WHAT DOES COMBINING MOLECULAR DATA SETS TELL US ABOUT DIATOM ORIGINS AND PHYLOGENY?

The past several years have seen an abundance of molecular sequence data gathered on heterokont algae and other stramenopiles with the goal of resolving phylogenetic relationships among major groups. The original focus was on SSU rDNA sequence, but lately a significant number of sequences of plastid and mitochondrial encoded genes (specifically rbcL and coxI) have been made available. Of particular interest to us has been the origin of diatoms and the relationship of diatoms to other stramenopiles. According to most claims based on morphological data, typically viewed from a non-rigorous evolutionary taxonomy standpoint (i.e. not with explicit cladistic or phylogenetic systematic methodology), diatoms are closely related to silica-scaled golden brown algae (chrysophytes or synurophytes). SSU rDNA sequence data, however, often place diatoms at the base of the heterokont alga tree, and chryso/synurophytes at the tip with eustigmatophytes, for example, as the chryso/synurophyte sister group. More recent analysis of rbcL sequences, however, supports the traditional classification. It is not automatically to be assumed that there is incongruence between the sequences, however. Taxon sampling is different in the different analyses, methods of analysis are often different, assumptions used to "filter" data are different, etc. Moreover, the relative strength of signal appears to be different in the data sets. We will present an analysis of combined SSU, rbcL and coxI data, an analysis of taxon-sampling issues, and review underlying assumptions and methodologies in an attempt to a) better understand the results of prior studies and b) reconcile the different hypotheses.     

A MOLECULAR PHYLOGENY OF THE HETEROKONT ALGAE BASED ON ANALYSES OF CHLOROPLAST-ENCODED rbcL SEQUENCE DATA1

Nearly complete ribulose-1,5-bisphosphate carboxylase/ oxygenase (rbcL)sequences from 27 taxa of heterokont algae were determined and combined with rbcL sequences obtained from GenBank for four other heterokont algae and three red algae. The phylogeny of the morphologically diverse haterokont algae was inferred from an unambiguously aligned data matrix using the red algae as the root, Significantly higher levels of mutational saturation in third codon positions were found when plotting the pair-wise substitutions with and without corrections for multiple substitutions at the same site for first and second codon positions only and for third positions only. In light of this observation, third codon positions were excluded from phylogenetic analyses. Both weighted-parsimony and maximum-likelihood analyses supported with high bootstrap values the monophyly of the nine currently recognized classes of heterokont algae. The Eustigmatophyceae were the most basal group, and the Dictyochophyceae branched off as the second most basal group. The branching pattern for the other classes was well supported in terms of bootstrap values in the weightedparsimony analysis but was weakly supported in the maximum-likelihood analysis (<50%). In the parsimony analysis, the diatoms formed a sister group to the branch containing the Chrysophyceae and Synurophyceae. This clade, charactetized by siliceous structures (frustules, cysts, scales), was the sister group to the Pelagophyceae/Sarcinochrysidales and Phaeo-/Xantho-/ Raphidophyceae clades. In the latter clade, the raphido-phytes were sister to the Phaeophyceae and Xanthophyceae. A relative rate test revealed that the rbcL gene in the Chrysophyceae and Synurophyceae has experienced a significantly different rate of substitutions compared to other classes of heterokont algae. The branch lengths in the maximum-likelihood reconstruction suggest that these two classes have evolved at an accelerated rate. Six major carotenoids were analyzed cladistically to study the usefulness of carotenoid pigmentation as a class-level character in the heterokont algae. In addition, each carotenoid was mapped onto both the rbcL tree and a consensus tree derived from nuclear-encoded small-subunit ribosomal DNA (SSU rDNA) sequences. Carotenoid pigmentation does not provide unambiguous phylogenetic information, whether analyzed cladistically by itself or when mapped onto phylogenetic trees based upon molecular sequence data.     

Complete large subunit ribosomal RNA sequences from the heterokont algae ochromonas danica, nannochloropsis salina, and tribonema aequale, and phylogenetic analysis

The large subunit ribosomal RNA sequences from the heterokont algae Ochromonas danica, Nannochloropsis salina, and Tribonema aequale were determined. These sequences were combined with small subunit ribosomal RNA sequences in order to carry out a phylogenetic analysis based on neighbor-joining, maximum parsimony, and maximum likelihood methods. Our results indicate that heterokont fungi and heterokont algae each are monophyletic, and confirm that they together form a monophyletic group called ``stramenopiles.' Within the heterokont algae, the eustigmatophyte Nannochloropsis salina either clusters with the chrysophyte Ochromonas danica or forms a sister group to a cluster comprising the phaeophyte Scytosiphon lomentaria and the xanthophyte Tribonema aequale. The alveolates were identified as the closest relatives of the stramenopiles, but the exact order of divergence between the eukaryotic crown taxa could not be established with confidence. Received: 22 November 1996 / Accepted: 14 February 1997     

Phylogenetic analysis of the Metastrongyloidea (Nematoda: Strongylida) inferred from ribosomal RNA gene sequences

Phylogenetic relationships among nematodes of the strongylid superfamily Metastrongyloidea were analyzed using partial sequences from the large-subunit ribosomal RNA (LSU rRNA) and small-subunit ribosomal RNA (SSU rRNA) genes. Regions of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction, directly sequenced, aligned, and phylogenies inferred using maximum parsimony. Phylogenetic hypotheses inferred from the SSU rRNA gene supported the monophyly of representative taxa from each of the 7 currently accepted metastrongyloid families. Metastrongyloid taxa formed the sister group to representative trichostrongyloid sequences based on SSU data. Sequences from either the SSU or LSU RNA regions alone provided poor resolution for relationships within the Metastrongyloidea. However, a combined analysis using sequences from all rDNA regions yielded 3 equally parsimonious trees that represented the abursate Filaroididae as polyphyletic, Parafilaroides decorus as the sister species to the monophyletic Pseudaliidae, and a sister group relationship between Oslerus osleri and Metastrongylus salmi. Relationships among 3 members of the Crenosomatidae, and 1 representative of the Skrjabingylidae (Skrjabingylus chitwoodorum) were not resolved by these combined data. However, members of both these groups were consistently resolved as the sister group to the other metastrongyloid families. These relationships are inconsistent with traditional classifications of the Metastrongyloidea and existing hypotheses for their evolution.     

Phylogenetic relationships of the Raphidophyceae and Xanthophyceae as inferred from nucleotide sequences of the 18S ribosomal RNA gene

Some earlier studies suggested an evolutionary relationship between the Raphidophyceae (chloromonads) and Xanthophyceae (yellow-green algae), whereas other studies suggested relationships with different algal classes or the öomycete fungi. To evaluate the relationships, we determined the complete nucleotide sequences of the 18S ribosomal RNA gene from the raphidophytes Vacuolaria virescens, Chattonella subsalsa, and Heterosigma carterae, and the xanthophytes Vaucheria bursata, Botrydium stoloniferum, Botrydiopsis intercedens, and Xanthonema debile. The results showed that the Xanthophyceae were most closely related to the Phaeophyceae. A cladistic analysis of combined data sets (nucleotide sequences, ultrastructure, and pigments) suggested the Raphidophyceae are the sister taxon to the Phaeophyceae-Xanthophyceae clade, but the bootstrap value was low (40%). The raphidophyte genera were united with high (100%) bootstrap values, supporting a hypothesis based upon ultrastructural features that marine and freshwater raphidophytes form a monophyletic group. We examined the relationship between Vaucheria, a siphoneous xanthophyte alga, and the öomycetes, and we confirmed that Vaucheria is a member of the class Xanthophyceae. Partial nucleotide sequences of the 18S rRNA gene from eight xanthophytes (including Bumillariopsis filiformis, Heterococcus caespitiosus, and Mischococcus sphaerocephalus) produce a phylogeny that is not congruent with the current morphology-based classification scheme.     

The phylogeny of the Hyphochytriomycota as deduced from ribosomal RNA sequences of Hyphochytrium catenoides

Based on biochemical and ultrastructural data, hyphochytriomycetes are believed to share an ancestor with oomycetes and heterokont algae. In order to study the phylogeny of the hyphochytriomycetes, we determined both the small- and large-subunit ribosomal RNA sequence of Hyphochytrium catenoides. Phylogenetic trees were constructed using the neighbor-joining and maximum-parsimony method and include representatives of Chlorobionta, Fungi, Metazoa, Alveolata, and all known Heterokonta. Our main conclusion is that the hyphochytriomycetes form a monophyletic group with the oomycetes and heterokont algae and that they are probably the closest relatives of the oomycetes. However, the order of divergence between the various heterokont algal phyla and the oomycete-hyphochytriomycete cluster remains uncertain.      

Siliceous structures and silicification in flagellated protists

Summary Flagellated protists produce a diverse range of siliceous structures, such as internal and external skeletons, scales, spines, bristles, cell walls, cyst walls, and loricae. The different groups of silica-depositing flagellates, i.e., chrysophytes/synurophytes, choanoflagellates, dinoflagellates, ebriids, silicoflagellates, thaumatomastigids, and the genusPetasaria are reviewed. Brief mention is also given to those algal groups in which silicification is uncommon and rare (i.e., chlorophytes, euglenophytes, haptophytes/prymnesiophytes, xanthophytes/tribophytes), but in which silicified structures nevertheless occur in few flagellate genera. Special attention is given to aspects of morphology and development of the different siliceous structures as well as on aspects of systematics and taxonomy.     

Characteristics and utility of nuclear-encoded large-subunit ribosomal gene sequences in phylogenetic studies of red algae

Primer sequences are described for amplifying and sequencing a large fragment (approximately 2500 b.p.) of the nuclear-encoded large-subunit ribosomal RNA gene (LSU) from red algae. In comparison to RuBisCo large-subunit gene (rbcL) and nuclear-encoded small-subunit ribosomal RNA gene (SSU) sequence data, LSU sequence data was intermediate in the number of phylogenetically informative positions and sequence divergence. Parsimony analysis of LSU sequences for 16 Gelidiales species resolved some nodes unresolved in rbcL and SSU parsimony trees. An analysis of LSU sequences from 13 species of red algae classified in 11 orders suggests that this gene may be useful in studies of higher-level relationships of red algae.     

Distribution of myo-inositol dehydrogenase in algae

The enzyme myo-inositol dehydrogenase (InDH; EC 1.1.1.18) catalyses the NAD-dependent oxidation of myo-inositol to scyllo-inosose (2-keto-inositol). A survey within different algal groups showed that this enzyme is present in rhodophytes, glaucocystophytes, phaeophytes, xanthophytes and haptophytes but not in green algae, euglenophytes and chrysophytes. Anion-exchange chromatography of crude homogenates resulted in two distinct peaks of activity. Both isoenzymes were specific for myo-inositol and scyllo-inosose. epi- and scyllo-inositol as well as epi-inosose were only poor substrates, while all other polyols and sugars tested did not serve as substrates. A possible role of the InDH isoenzymes is the shuttling of reducing power between the mitochondrion and the cytosol.     

DISTRIBUTION OF THE MITOCHONDRIAL DEVIANT GENETIC CODE AUA FOR METHIONINE IN HETEROKONT ALGAE

The DNA sequence of the cytochrome oxidase subunit I ( COX I) gene (1059 bp), was determined in a number of heterokont algae, including five species of the Phaeophyceae [ Chorda filum (Linnaeus) Stackhouse, Colpomenia bullosa (Saunders) Yamada, Ectocarpus sp., Pseudochorda nagaii (Tokida) Inagaki, Undaria pinnatifida (Harvey) Suringar], and a member of the Raphidophyceae [ Chattonella antiqua (Hada) Ono]. The distribution of a deviant mitochondrial code, the AUA codon for methionine (AUA/Met), which was previously reported in the Xanthophyceae, was inferred from these COX I sequences. Comparative analyses of these sequences revealed that all the algae described above bear the universal genetic code, including the assignment for the AUA codon. A phylogenetic tree was constructed using the obtained sequences along with already-published COX I sequences of various heterokont algae. The clusters of the Xanthophyceae and the Phaeophyceae were resolved as sister groups with high bootstrap support, excluding a bacillariophycean species, a raphidophycean species, and three species of the Eustigmatophyceae. Taking the distribution of the deviant code and the COX I phylogenetic tree together, the genetic code change most probably occurred in an ancestor of the Xanthophyceae after it had branched off from the Phaeophyceae.     

A case of tongueworms (Pentastomida): a specific problem in context of the modern phylogenetics

A short essay on anatomy, ultrastructure and larval development, life cycles, classification, palaeontology and phylogenetic relationships of Pentastomida is given. Currently, the Pentastomida are usually placed within Crustacea, as sister group of Branchiura (Crustacea, Maxillopoda). The grounds are striking similarities in ultrastructures of spermatozoa and congruence in the 18S rRNA nucleotide sequence. Both taxa are however sharply disparate in all other characters concerning morphology, embryology, life cycles and geological history. A direct introduction of Pentastomida in the system of Crustacea implies an unjustified inflation of the taxonomic diagnosis of the latter group; hence it is unacceptable. Two alternative hypotheses are suggested as tentative phylogenetic relationships to be tested. Each hypothesis infers however a very complex and unlikely evolutionary scenario. 1) Pentastomida and Branchiura are true sister groups as justified by coincidence in spermatozoan ultrastructure and sequence of ribosomal RNA. Since their divergence, the evolutions of pentastomids and branchiurans ran in different milieus, in different shapes and with different rates. To present time, the pentastomids lost nearly all characters of maxillopods and crustaceans as well as majority of anthropod features. 2) Pentastomida and Branchiura are not close related. The similarity in ultrastructural details of spermatozoa may be occasional or resulted from convergent evolution with unknown factors. Coincidence in nucleotide sequences or ribosomal RNA should be reexamined and tested with other pentastomid species. The second hypothesis seems to be more likely.     

Phylogenetic Analysis of Ruminant Theileria spp. from China Based on 28S Ribosomal RNA Gene

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.     

Phylogenetic analyses of the rbcL sequences from haptophytes and heterokont algae suggest their chloroplasts are unrelated

Using the large subunit of RuBisCo (rbcL) sequences from cyanobacteria, proteobacteria, and diverse groups of algae and green plants, we evaluated the plastid relationship between haptophytes and heterokont algae. The rbcL sequences were determined from three taxa of heterokont algae (Bumilleriopsis filiformis, Pelagomonas calceolata, and Pseudopedinella elastica) and added to 25 published sequences to obtain a data set comprising 1,434 unambiguously aligned sites (approximately 98% of the total rbcL gene). Higher levels of mutational saturation in third codon positions were observed by plotting the pairwise substitutions with and without corrections for multiple substitutions at the same site for first and second codon positions only and for third positions only. In accordance with this finding phylogeny reconstructions were completed by omitting third codon positions, thus using 956 bp in weighted-parsimony and maximum-likelihood analyses. The midpoint-rooted phylogenies showed two major clusters, one containing cyanobacteria, glaucocystophytes, a phototrophic euglenoid, chlorophytes, and embryophytes (the green lineage), the other containing proteobacteria, haptophytes, red algae, a cryptophyte, and heterokont algae (the non-green lineage). In the nongreen lineage, the haptophytes formed a sister group to the clade containing heterokont algae, red algae, and the cryptophyte Guillardia theta. This branching pattern was well supported in terms of bootstrap values in weighted- parsimony and maximum-likelihood analyses (100% and 92%, respectively). However, the phylogenetic relationship among red algae, heterokonts, and a cryptophyte taxon was not especially well resolved. A four- cluster analysis was performed to further explore the statistical significance of the relationship between proteobacteria, red algae (including and excluding Guillardia theta), haptophytes, and heterokont algae. This test strongly favored the hypothesis that the heterokonts and red algae are more closely related to each other than either is to proteobacteria or haptophytes. Hence, this molecular study based on a plastid-encoded gene provides additional evidence for a distant relationship between haptophytes and the heterokont algae. It suggests an evolutionary scenario in which the ancestor of the haptophyte lineage engulfed a phototrophic eukaryote and, more recently, the heterokont lineage became phototrophic by engulfing a red alga.      

Evaluating hypotheses of deuterostome phylogeny and chordate evolution with new LSU and SSU ribosomal DNA data

We investigated evolutionary relationships among deuterostome subgroups by obtaining nearly complete large-subunit ribosomal RNA (LSU rRNA)-gene sequences for 14 deuterostomes and 3 protostomes and complete small-subunit (SSU) rRNA-gene sequences for five of these animals. With the addition of previously published sequences, we compared 28 taxa using three different data sets (LSU only, SSU only, and combined LSU + SSU) under minimum evolution (with LogDet distances), maximum likelihood, and maximum parsimony optimality criteria. Additionally, we analyzed the combined LSU + SSU sequences with spectral analysis of LogDet distances, a technique that measures the amount of support and conflict within the data for every possible grouping of taxa. Overall, we found that (1) the LSU genes produced a tree very similar to the SSU gene tree, (2) adding LSU to SSU sequences strengthened the bootstrap support for many groups above the SSU-only values (e.g., hemichordates plus echinoderms as Ambulacraria; lancelets as the sister group to vertebrates), (3) LSU sequences did not support SSU-based hypotheses of pterobranchs evolving from enteropneusts and thaliaceans evolving from ascidians, and (4) the combined LSU + SSU data are ambiguous about the monophyly of chordates. No tree-building algorithm united urochordates conclusively with other chordates, although spectral analysis did so, providing our only evidence for chordate monophyly. With spectral analysis, we also evaluated several major hypotheses of deuterostome phylogeny that were constructed from morphological, embryological, and paleontological evidence. Our rRNA-gene analysis refutes most of these hypotheses and thus advocates a rethinking of chordate and vertebrate origins.     

PHYLOGENY, BIOGEOGRAPHY AND LIFE HISTORY EVOLUTION IN THE RED ALGAL FAMILY PHYLLOPHORACEAE (GIGARTINALES)

Red algae are exceptional for the great diversity in reproductive morphology and for their complex life histories. In particular, the family Phyllophoraceae, consisting of ∼100 species worldwide, stands out in exhibiting a wide spectrum of unique life history types that makes it unusually interesting for assessing the phylogenetic importance of reproductive traits relative to classification criteria. Type of life history and position of the reproductive structures on the plant body have traditionally formed the basis for separating eleven genera in the Phyllophoraceae; however, phylogenetic analyses inferred from three sets of DNA sequences [chloroplast-encoded rbcL , nuclear large-subunit ribosomal RNA gene (LSU), and internal transcribed spacer regions (ITS) of nuclear ribosomal DNA], instead indicate a lack of correlation between type of life history and phylogenetic relationships among the established taxa. This lack of correlation dramatically challenges all of the traditional taxonomy. The study will answer the question which morphological features and which aspects of life history evolution can be used as meaningful indicators of phylogenetic relationships in the Phyllophoraceae. The results are addressed in light of global biogeographic hypotheses for the family.     

Heliobacterium and the origin of chrysoplasts

Chrysoplasts, golden-yellow and brown photosynthetic membrane-bounded plastids, photosynthetic organelles of algae such as phaeophytes (brown seaweeds), bacillariophytes (diatoms) and chrysophytes (golden-yellow algae including silicoflagellates), are hypothesized to have originated from brownish photoheterotrophic bacteria such as the newly discovered anaerobic nitrogen-fixing Heliobacterium. The consequences of this hypothesis as well as the data required to verify or disprove it are presented.     

Evolutionary Relationships Among the Eukaryotic Crown Taxa Taking into Account Site-to-Site Rate Variation in 18S rRNA

In this study we constructed a bootstrapped distance tree of 500 small subunit ribosomal RNA sequences from organisms belonging to the so-called crown of eukaryote evolution. Taking into account the substitution rate of the individual nucleotides of the rRNA sequence alignment, our results suggest that (1) animals, true fungi, and choanoflagellates share a common origin: The branch joining these taxa is highly supported by bootstrap analysis (bootstrap support [BS] > 90%), (2) stramenopiles and alveolates are sister groups (BS = 75%), (3) within the alveolates, dinoflagellates and apicomplexans share a common ancestor BS > 95%), while in turn they both share a common origin with the ciliates (BS > 80%), and (4) within the stramenopiles, heterokont algae, hyphochytriomycetes, and oomycetes form a monophyletic grouping well supported by bootstrap analysis (BS > 85%), preceded by the well-supported successive divergence of labyrinthulomycetes and bicosoecids. On the other hand, many evolutionary relationships between crown taxa are still obscure on the basis of 18S rRNA. The branching order between the animal-fungal-choanoflagellates clade and the chlorobionts, the alveolates and stramenopiles, red algae, and several smaller groups of organisms remains largely unresolved. When among-site rate variation is not considered, the inferred tree topologies are inferior to those where the substitution rate spectrum for the 18S rRNA is taken into account. This is primarily indicated by the erroneous branching of fast-evolving sequences. Moreover, when different substitution rates among sites are not considered, the animals no longer appear as a monophyletic grouping in most distance trees. Received: 11 June 1997 / Accepted: 21 July 1997     

GENOMIC INSIGHTS INTO EVOLUTIONARY RELATIONSHIPS AMONG HETEROKONT LINEAGES EMPHASIZING THE PHAEOPHYCEAE1

Heterokonts comprise a large and diverse group of organisms unified by the heterokont biflagellate condition. Monophyly of many of these lineages is well established, but evolutionary relationships among the various lineages remain elusive. Among these lineages, the brown algae (Phaeophyceae) are a monophyletic, taxonomically diverse, and ecologically critical group common to marine environments. Despite their biological and scientific importance, consensus regarding brown algal phylogeny and taxonomic relationships is missing. Our long‐term research goal is to produce a well‐resolved taxon‐rich phylogeny of the class to assess evolutionary patterns and taxonomic relationships among brown algal lineages and their relationship to other closely related heterokont groups. To accomplish this goal and augment existing loci for phaeophycean‐wide systematic studies, we generated expressed sequence tags (ESTs) from several major brown algal lineages and from the heterokont lineage representing the closest sister group to brown algae. To date, we have successfully constructed cDNA libraries for two lineages (Choristocarpus tenellus Zanardini and Schizocladia ischiensis E. C. Henry, Okuda et H. Kawai) and in the library test phase obtained up to 1,600 ESTs per organism. Annotation results showed a gene discovery rate of 45%–50% for each library revealing 500–700 unique genes from each organism. We have identified several potential genes for phylogenetic inference and used these loci for preliminary molecular clock analyses. Our molecular clock analysis suggests that the basal divergence in brown algae occurred around the time of the pennate‐centric diatom divergence. Here we report this analysis and other uses of ESTs in brown algal phylogenomics and the utility of these data for resolving the phylogeny of this group.     

PHYLOGENETIC AFFINITIES OF CRYSONEPHELE PALUSTRIS (CHRYSOPHYCEAE) BASED ON INFERRED NUCLEAR SMALL-SUBUNIT RIBOSOMAL RNA SEQUENCE

The nuclear small-subunit ribosomal DNA sequence was determined from the colonial chromophytic alga Chrysonephele palustris Pipes, Tyler, et Leedale using polymerase chain reaction methods. The inferred ribosomal RNA sequence was included in a multiple alignment, containing heterokont chromophytes, and subjected to molecular systematic analyses in order to determine the phylogenetic relationships of this alga . Chrysonephele palustris grouped within the Chrysophyceae and not in an intermediate position between the Chrysophyceae and Eustigmatophyceae, suggesting that Chrysonephele is not a phylogenetic link between these algal classes .     

TWO CHLOROPLAST TRANSFER RNA INTRONS FOUND IN CHAETOSPHAERIDIUM SUPPORT A MONOPHYLETIC COLEOCHAETALES

Lewandowski, J. D. & Delwiche, C. F. Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742 USA The evolutionary relationships of the algal genera Mesostigma and Chaetosphaeridium to other algae and land plants are currently controversial. A close evolutionary relationship between land plants and two orders of the charophycean algae, the Charales and Coleochaetales, is supported by morphological, ultrastructural, biochemical, genomic, and phylogenetic data. A number of phylogenetic analyses support a monophyletic Coleochaetales, with Coleochaete and Chaetosphaeridum as sister groups. Mesostigma was traditionally viewed as a member of the prasinophytes and has recently been considered as a lineage possibly basal to the charophycean algae, or sister to all green algae. By contrast, recent analyses of small subunit ribosomal RNA gene sequences have been interpreted as evidence of an alternative classification with Mesostigma forming a clade with Chaetosphaeridium to the exclusion of Coleochaete and other charophycean lineages. The shared presence of introns in two chloroplast tRNA genes (tRNAAla and tRNAIle) among charophytes Coleochaete and Nitella and the liverwort Marchantia supports a monophyletic group containing the Coleochaetales, the Charales, and land plants. Through isolation and sequence analysis of the tRNAAla and tRNAIle genes in Chaetosphaeridium, we have identified introns similar in sequence and position to those found in Coleochaete. These data and the published absence of these introns in Mesostigma lend new support to a monophyletic Coleochaetales including the genera Coleochaete and Chaetosphaeridium.