGG)-like sequence. We identified 12 open reading frames by homology of their coding sequences to a 4Fe-4S-type ferredoxin protein, a bacterial nitrogenase reductase component (Fe-protein), five human mitochondrial components of NADH dehydrogenase (ND1, ND4, ND4L, ND5 and ND6), two Escherichia coli ribosomal proteins (S15 and L21), two putative proteins encoded in the kinetoplast maxicircle DNA of Leishmania tarentolae (LtORF 3 and LtORF 4), and a bacterial permease inner membrane component (encoded by malF in E. coli or hisQ in Salmonella typhimurium).     

Phylogenetic relationships of silver crusian carp Carassius auratus gibelio, C. auratus cuvieri, crucian carp Carassius carassius, and common carp Cyprinus carpio as inferred from mitochondrial DNA variation

PCR-RFLP analysis of the ND3/ND4L/ND4 and 12S/16S rRNA regions and nucleotide sequence variation of the cytochrome b gene were used to study the mtDNA divergence in species of the family Cyprinidae, to examine the phylogenetic relationships of the species, and to identify their taxonomic status. The results indicated that an ancestral form diverged into silver crucian carp and crucian carp after its separation from the common carp lineage. The divergence of continental Carassius auratus gibelio and Japanese C. auratus cuvieri occurred more recently. Two well distinguishable mtDNA phylogroups, suggesting divergent evolution, were observed in continental C. auratus gibelio populations. The divergence was possibly related to the formation of two silver crucian carp groups with different types of reproduction, triploid gynogenetic and diploid gonochoric. At the same time, the results supported the high probability of current genetic exchange between the forms. In view of these findings and high morphological similarity of the two forms, they were not considered to be separate species.     

Limitations of Metazoan 18S rRNA Sequence Data: Implications for Reconstructing a Phylogeny of the Animal Kingdom and Inferring the Reality of the Cambrian Explosion

We document the phylogenetic behavior of the 18S rRNA molecule in 67 taxa from 28 metazoan phyla and assess the effects of among-site rate variation on reconstructing phylogenies of the animal kingdom. This empirical assessment was undertaken to clarify further the limits of resolution of the 18S rRNA gene as a phylogenetic marker and to address the question of whether 18S rRNA phylogenies can be used as a source of evidence to infer the reality of a Cambrian explosion. A notable degree of among-site rate variation exists between different regions of the 18S rRNA molecule, as well as within all classes of secondary structure. There is a significant negative correlation between inferred number of nucleotide substitutions and phylogenetic information, as well as with the degree of substitutional saturation within the molecule. Base compositional differences both within and between taxa exist and, in certain lineages, may be associated with long branches and phylogenetic position. Importantly, excluding sites with different degrees of nucleotide substitution significantly influences the topology and degree of resolution of maximum-parsimony phylogenies as well as neighbor-joining phylogenies (corrected and uncorrected for among-site rate variation) reconstructed at the metazoan scale. Together, these data indicate that the 18S rRNA molecule is an unsuitable candidate for reconstructing the evolutionary history of all metazoan phyla, and that the polytomies, i.e., unresolved nodes within 18S rRNA phylogenies, cannot be used as a single or reliable source of evidence to support the hypothesis of a Cambrian explosion. Received: 9 December 1997 / Accepted: 23 March 1998     

Variation in Coding (NADH Dehydrogenase Subunits 2, 3, and 6) and Noncoding Intergenic Spacer Regions of the Mitochondrial Genome in Octocorallia (Cnidaria: Anthozoa)

Low rates of evolution in cnidarian mitochondrial genes such as COI and 16S rDNA have hindered molecular systematic studies in this important invertebrate group. We sequenced fragments of 3 mitochondrial protein-coding genes (NADH dehydrogenase subunits ND2, ND3 and ND6) as well as the COI-COII intergenic spacer, the longest noncoding region found in the octocoral mitochondrial genome, to determine if any of these regions contain levels of variation sufficient for reconstruction of phylogenetic relationships among genera of the anthozoan subclass Octocorallia. Within and between the soft coral families Alcyoniidae and Xeniidae, sequence divergence in the genes ND2 (539 bp), ND3 (102 bp), and ND6 (444 bp) ranged from 0.5% to 12%, with the greatest pairwise distances between the 2 families. The COI-COII intergenic spacer varied in length from 106 to 122 bp, and pairwise sequence divergence values ranged from 0% to 20.4%. Phylogenetic trees constructed using each region separately were poorly resolved. Better phylogenetic resolution was obtained in a combined analysis using all 3 protein-coding regions (1085 bp total). Although relationships among some pairs of species and genera were well supported in the combined analysis, the base of the alcyoniid family tree remained an unresolved polytomy. We conclude that variation in the NADH subunit coding regions is adequate to resolve phylogenetic relationships among families and some genera of Octocorallia, but insufficient for most species - or population-level studies. Although the COI-COII intergenic spacer exhibits greater variability than the protein-coding regions and may contain useful species-specific markers, its short length limits its phylogenetic utility.     

The interrelationships of Acanthomorph fishes: A total evidence approach using molecular and morphological data

DNA sequence and morphological data were analyzed for specimens of twenty-five species of acanthomorph fishes and two specimens representing the outgroups Aulopiformes and Myctophiformes. A 572 base-pair (bp) segment of the 12S ribosomal mitochondrial gene, 1112 bp from three regions of the 28S ribosomal nuclear gene, and 38 morphological transformation series were analyzed under the criterion of maximum parsimony. The total evidence analysis resulted in a set of four most parsimonious trees. Relationships common to all trees are largely congruent with the hypothesis articulated by Johnson and Patterson (1993. Bull. Mar. Sci. 52, 554-626).