J. Eukaryot. Microbiol., 57(1), 2010 pp. 19–32 r 2009 The Author(s) Journal compilation r 2009 by the International Society of Protistologists DOI: 10.1111/j.1550-7408.2009.00447.x Evolution of Distorted Pellicle Patterns in Rigid Photosynthetic Euglenids (Phacus Dujardin) HEATHER J. ESSONa,1 and BRIAN S. LEANDERa,b aDepartment of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, Canada V6T 1Z4, and bDepartment of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, Canada V6T 1Z4 ABSTRACT. Members of the euglenid genus Phacus are morphologically differentiated from other photosynthetic species by the pres- ence of a rigid cytoskeleton (pellicle) and predominantly dorsoventrally flattened, leaf-shaped cells. In order to better understand the evolutionary history of this lineage, we used scanning electron microscopy to examine patterns of pellicle strips in Phacus acuminatus, Phacus longicauda var. tortus, Phacus triqueter, Phacus segretii, Phacus pleuronectes, Phacus similis, Phacus pusillus, Phacus orbic- ularis, Phacus warszewiczii, and Discoplastis spathirhyncha, a putative close relative of Phacus and Lepocinclis. Our observations showed that while the earliest diverging species in our analyses, namely P. warszewiczii, has three whorls of exponential reduction, most members of Phacus have clustered patterns of posterior strip reduction that are bilaterally symmetrical distortions of the radially sym- metrical ‘‘whorled’’ patterns found in other photosynthetic euglenids. Comparative morphology, interpreted within the context of mo- lecular phylogenetic analyses of combined nuclear small subunit rDNA and partial nuclear large subunit rDNA sequences, demonstrates that clustered patterns of posterior strip reduction arose after the divergence of Phacus from other photosynthetic euglenids and are the result of developmental processes that govern individual strip length. Clustered patterns of pellicle strips in Phacus do not appear to be adaptively significant themselves; they evolved in association with the origin of cell flattening and cell rigidity, which may be adaptations to a planktonic lifestyle. Key Words. Character evolution, cytoskeleton, Discoplastis, morphology, phylogeny. While the leaf-like morphology described by Dujardin (1841) is HACUS (Dujardin, 1841) is a morphologically distinctive predominant in Phacus sensu stricto, a number of Phacus taxa that P clade of photosynthetic euglenids that includes rigid, dorso- are not yet placed in other genera based on phylogenetic analyses ventrally flattened cells. Most species have an elongated caudal deviate from it in one or more characters. For example, process and longitudinally arranged pellicle strips (Fig. 1–13). P. triqueter and Phacus warszewiczii are conspicuously tri-lobed Several species of Phacus consist of three lobes and are deltoid in rather than being dorsoventrally flattened per se (Fig. 8–10; Hub- transverse section, while other species have become twisted er-Pestalozzi 1955; Leander and Farmer 2001b), and P. wars- around their longitudinal axis in a corkscrew fashion (e.g. Phacus zewiczii is illustrated with helically arranged pellicle strips inflexus and Phacus similis [ 5 P. smulkowskianus Zakrys], Fig. 5; (Huber-Pestalozzi 1955). Moreover, taxa such as Phacus segretii Huber-Pestalozzi 1955). Molecular phylogenetic analyses have (Fig. 7) and Phacus stokesii, lack a caudal process and instead demonstrated that the genus was polyphyletic, and several species have rounded posterior ends (Huber-Pestalozzi 1955). Other taxa, formerly grouped within Phacus based on light microscopical ob- such as Phacus parvulus and Phacus pusillus, are described as servations have subsequently been moved to other rigid photo- having extremely blunt caudal processes (Huber-Pestalozzi 1955; synthetic genera, namely Monomorphina and Cryptoglena (Marin Fig. 11). To date, very few of these atypical taxa (P. triqueter, et al. 2003). The phylogenetic relationships within Phacus sensu P. parvulus, and P. pusillus) have been included in molecular or stricto, however, remain poorly understood (Brosnan et al. 2003; morphological phylogenetic analyses (Leander and Farmer Kosmala et al. 2007; Linton et al. 2000; Marin et al. 2003; Mu¨llner 2001b; Marin et al. 2003; Triemer et al. 2006). et al. 2001; Nudelman et al. 2003; Triemer et al. 2006). One pellicle character that has been informative in previous Comparative analyses of morphological data, particularly pell- studies of euglenid evolution and taxonomy is posterior strip re- icle characters, are expected to help build a phylogenetic frame- duction: patterns formed on the posterior cell surface by pellicle work for understanding the overall diversity of Phacus. Kosmala strips of different lengths (e.g. Leander and Farmer 2000a). The et al. (2007) found that characters visible using light microscopy, presence of uniquely modified patterns of posterior reduction in such as the presence or absence of transverse struts, were good some species of Phacus indicate that it may be particularly useful taxonomical characters in delimiting Phacus pleuronectes and in resolving relationships within the genus and forming inferences Phacus orbicularis. Leander and Farmer (2000a, b, 2001a, b) and regarding pellicle character evolution (Leander and Farmer Leander, Witek and Farmer (2001) used scanning and transmis- 2001b). We were interested in exploring how the unusual cell sion electron microscopy (SEM and TEM, respectively) to de- shapes observed in Phacus affect the whorled patterns of pellicle scribe pellicle characters which, when incorporated into cladistic strips that have been characterized in other euglenid lineages. Our analyses and compared with molecular data, provided robust in- knowledge of Phacus surface morphology is extremely spotty and ferences about euglenid phylogeny. Their sampling of Phacus, in the vast majority of cases, non-existent, and this research takes however, turned out to include only three members of Phacus the initial steps needed to help illuminate this area of uncertainty. sensu stricto: Phacus oscillans, Phacus triqueter, and Phacus Because of the complex evolutionary history and developmental brachykentron (reidentified as Phacus acuminatus; other taxa be- processes underlying the formation of these patterns (Esson and longed to Lepocinclis and Monomorphina; Leander and Farmer Leander 2006, 2008), a brief review of their diversity and struc- 2001b; Marin et al. 2003; Triemer et al. 2006). ture is included below. Corresponding Author: H. J. Esson, Department of Botany, Univer- sity of British Columbia, 6270 University Boulevard, Vancouver, Can- ada V6T 1Z4—Telephone number: 143 0664 60277 24032; FAX Evolutionary significance of posterior strip reduction. In number: 143 1 4277 9240; e-mail: [email protected] addition to a corset of microtubules and a network of endoplasmic 1Present Address: Max F. Perutz Laboratories, University of Vienna reticulum, the peripheral cytoskeleton of euglenids is reinforced and Medical University of Vienna. Dr. Bohr-Gasse 9, A-1030 Vienna, by 4–120 proteinaceous strips that lie beneath the plasma mem- Austria. brane and extend longitudinally or helically from the anterior 19 20 J. EUKARYOT. MICROBIOL., 57, NO. 1, JANUARY– FEBRUARY 2010 Fig. 1–13. Scanning electron micrographs showing the diversity of Phacus. 1. Discoplastis spathirhyncha, a closely related lineage to Phacus with 32 pellicle strips. 2. Phacus pleuronectes. 3. Phacus longicauda var. tortus. 4. Phacus oscillans. 5. Phacus similis. 6. Phacus orbicularis. 7. Phacus segretii, showing the rounded posterior end of the cell. 8. Phacus triqueter. 9. Phacus warszewiczii. 10. Posterior view of Phacus warszewiczii showing three lobes of the deltoid shaped cell. 11. Phacus pusillus. 12. Phacus acuminatus, UBC isolate. 13. P. acuminatus (brachykentron), UTEX LB 1317. All images at same scale (bar 5 20 mm). ESSON & LEANDER—PELLICLE PATTERNS IN PHACUS 21 canal region to the posterior end of the cell (Leander, Esson and Table 1. Taxon names, strain identification, and accession numbers of Breglia 2007). The number of pellicle strips around the cell sequences used for molecular phylogenetic analyses in this study. periphery is more or less consistent within species and is referred to using the variable ‘‘P’’ (Leander and Farmer 2000a). In pho- Taxon Strain GenBank accession tosynthetic euglenids, however, some strips are too short to reach identification numbers the posterior end of the cell and instead terminate at a certain point along the length of the cell. The length of any particular strip de- SSU LSU pends on its relative age: pellicle strips are duplicated and inher- Euglena viridis SAG 1224-17c AY523037 DQ140125 ited semi-conservatively, where existing strips resume and Discoplastis spathirhynchaa SAG 1224-42 AJ532454 DQ140100 terminate growth with each subsequent round of cytokinesis (Es- Colacium mucronatumb UTEX 2524 AF326232 AY130224 b son and Leander 2006). Just before cytokinesis, a new strip forms Monomorphina pyrum UTEX 2354 AF112874 AY130238 between every pair of existing strips, and these are the youngest Trachelomonas lefevrei SAG 1283-10 DQ140136 AY359949 Lepocinclis ovum strips on the pellicle of any cell. The youngest strips are shorter SAG 1244-8 AF110419 AY130235 Lepocinclis steinii UTEX 523 AF096993 AY130815 than all other pellicle strips, while the oldest strips reach the pos- (L. buetschlii in Leander terior tip of the cell (Bouck and Ngo 1996; Esson