MOLECULAR SYSTEMATICS OF ECTOCARPUS AND KUCKUCKIA (ECTOCARPALES, PHAEOPHYCEAE) INFERRED FROM PHYLOGENETIC ANALYSIS OF NUCLEAR- AND PIASTTD-ENCODED DNA SEQUENCES

The phylogeny of Ectocarpus and Kuckuckia strains representing widely separated populations from both hemispheres was inferred from sequence analysis of the internal transcribed spacers of the nuclear ribosomal DNA (ITS 1—5.8S-ITS 2) and the spacer region in the plastid-encoded ribulose- bis -phosphate-carboxylase (RUBISCO) cistron (partial rbc L-spacer-partial rbc S ). Both sequences resulted in matching phylogenies, with the RUBISCO spacer region being most informative at the level of genera and species and the internal transcribed spacer sequences at the level of species and populations. Three major clades were formed by strains previously described by morphology and physiology as Kuckuckia, E. fasciculatus, and E. siliculosus, confirming the validity of these taxa . Ectocarpus and Kuckuckia are regarded as sibling taxa with respect to the outgroup species Feldmannia simplex, Hincksia mitchelliae, and Pilayella littoralis. The clade formed by sexual E. siliculosus strains and most asexual Ectocarpus strains was subdivided into several clades that are consistent with geographical races within E. siliculosus. The inferred phylogeny of Ectocarpus corresponds generally with results from cross-fertilization experiments, morphology, and lipid analysis. A hypothesis on the origin and dispersal of E. siliculosus suggests several natural dispersal events during periods of global cooling as well as recent and possibly anthropogenic dispersal events .     

rDNA片段的序列分析在苦苣苔亚科系统学研究中的应用

本文测定了苦苣苔亚科4族、5属、5种植物的核糖体DNA中的内转录间隔区(ITS)序列及5．8s rRNA基因的3′端序列。这几种苦苣苔亚科植物的ITS-1的长度范围为234～258 bp,ITS-2的长度范 围为218～246bp。Whytockia bijieensis的ITS-1(258bP)和ITS-2(218 bp)在长度、序列及GC含量上 均与其它几个种有较大差异,其代表的尖舌苣苔族可能很早就自苦苣苔亚科的祖先沿单独的一个分支 演化。以w．bijieensis作为功能性外类群,运用PAUP软件分析仅得到一个最简约树。在简约树上, Cyrtandra umbelliferm、Briggsia longipes和Anna mollifolia形成一个单系群,bootstrap分析对该分支的 支持强度达97％,Chirita crasslfolia位于该分支的基部。由于系统树上Cyrtandra umbellifera代表的 浆果苣苔族和Anna mollifolid代表的芒毛苣苔族均起源于长蒴苣苔族,结合这3个族在形态上存在过渡系列,建议将浆果苣苔族和芒毛苣苔族均并入长蒴苣苔族。     

Phylogenetic relationships ofPelvetia andPelvetiopsis (Phaeophyceae) based on small subunit ribosomal DNA sequences

Nucleotide sequences of the small-subunit (SSU) ribosomal DNA were determined forPelvetia babingtonii, P. canaliculate, Pelvetiopsis limitata, andAscophyllum nodosum in the family Fucaceae. A total of 1755 positions were aligned for the whole sequence. The positional differences in the primary structure among the taxa ranged from 16 to 30 nucleotide changes in pairwise comparisons. There was a minimum divergence betweenPs. limitata andP. babingtonii while a maximum betweenPs. limitata andP. canaliculata. The SSU rDNA trees showed that the genusPelvetia was not monophyletic and the genusPelvetiopsis was not closely related toPelvetia. Our results suggest that the taxonomic revision of the genusPelvetia as well as the family Fucaceae is needed based on detailed morphological observations.     

THE EVOLUTION OF PARASITES FROM THEIR HOSTS: A CASE STUDY IN THE PARASITIC RED ALGAE

Morphological similarities of many parasites and their hosts have led to speculation that some groups of plant, animal, fungal, and algal parasites may have evolved directly from their hosts. These parasites, which have been termed adelphoparasites in the botanical literature, and more recently, agastoparasites in the insect literature, may evolve monophyletically from one host and radiate secondarily to other hosts or, these parasites may arise polyphyletically, each arising from its own host. In this study we compare the internal transcribed spacer regions of the nuclear ribosomal repeats of species and formae specialis (host races) included in the red algal parasite genus Asterocolax with its hosts, which all belong to the Phycodrys group of the Delesseriaceae and with closely related nonhost taxa of the Delesseriaceae. These analyses reveal that species of Asterocolax have evolved polyphyletically. Asterocolax erythroglossi from the North Atlantic host Erythroglossum laciniatum appears to have evolved from its host, whereas taxa included in the north Pacific species Asterocolax gardneri have had two independent origins. Asterocolax gardneri from the host Polyneura latissima probably arose directly from this host. In contrast, all other A. gardneri formae specialis appear to have originated from either Phycodrys setchellii or P. isabelliae and radiated secondarily onto other closely related taxa of the Phycodrys group, including Nienburgia andersoniana and Anisocladella pacifica. Gamete crossing experiments confirm that A. gardneri from each host is genetically isolated from both its host, and from other A. gardneri and their hosts. Cross-infection experiments reveal that A. gardneri develops normally only on its natural host, although some abberrant growth may occur on alternate hosts. The ability of red algal parasites to radiate secondarily to other red algal taxa, where they may become isolated genetically and speciate, suggests that this process of speciation is not a “genetic dead end” but one that may give rise to related clusters of parasite species.     

IMMUNOLOCALIZATION OF CENTRIN DURING FERTILIZATION AND THE FIRST CELL CYCLE IN FUCUS DISTICHUS AND PELVETIA COMPRESSA (FUCALES,PHAEOPHYCEAE)1

Antibodies that recognize the centrosome-associated protein centrin were used to characterize centrosomal origin and positioning during fertilization and the first cell cycle in Fucus distichus subsp. evanescens (C. Agardh) Powell and Pelvetia compressa (J. Agardh) De Toni. Centrin was identified in sperm, eggs, and zygotes on protein blots, indicating the protein is present in both gametes. Using immunofluorescence microscopy, centrin was found in discrete foci in sperm. In contrast, eggs lack centrosomes and centrin was not detectable by immunofluorescence, indicating that centrin was probably dispersed in the cytoplasm. Two foci of centrin were present on the nuclear envelope of zygotes, but microtubules remained dispersed over the zygotic nucleus. Centrin foci separated over the nuclear envelope as the first cell cycle progressed. Microtubules became concentrated at the centrin foci to form centrosomes that gave rise to the spindle poles at mitosis.     

PROPERTIES OF PHOSPHOENOLPYRUVATE CARBOXYKINASE FROM ASCOPHYLLUM NODOSUM (PHAEOPHYCEAE)1

Phosphoenolpyruvate carboxykinase was purified to electrophoretic homogeneity from the brown alga. Ascophyllum nodosum (L.) le Jol. Its molecular mass was 60 kDa as determined by gel filtration. The pH optimum of the carboxylating reaction was 7.9 and the apparent K_m for PEP⁴, ADP and HCO₃^- were 0.036, 0.0116 and 50 mol · m^-3, respectively. Rates of light and dark carbon fixation are also reported for A. nodosum apices and it is shown that rates of in vitro PEPCK activity would account for the observed rate of dark fixation. The physiological role of PEPCK is discussed in relation to light-independent carbon fixation.     

EVOLUTION OF PHACUS (EUGLENOPHYCEAE) AS INFERRED FROM PELLICLE MORPHOLOGY AND SSU rDNA

This research integrates a large morphological data set into a molecular context. Nineteen pellicle characters and 62 states from 13 euglenid taxa were analyzed cladistically. The pellicle morphology of Euglena tripteris (Klebs), Lepocinclis ovata (Conrad), Phacus brachykentron (Pochmann), P. oscillans (Klebs), P. pyrum (Stein), and P. triqueter (Dujardin) is described comprehensively. These data are compared with new information on the pellicle morphology of Euglena acus (Ehrenberg), E. stellata (Mainx), and Peranema trichophorum (Stein) in addition to published data on Entosiphon sulcatum (Dujardin), Euglena gracilis (Klebs), Distigma proteus (Pringsheim), and Petalomonas cantuscygni (Cann and Pennick). Nuclear small subunit (SSU) rDNA sequences provided an independent test for establishing a robust organismal pedigree of the same taxa. A synthetic tree derived from the combined phylogenetic analyses of pellicle morphology and SSU rDNA enabled us to parsimoniously map morphological character states. This approach demonstrated the utility of pellicle morphology for inferring phylogenetic relationships of euglenids and establishing apomorphy-based clade definitions. Three robust clades with unambiguous pellicle-based apomorphies can be recognized within taxa traditionally classified as Phacus : (1) L. ovata and P. pyrum , (2) E. tripteris and P. triqueter , and (3) P. brachykentron and P. oscillans. Taxonomic concerns that emerged from these results are discussed.     

AMINO ACID SEQUENCE ANALYSIS OF THE SMALL SUBUNIT OF RIBULOSE BISPHOSPHATE CARBOXYLASE FROM FUCUS (PHAEOPHYCEAE)1

This paper reports for the first time amino acid sequence information for the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) from a non-green alga. N-terminal sequences are presented for the polypeptide from three species of the genus Fucus (Phaeophyceae). Although homologous to small subunit polypeptides from other organisms, the Fucus sequences exhibit a unique N-terminal section resembling neither cyanobacterial nor chlorophytic sequences. This difference may be a consequence of the plastid DNA coding arrangement for the small subunit in chromophytes, a situation reported for the related organism Olisthodiscus but not previously investigated at the amino acid sequence level.     

ORIGIN AND BIOGEOGRAPHY OF AESCULUS L. (HIPPOCASTANACEAE): A MOLECULAR PHYLOGENETIC PERSPECTIVE

Sequences of chloroplast gene matK and internal transcribed spacers of nuclear ribosomal RNA genes were used for phylogenetic analyses of Aesculus, a genus currently distributed in eastern Asia, eastern and western North America, and southeastern Europe. Phylogenetic relationships inferred from these molecular data are highly correlated with the geographic distributions of species. The identified lineages closely correspond to the five sections previously recognized on the basis of morphology. Ancestral character-state reconstruction, a molecular clock, and fossil evidence were used to infer the origin and biogeographic history of the genus within a phylogenetic framework. Based on the molecular phylogenetic reconstruction of the genus, sequence divergence, and paleontological evidence, we infer that the genus originated during the transition from the Cretaceous to the Tertiary (~65 M.Y.B.P.) at a high latitude in eastern Asia and spread into North America and Europe as an element of the “boreotropical flora”; the current disjunct distribution of the genus resulted from geological and climatic changes during the Tertiary.     

ISOLATION AND CHARACTERIZATION OF GOLGI APPARATUS FROM EGGS OF FUCUS (PHAEOPHYCEAE)1

A golgi-rich cell-free fraction has been obtained from eggs of Fucus serratus L. and characterized by enzyme markers (IDP-ase and TPP-ase) and electron microscopy. The results are correlated with cytochemical localization of IDP-ase and TPP-ase in situ.     

Origin and relationships of Saintpaulia (Gesneriaceae) based on ribosomal DNA internal transcribed spacer (ITS) sequences

Phylogenetic relationships of eight species of Saintpaulia H. Wendl., 19 species of Streptocarpus Lindl. (representing all major growth forms within the genus), and two outgroups (Haberlea rhodopensis Friv., Chirita spadiciformis W. T. Wang) were examined using comparative nucleotide sequences from the two internal transcribed spacers (ITS) of nuclear ribosomal DNA. The length of the ITS 1 region ranged from 228 to 249 base pairs (bp) and the ITS 2 region from 196 to 245 bp. Pairwise sequence divergence across both spacers for ingroup and outgroup species ranged from 0 to 29%. Streptocarpus is not monophyletic, and Saintpaulia is nested within Streptocarpus subgenus Streptocarpella. Streptocarpus subgenus Streptocarpus is monophyletic. The ITS sequence data demonstrate that the unifoliate Streptocarpus species form a clade, and are also characterized by a unique 47-bp deletion in ITS 2. The results strongly support the monophyly of (1) Saintpaulia, and (2) Saintpaulia plus the African members of the subgenus Streptocarpella of Streptocarpus. The data suggest the evolution of Saintpaulia from Streptocarpus subgenus Streptocarpella. The differences in flower and vegetative characters are probably due to ecological adaptation leading to a relatively rapid radiation of Saintpaulia.     

A PCR IMMUNOASSAY METHOD FOR THE DETECTION OF ALEXANDRIUM (DINOPHYCEAE) SPECIES

PCR primers targeting the internal transcribed spacer (ITS)-5.8S rDNA regions specific for the genus Alexandrium were used to develop an ELISA assay method to detect and enumerate this genus in cultured isolates. The solid-phase ELISA involves the application of a biotinylated labeled primer to target the specific ITS-5.8S rDNA region; the PCR-amplified products, generated in the presence of digoxigenin-11-deoxiuracil triphosphate nucleotide, are captured on the streptavidin-coated microplate. The captured molecules were hybridized to an anti-digoxigenin antibody conjugated with alkaline phosphatase. The presence and number of the Alexandrium cells in the samples resulted in a proportional appearance of color generated by the phosphatase activity in the presence of a chromogenic substrate and measured in a plate reader. This PCR and immunoassay solid-phase assay proved to be a useful technique to detect the presence of Alexandrium sp. in cultured isolates and seawater samples.     

Evolution and biogeography of the woody Hawaiian violets (Viola, Violaceae): arctic origins, herbaceous ancestry and bird dispersal

Specialists studying the genus Viola have consistently allied the Hawaiian violets comprising section Nosphinium--most of which are subshrubs or treelets--with putatively primitive subshrubs in certain South American violet groups. Hawaiian violets also possess inflorescences, a floral disposition otherwise found only in other genera of the Violaceae, thus strengthening the hypothesis of a very ancient origin for the Hawaiian species. A survey of phylogenetic relationships among infrageneric groups of Viola worldwide using nuclear rDNA internal transcribed spacer (ITS) sequences revealed a dramatically different biogeographic origin for the Hawaiian violets: A monophyletic Hawaiian clade was placed in a close sister relationship with the amphi-Beringian tundra violet, V. langsdorffii s. 1., in a highly derived position. This remarkable and unforeseen relationship received strong clade support values across analyses, and monophyly of the Hawaiian lineage was further indicated by a unique 26-base-pair deletion in section Nosphinium. The high polyploid base chromosome number (n approximately equal to 40) in the Hawaiian violets relates them to Alaskan and eastern Siberian populations in the polyploid V. langsdorffii complex. More than 50 species of the 260 allochthonous birds wintering in the Hawaiian Islands are found to breed in the Arctic, occupying habitats in which individual birds might have encountered ancestral V. langsdorffii populations and served as dispersers to the central Pacific region. Acquisition of derived morphological traits (e.g., arborescence and inflorescences), significance of a confirmed Arctic origin for a component of the Hawaiian flora, and the likelihood of other "cryptic" Arctic elements in the Hawaiian flora deserving independent molecular phylogenetic corroboration are discussed.     

REPRODUCTIVE SUCCESS OF FUCUS VESICULOSUS (PHAEOPHYCEAE) IN THE BALTIC SEA

Marine organisms colonizing brackish habitats such as the Baltic Sea must cope with the negative effects of low salinities on reproductive success because these may reduce gamete viability and/or increase polyspermy. Reproductive characteristics of the marine seaweed Fucus vesiculosus L. were studied in several brackish habitats, particularly in the northern Baltic Sea, to understand its ability to reproduce where few other marine species survive. Polyspermy and fertilization success were variable at the boundary of the continuous distribution of F. vesiculosus in the Baltic Sea, and polyspermy was high (10%–30%) when fertilization was successful. A strong female bias (80%–86%, ca. 5.5:1) was found at the northernmost limit of Baltic F. vesiculosus. Electrophysiological studies showed that many eggs have a high input resistance (519 ± 150 MΩ[mean ± SE, n = 14] at Drivan, 1995), which may be helpful in preventing polyspermy in this brackish habitat. The polyspermy block remains sodium-dependent in the northern Baltic. Sperm bound quickly to northern Baltic eggs in natural water, but fertilization was delayed compared to marine F. vesiculosus. A subset of northern Baltic eggs studied during an optimal reproductive period (7–11 July 1995) had a membrane potential (E_m) of ca. −100 mV and an effective fertilization potential (FP) of ca. 2 min with a plateau of −25 mV, but repolarized too rapidly for the FP to be protective. Pronuclear migration and cell wall secretion occurred more slowly in Baltic than in marine zygotes. The reproductive success of theseboundary populations may be dependent upon windows of opportunity when there are favorable combinations of the levels of salinity, water motion, population density, and sex ratio. These factors and the short duration of the reproductive season in the northern Baltic Sea may result in reproductive failure in some years.     

THE RELEASE OF COPPER-COMPLEXING LIGANDS BY THE BROWN ALGA FUCUS VESICULOSUS (PHAEOPHYCEAE) IN RESPONSE TO INCREASING TOTAL COPPER LEVELS

The growth of Fucus vesiculosus L. germlings in chemically defined culture media containing a range of Cu concentrations (20–1000 nM) was monitored simultaneously with measurement of the Cu speciation in the media by competitive equilibrium-adsorptive cathodic stripping voltammetry. Fucus vesiculosus germlings were found to exude Cu-complexing ligands with conditional stability constants of the order of 1.6 × 10¹¹. Ligand concentrations increased with increasing total dissolved Cu concentrations (Cu_T) until a concentration of 500–800 neq Cu·L⁻¹ was reached. Concentrations of the ligand exceeded Cu_T in treatments containing 20 and 100 nM Cu, were similar to Cu_T in the 500-nM Cu treatment, but were less than Cu_T in the 1000-nM treatment. Therefore, [Cu²⁺] were calculated to be at concentrations of 10⁻¹¹− 10⁻¹⁰ M in the 20- and 100-nM treatments, 10⁻⁹ M in the 500-nM treatment, and 10⁻⁷ M in the 1000-nM treatment. Growth rates were lowest at Cu²⁺ concentration > 10⁻⁹. These results are discussed within the context of the potential roles for exuded copper-complexing ligands.     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

PHYLOGENY OF THE GENUS PYRAMIMONAS (PRASINOPHYCEAE,CHLOROPHYTA) INFERRED FROM THE rbcL GENE1

A 1089-basepair fragment (approx. 75%) of the large subunit of the chloroplast-encoded gene, ribulose-1,5-bis-phosphate carboxylase/oxygenase (rbcL), was sequenced from 16 species of the genus Pyramimonas Schmarda. Electron microscopic and biochemical studies of Pyramimonas, one of the most morphologically diverse genera within the potential sister groups to the chlorophyll a- and b-containing plants, suggest that this genus consists of at least four separate subgenera. Using the homologous sequence of rbcL from Cymbomonas tetramitiformis Schiller (Halosphaeraceae) as an outgroup and applying the maximum likelihood method, we show that the inferred topology is congruent with traditional delimitations of the taxa based on observations of periplast, internal ultrastructure, and biochemical features. A bootstrap analysis also supports division at the subgeneric level; however, the low bootstrap support associated with the deep nodes precludes resolution of these branches. A maximum likelihood relative rate test revealed that the rbcL gene in these single-celled green flagellates has a heterogeneous rate of substitution. The rbcL gene in species of the subgenus Pyramimonas has evolved at an accelerated rate relative to that of congenerics.     

MOLECULAR PHYLOGENY OF THE GENUS CAULERPA (CAULERPALES,CHLOROPHYTA) INFERRED FROM CHLOROPLAST tufA GENE1

The genus Caulerpa consists of about 75 species of tropical to subtropical siphonous green algae. To better understand the evolutionary history of the genus, a molecular phylogeny was inferred from chloroplast tufA sequences of 23 taxa. A sequence of Caulerpella ambigua was included as a potential outgroup. Results reveal that the latter taxon is, indeed, sister to all ingroup sequences. Caulerpa itself consists of a series of relatively ancient and species‐poor lineages and a relatively modern and rapidly diversifying clade, containing most of the diversity. The molecular phylogeny conflicts with the intrageneric sectional classification based on morphological characters and an evolutionary scheme based on chloroplast ultrastructure. High bootstrap values support monophyly of C. mexicana, C. sertularioides, C. taxifolia, C. webbiana, and C. prolifera, whereas most other Caulerpa species show para‐ or polyphyly.     

利用核核糖体DNA内转录间隔区(ITS)探讨广义羽藓科系统发育

利用核核糖体DNA ITS序列,探讨了苔藓植物广义羽藓科的系统发育,摸索出适于扩增ITS片段的最适反应条件。实验共得到广义羽藓科6个种的ITS序列,它们分别是：Abieti-nela abietina(AJ417494),Anomodon minar(AJ344145),Chaopodium aciculum(AJ315968),Tuidium pristocalyx(AJ416443),Thuidium assimile(AJ416442),Herpetineuron toccoae(AJ315967),其中后5个种是国际上首次得到的。本文利用ITS序列构建羽藓科7属、11种植物的系统发育树,据Bootstrap严格一致树表明：广义的羽藓科为并系发育,可分为两个主要的分支,牛舌藓属Anomodon,羊角藓属Herpetineuron和多枝藓属Happohymenium等为一支,而山羽藓属Abietinella,羽藓属Thuuidium,沼羽藓属Helodium和麻羽藓属Claopodium等为另一主要分支,从分子水平上支持了据形态特征把原牛舌藓亚科的牛舌藓属,羊角藓属,多枝藓属提升为牛舌藓科的结论。     

PHYLOGENY OF THE EUGLENALES INFERRED FROM PLASTID LSU rDNA SEQUENCES1

To gain insights into the phylogeny of the Euglenales, we analyzed the plastid LSU rDNA sequences from 101 strains of the photosynthetic euglenoids belonging to nine ingroup genera (Euglena, Trachelomonas, Strombomonas, Monomorphina, Cryptoglena, Colacium, Discoplastis, Phacus, and Lepocinclis) and two outgroup genera (Eutreptia and Eutreptiella). Bayesian and maximum‐likelihood (ML) analyses resulted in trees of similar topologies and four major clades: a Phacus and Lepocinclis clade; a Colacium clade; a Trachelomonas, Strombomonas, Monomorphina, and Cryptoglena clade; and a Euglena clade. The Phacus and Lepocinclis clade was the sister group of all other euglenalian genera, followed by Discoplastis spathirhyncha (Skuja) Triemer and the Colacium clade, respectively, which was inconsistent with their placement based on nuclear rDNA genes. The Trachelomonas, Strombomonas, Monomorphina, and Cryptoglena clade was sister to the Euglena clade. The loricate genera, Trachelomonas and Strombomonas, were closely related to each other, while Monomorphina and Cryptoglena also grouped together. The Euglena clade formed a monophyletic lineage comprising most species from taxa formerly allocated to the subgenera Calliglena and Euglena. However, within this genus, none of the subgenera was monophyletic.