J. Phycol. 37, 143–159 (2001) EVOLUTION OF PHACUS (EUGLENOPHYCEAE) AS INFERRED FROM PELLICLE MORPHOLOGY AND SSU rDNA1 Brian S. Leander 2 and Mark A. Farmer Center for Advanced Ultrastructural Research, 154 Barrow Hall, The University of Georgia, Athens, Georgia, 30602 This research integrates a large morphological cal information and ultrastructural data. Even though data set into a molecular context. Nineteen pellicle the classification system proposed by Leedale (1967) characters and 62 states from 13 euglenid taxa were was a significant improvement over previous schemes, analyzed cladistically. The pellicle morphology of Eu- limited awareness of euglenid characteristics contin- glena tripteris (Klebs), Lepocinclis ovata (Conrad), Phacus ues to forestall phylogenetic hypotheses with predic- brachykentron (Pochmann), P. oscillans (Klebs), P. pyrum tive power. New molecular and morphological data (Stein), and P. triqueter (Dujardin) is described compre- have accrued over the past 15 years, indicating that hensively. These data are compared with new informa- the current set of taxonomic problems within the Eu- tion on the pellicle morphology of Euglena acus (Ehren- glenida is profound (Farmer 1988, Linton et al. 1999, berg), E. stellata (Mainx), and Peranema trichophorum Linton et al. 2000, Preisfeld et al. 2000). (Stein) in addition to published data on Entosiphon sulca- Euglenids have been lumped informally into two tum (Dujardin), Euglena gracilis (Klebs), Distigma proteus groups depending on the general state of the pellicle, (Pringsheim), and Petalomonas cantuscygni (Cann and namely the “aplastic” and the “plastic” euglenids (Triemer Pennick). Nuclear small subunit (SSU) rDNA sequences and Farmer 1991). The plastics (e.g. Euglena, Peranema, provided an independent test for establishing a robust and Distigma) are either heterotrophic or phototrophic, organismal pedigree of the same taxa. A synthetic tree are suspected to be monophyletic (Montegut-Felkner and derived from the combined phylogenetic analyses of Triemer 1997, Linton et al. 1999, Linton et al. 2000, Preis- pellicle morphology and SSU rDNA enabled us to parsi- feld et al. 2000), and have many pellicle strips (greater moniously map morphological character states. This ap- than 16) that are arranged helically and often permit the proach demonstrated the utility of pellicle morphology cell to undergo “euglenoid movement” (Gallo and for inferring phylogenetic relationships of euglenids Shrével 1982, Suzaki and Williamson 1985). The aplas- and establishing apomorphy-based clade definitions. tics (e.g. Petalomonas, Ploeotia, and Entosiphon) are all Three robust clades with unambiguous pellicle-based heterotrophic, have a rigid pellicle of few longitudi- apomorphies can be recognized within taxa tradition- nally arranged strips (usually less than 12), and mo- ally classified as Phacus: (1) L. ovata and P. pyrum, (2) lecular data indicate that they may encompass the E. tripteris and P. triqueter, and (3) P. brachykentron and most recent common ancestor of euglenids (Triemer P. oscillans. Taxonomic concerns that emerged from and Farmer 1991, Montegut-Felkner and Triemer these results are discussed. 1997, Linton et al. 1999). Therefore, the aplastics may Key index words: Euglena; Euglenophyta; Euglenozoa; tag a paraphyletic group that, if made monophyletic evolution; Lepocinclis; pellicle; Phacus; phylogeny; tax- within a strictly cladistic framework, would be synony- onomy mous with the Euglenida. Comparative data from small subunit (SSU) rDNA Abbreviations: P, the maximum number of strips indicate that some phototrophic taxa within the plas- around the cell periphery; S, the number of strips be- tics have secondarily evolved a rigid pellicle (Linton tween consecutively terminating strips; SSU, small sub- et al. 1999, Linton et al. 2000). Traditionally, cells like unit; T, the number of strips that converge at the poste- these that were laterally flattened were classified as rior tip; WA, the number of whorls at the anterior end; Phacus (Dujardin 1841) and those that were not flat- WP, the number of whorls at the posterior end tened were placed into Lepocinclis (Perty 1849). Some members within these two taxa have acquired a pelli- cle with strips arranged longitudinally (e.g. L. ovum The taxonomic history of euglenids has included Lemmermann and P. oscillans), whereas others have many classification systems that were based primarily retained helically arranged strips (e.g. L. ovata and P. on a limited number of morphological characteristics pyrum). A close examination of whether pellicular fea- observable with light microscopy (Klebs 1883, Bütschli tures like rigidity, cell flatness, and strip orientation 1884, Senn 1900, Lemmermann 1913, Calkins 1933, Hol- are homologous between members of Lepocinclis and lande 1942, 1952). The classification most often used to- Phacus has yet to take place. It is clear that the pelli- day (Leedale 1967, 1978) incorporates some physiologi- cle morphology within these taxa is very diverse, but how this diversity reflects phylogeny remains largely unexplored (Pochmann 1942, Huber-Pestalozzi 1955, Bourrelly and Couté 1981, Conforti and Tell 1983, 1Received 24 July 2000. Accepted 30 October 2000. 1989, Leedale 1985, Tell and Conforti 1986, Couté 2Author for correspondence: e-mail [email protected]. and Thérézien 1994, Zakrys and Walne 1994). 143 144 BRIAN S. LEANDER AND MARK A. FARMER One molecular analysis indicates that neither Phacus actually P. oscillans. Because we examined cells from the same nor Lepocinclis are monophyletic and both are inter- source, we also refer to this culture as P. oscillans. Peranema tri- chophorum was obtained from the Carolina Biological Supply mixed within Euglena Ehrenberg (Linton et al. 2000). Company (WW-13-1838). Entosiphon sulcatum was isolated from Moreover, comparative analyses of other euglenids the Delaware–Raritan canal in New Brunswick, New Jersey, and demonstrate that details of the pellicle provide impor- a unialgal culture was temporarily maintained in soil–water me- tant phylogenetic information (Buetow 1968, Leedale dium that unfortunately is no longer available. and Hibberd 1974, Cann 1986, Conforti and Tell 1989, Electron microscopy. Cells from each culture were concentrated by slow centrifugation, chemically fixed, and prepared for trans- Dragos et al. 1997, Angeler et al. 1999, Leander and mission and scanning electron microscopy by the protocols de- Farmer 2000, 2001). We tested these hypotheses by pro- scribed in Leander and Farmer (2000). viding data on the pellicle morphology of Peranema Morphological descriptions and replicate observations. The termi- trichophorum, four Phacus taxa, one Lepocinclis taxon, nology used to describe pellicle morphology has been defined by Leander and Farmer (2000, 2001); terms dealing with strip and three Euglena taxa. Six of these taxa have rigid substructure are reviewed in Table 1. To determine whether pellicles, and we suspect that they represent three sep- distinct surface patterns of strips were consistent within taxa, 30 arate clades that are not reflected in the current classi- different cells were scored and the mode and range of variation fication: (1) L. ovata and P. pyrum, (2) E. tripteris and were presented. P. triqueter, and (3) P. oscillans and P. brachykentron. Phylogenetic analysis of morphological characters. Nineteen pelli- cle characters containing 62 character states were scored for 13 The morphological data set was compared with previ- euglenid taxa. The matrix of character states was analyzed with ously published data for Petalomonas cantuscygni, Dis- PAUP* 4.0 using the branch-and-bound algorithm (Swofford tigma proteus, Entosiphon sulcatum, and Euglena gracilis; 1999). States for two characters were ordered, and the remain- analyzed cladistically; and coupled with comparative der was left unordered. The two ordered characters possess po- lar states that are linked by clear intermediate states (see Re- analyses of SSU rDNA sequences. We uncovered char- sults). The robustness of each node on the most parsimonious acters associated with the pellicle that provide robust tree(s) was investigated with decay indices using AutoDecay apomorphy-based definitions for clades at different 4.0.2 (Eriksson 1998) and nonparametric bootstrap values us- levels in the macroevolutionary hierarchy. ing PAUP* 4.0. The tree length, number of informative charac- ters, retention index, and consistency index were presented. Character state changes were phylogenetically mapped using materials and methods MacClade 3.03. Culture conditions. Cultures of Euglena acus Ehrenberg (UTEX DNA isolation, amplification, and sequencing. Genomic DNA was LB 1316), E. stellata (UTEX 372), E. tripteris (UTEX LB 1311), extracted from E. tripteris, P. brachykentron, and P. triqueter using Lepocinclis ovata (UTEX LB 1305), Phacus brachykentron (UTEX LB a standard hexadecyltrimethylammonium bromide (CTAB) ex- 1317), P. caudata Hübner (UTEX LB 1285), P. pyrum (UTEX LB traction protocol (Zolan and Pukkila 1986). After RNAse diges- 2345), and P. triqueter (UTEX LB 1286) were obtained from the Cul- tion, sequences of SSU rDNA were amplified using PCR prim- ture Collection of Algae at the University of Texas at Austin ers and a thermocycling protocol established previously for the (UTEX). Euglena stellata was grown in Proteose medium, and the group (Elwood et al. 1985, Montegut-Felkner and Triemer 1997, ϩ remaining cultures were grown in a soil–water (GR / NH4) me-