Matthew S. Bennett

Phylogeny of the euglenales based upon combined SSU and LSU rDNA sequence comparisons and description of Discoplastis gen. nov. (Euglenophyta)

A Bayesian analysis, utilizing a combined data set developed from the small subunit (SSU) and large subunit (LSU) rDNA gene sequences, was used to resolve relationships and clarify generic boundaries among 84 strains of plastid‐containing euglenophytes representing 11 genera. The analysis produced a tree with three major clades: a Phacus and Lepocinlis clade, a Discoplastis clade, and a Euglena, Colacium, Trachelomonas, Strombomonas, Monomorphina, and Cryptoglena clade. The majority of the species in the genus Euglena formed a well‐supported clade, but two species formed a separate clade near the base of the tree. A new genus, Discoplastis, was erected to accommodate these taxa, thus making the genus Euglena monophyletic. The analysis also supported the monophyly of Colacium, Trachelomonas, Strombomonas, Monomorphina, and Cryptoglena, which formed two subclades sister to the Euglena clade. Colacium, Trachelomonas, and Strombomonas, all of which produce copious amounts of mucilage to form loricas or mucilaginous stalks, formed a well‐supported lineage. Our analysis supported retaining Strombomonas and Trachelomonas as separate genera. Monomorphina and Cryptoglena formed two well‐supported clades that were sister to the Colacium, Trachelomonas, and Strombomonas clade. Phacus and Lepocinclis, both of which have numerous small discoid chloroplasts without pyrenoids and lack peristaltic euglenoid movement (metaboly), formed a well‐supported monophyletic lineage that was sister to the larger Euglena through Cryptoglena containing clade. This study demonstrated that increased taxon sampling, multiple genes, and combined data sets provided increased support for internal nodes on the euglenoid phylogenetic tree and resolved relationships among the major genera in the photosynthetic euglenoid lineage.

Evolution of the chloroplast genome in photosynthetic euglenoids: a comparison of Eutreptia viridis and Euglena gracilis (Euglenophyta).

The chloroplast genome of Eutreptia viridis Perty, a basal taxon in the photosynthetic euglenoid lineage, was sequenced and compared with that of Euglena gracilis Ehrenberg, a crown species. Several common gene clusters were identified and gene order, conservation, and sequence similarity was assessed through comparisons with Euglena gracilis. Significant gene rearrangements were present between Eutreptia viridis and Euglena gracilis chloroplast genomes. In addition, major expansion has occurred in the Euglena gracilis chloroplast accounting for its larger size. However, the key chloroplast genes are present and differ only in the absence of psaM and roaA in Eutreptia viridis, and psaI in Euglena gracilis, suggesting a high level of gene conservation within the euglenoid lineage. Further comparisons with the plastid genomes of closely related green algal taxa have provided additional support for the hypothesis that a Pyramimonas-like alga was the euglenoid chloroplast donor via secondary endosymbiosis.

Highly conserved organellar genomes in the Gracilariales as inferred using new data from the Hawaiian invasive alga Gracilaria salicornia (Rhodophyta)

Absrtact: The phylum Rhodophyta contains of a wide range of ecologically and economically important species distributed worldwide. One taxon, Gracilaria salicornia, was an introduced and invasive species in the Hawaiian Islands that had displaced native flora and fauna, and caused widespread ecological damage. We presented the chloroplast and mitochondrial genomic sequences of G. salicornia from Hawaiian material and compared them to sequences of closely related species. We found that the gene content and synteny of both organellar genomes were very similar to the sequences of other closely related Gracilariales (Gracilaria tenuistipitata var. liui for the chloroplast and Gracilariopsis andersonii and Gracilariophila oryzoides for the mitochondrion). Minor changes included the absence of the horizontally transferred leuC/D operon in the G. salicornia chloroplast genome and the absence in the G. salicornia mitochondrial genome of an approximately 2Kb region found in Gp. andersonii. Overall, this study indicated that organellar genome structures in the order Gracilariales were remarkably well maintained.

Chloroplast Genome Evolution in the Euglenaceae

Over the last few years multiple studies have been published outlining chloroplast genomes that represent many of the photosynthetic euglenid genera. However, these genomes were scattered throughout the euglenophyceaean phylogenetic tree, and focused on comparisons with Euglena gracilis. Here, we present a study exclusively on taxa within the Euglenaceae. Six new chloroplast genomes were characterized, those of Cryptoglena skujai, E. gracilis var. bacillaris, Euglena viridis, Euglenaria anabaena, Monomorphina parapyrum, and Trachelomonas volvocina, and added to six previously published chloroplast genomes to determine if trends existed within the family. With this study: at least one genome has now been characterized for each genus, the genomes of different strains from two taxa were characterized to explore intraspecific variability, and a second taxon has been characterized for the genus Monomorphina to examine intrageneric variability. Overall results showed a large amount of variability among the genomes, though a few trends could be identified both within Euglenaceae and within Euglenophyta. In addition, the intraspecific analysis indicated that the similarity of a genome sequence between strains was taxon dependent, and the intrageneric analysis indicated that the majority of the evolutionary changes within the Euglenaceae occurred intergenerically.

Characterization of Euglenaformis gen. nov. and the chloroplast genome of Euglenaformis (Euglena) proxima (Euglenophyta)

Abstract: Euglena proxima is a common, globally dispersed, and easily identified photosynthetic euglenoid. Previous phylogenetic analyses using nuclear-encoded small subunit (SSU) and large subunit (LSU) rRNA genes revealed that this taxon was paraphyletic with other Euglena species and was positioned as sister to all Euglenaceae. Despite this, authors were reluctant to remove this taxon from Euglena until additional data, or taxa pairing with E. proxima, were obtained because it created a monotypic genus. To clarify the taxonomy, the chloroplast genome of E. proxima was sequenced and compared with those of other photosynthetic euglenoids. Phylogenomic analyses were performed comparing 79 chloroplast-encoded genes from E. proxima with those found in seven photosynthetic euglenoids and three prasinophytes, the group from which the euglenoid chloroplast was probably derived. These analyses resulted in highly supported phylogenomic trees with topologies that were consistent with all previous phylogenetic analyses, i.e. they positioned E. proxima as sister to all of the Euglenaceae. In addition, a syntenic comparison was conducted between E. proxima and the chloroplast genomes of two Euglena taxa in order to determine their similarities. This analysis showed that the construction of the E. proxima chloroplast genome was very different from that of the two Euglena chloroplast genomes, which were extremely similar to each other. Based on these data, E. proxima was removed from Euglena, and a new genus, Euglenaformis, was erected to clarify the true relationship of this taxon to the rest of the photosynthetic euglenoids.

Tracing Patterns of Chloroplast Evolution in Euglenoids: Contributions from Colacium vesiculosum and Strombomonas acuminata (Euglenophyta)

The chloroplast genomes of two photosynthetic euglenoids, Colacium vesiculosum Ehrenberg (128,889 bp), and Strombomonas acuminata (Schmarda) Deflandre (144,167 bp) have been sequenced. These chloroplast genomes in combination with those of Euglena gracilis, Eutreptia viridis, and Eutreptiella gymnastica provide a snapshot of euglenoid chloroplast evolution allowing comparisons of gene content, arrangement, and expansion. The gene content of the five chloroplast genomes is very similar varying only in the presence or absence of, rrn5, roaA, psaI, psaM, rpoA, and two tRNAs. Large gene rearrangements have occurred within the C. vesiculosum and S. acuminata chloroplast genomes. Most of these rearrangements represent repositioning of entire operons rather than single genes. When compared with previously sequenced genomes, C. vesiculosum and S. acuminata chloroplast genomes more closely resemble the E. gracilis chloroplast genome in size of the genome, number of introns, and gene order than they do those of the Eutreptiales. Overall, the chloroplast genomes of these five species show an evolutionary trend toward increased intron number, a decrease in gene density, and substantial rearrangement of gene clusters.

Comparative chloroplast genomics between Euglena viridis and Euglena gracilis (Euglenophyta)

Bennett M.S., Wiegert K.E. and Triemer R.E. 2012. Comparative chloroplast genomics between Euglena viridis and Euglena gracilis (Euglenophyta). Phycologia 51: 711–718. DOI: 10.2216/12-017.1 The chloroplast genomes of Euglena gracilis, Eutreptia viridis, Eutreptiella gymnastica, Colacium vesiculosum, Strombomonas acuminata and the colourless Euglena (Astasia) longa, which had secondarily lost the ability to photosynthesize, were previously reported. These studies had shown that there was a great diversity in the size of euglenoid chloroplast genomes and in the arrangement of gene clusters. However, while these genomes provided important insights into the evolution of the chloroplast genome across genera, they did not address genomic variability within a genus. In an effort to continue with these investigations, we sequenced the chloroplast genome of Euglena viridis and compared this to the E. gracilis chloroplast genome in order to explore intrageneric chloroplast evolution. The chloroplast genome of E. viridis was also compared to those of the other previously published euglenoid chloroplast genomes. Our results showed that while the chloroplast genome of E. viridis most closely resembled that of E. gracilis, the chloroplast genomes did show significant differences. The chloroplast genome of E. viridis was far more compact, had a gene cluster that was reversed in both gene order and strand orientation, had a region that was comprised almost entirely of open reading frames and had substantially fewer introns. However, despite these differences, it was clear from the chloroplast genome sequence that E. viridis and E. gracilis were very closely related and that the majority of euglenoid chloroplast evolution probably occurred before the divergence of the genus Euglena.

Phylogenetic Relationships and Morphological Character Evolution of Photosynthetic Euglenids (Excavata) Inferred from Taxon-rich Analyses of Five Genes

Photosynthetic euglenids acquired chloroplasts by secondary endosymbiosis, which resulted in changes to their mode of nutrition and affected the evolution of their morphological characters. Mapping morphological characters onto a reliable molecular tree could elucidate major trends of those changes. We analyzed nucleotide sequence data from regions of three nuclear‐encoded genes (nSSU, nLSU, hsp90), one chloroplast‐encoded gene (cpSSU) and one nuclear‐encoded chloroplast gene (psbO) to estimate phylogenetic relationships among 59 photosynthetic euglenid species. Our results were consistent with previous works; most genera were monophyletic, except for the polyphyletic genus Euglena, and the paraphyletic genus Phacus. We also analyzed character evolution in photosynthetic euglenids using our phylogenetic tree and eight morphological traits commonly used for generic and species diagnoses, including: characters corresponding to well‐defined clades, apomorphies like presence of lorica and mucilaginous stalks, and homoplastic characters like rigid cells and presence of large paramylon grains. This research indicated that pyrenoids were lost twice during the evolution of phototrophic euglenids, and that mucocysts, which only occur in the genus Euglena, evolved independently at least twice. In contrast, the evolution of cell shape and chloroplast morphology was difficult to elucidate, and could not be unambiguously reconstructed in our analyses.

A NEW METHOD FOR OBTAINING NUCLEAR GENE SEQUENCES FROM FIELD SAMPLES AND TAXONOMIC REVISIONS OF THE PHOTOSYNTHETIC EUGLENOIDS LEPOCINCLIS (EUGLENA) HELICOIDEUS AND LEPOCINCLIS (PHACUS) HORRIDUS (EUGLENOPHYTA)1

One of the foremost issues in the field of algal taxonomy is the inability to acquire, grow, and sequence new taxa. This problem is particularly true in the study of photosynthetic euglenoids where most of the distinct taxa in culture collections have been sequenced, and many other taxa of interest have been resistant to culturing, and thus, sequencing. In an effort to address this problem, we have utilized a new technique, novel to the field of taxonomy, which allows for the sequencing of nuclear genes from a very small number of cells. Through this procedure, a DNA extraction followed by a multiple displacement amplification (MDA), taxa obtained by field collection had their genomic DNA (gDNA) amplified many fold to microgram quantities. The DNA was then used as template DNA for PCR reactions, and multiple nuclear genes were amplified successfully from several different taxa. By applying this procedure, we were able to shed new light on taxa that have been historically difficult to classify, resulting in the assignment of Euglena helicoideus (C. Bernard) M. S. Benn. et Triemer and Phacus horridus (Pochm.) M. S. Benn. et Triemer to the genus Lepocinclis.

A new method for obtaining nuclear gene sequences from field samples and taxonomic revisions of the photosynthetic euglenoids lepocinclis (euglena) helicoideus and lepocinclis (phacus) horridus (euglenophyta)

One of the foremost issues in the field of algal taxonomy is the inability to acquire, grow, and sequence new taxa. This problem is particularly true in the study of photosynthetic euglenoids where most of the distinct taxa in culture collections have been sequenced, and many other taxa of interest have been resistant to culturing, and thus, sequencing. In an effort to address this problem, we have utilized a new technique, novel to the field of taxonomy, which allows for the sequencing of nuclear genes from a very small number of cells. Through this procedure, a DNA extraction followed by a multiple displacement amplification (MDA), taxa obtained by field collection had their genomic DNA (gDNA) amplified many fold to microgram quantities. The DNA was then used as template DNA for PCR reactions, and multiple nuclear genes were amplified successfully from several different taxa. By applying this procedure, we were able to shed new light on taxa that have been historically difficult to classify, resulting in the assignment of Euglena helicoideus (C. Bernard) M. S. Benn. et Triemer and Phacus horridus (Pochm.) M. S. Benn. et Triemer to the genus Lepocinclis.