Evolutionary relationships of Mycaureola dilseae (Agaricales), a basidiomycete pathogen of a subtidal rhodophyte

Mushroom-forming fungi (homobasidiomycetes) are major examples of morphological and ecological diversification in terrestrial habitats. Homobasidiomycetes includes only nine described species that are known from marine environments. Morphological traits that have concealed the ancestry of these fungi include reduced fruiting bodies with hairy surfaces and extremely modified spores, both of which may function as floating devices to aid successful dispersal and adhesion to various substrates such as driftwood. Our previous results suggested that all marine forms as yet investigated are placed in the Nia clade (euagarics) and that they have primarily evolved from cypelloid forms (minute, cup-shaped, terrestrial saprotrophs) via transitions through mangroves to fully marine habitats. We show here that Mycaureola dilseae, which parasitizes the red alga Dilsea carnosa, is a second independent lineage of marine fungi in the euagarics clade that is not related to cyphelloid forms. Phylogenetic reconstructions were based on two data sets: a partial four-region rDNA data set (nuc-ssu, nuc-lsu, mt-ssu, and mt-lsu) with inclusive sampling of 249 taxa and a densely sampled ITS data set including 32 taxa, which formed a clade with Mycaureola in the four-region rDNA analyses. Inferences using constrained and unconstrained six-parameter weighted parsimony, Bayesian Markov chain Monte Carlo methods, and maximum likelihood approaches place M. dilseae in the morphologically diverse /physalacriaceae clade next to Gloiocephala spp., a group of highly reduced stipitate-pileate saprotrophs.     

Evolution of complex fruiting-body morphologies in homobasidiomycetes

The fruiting bodies of homobasidiomycetes include some of the most complex forms that have evolved in the fungi, such as gilled mushrooms, bracket fungi and puffballs ('pileate-erect') forms. Homobasidiomycetes also include relatively simple crust-like 'resupinate' forms, however, which account for ca. 13-15% of the described species in the group. Resupinate homobasidiomycetes have been interpreted either as a paraphyletic grade of plesiomorphic forms or a polyphyletic assemblage of reduced forms. The former view suggests that morphological evolution in homobasidiomycetes has been marked by independent elaboration in many clades, whereas the latter view suggests that parallel simplification has been a common mode of evolution. To infer patterns of morphological evolution in homobasidiomycetes, we constructed phylogenetic trees from a dataset of 481 species and performed ancestral state reconstruction (ASR) using parsimony and maximum likelihood (ML) methods. ASR with both parsimony and ML implies that the ancestor of the homobasidiomycetes was resupinate, and that there have been multiple gains and losses of complex forms in the homobasidiomycetes. We also used ML to address whether there is an asymmetry in the rate of transformations between simple and complex forms. Models of morphological evolution inferred with ML indicate that the rate of transformations from simple to complex forms is about three to six times greater than the rate of transformations in the reverse direction. A null model of morphological evolution, in which there is no asymmetry in transformation rates, was rejected. These results suggest that there is a 'driven' trend towards the evolution of complex forms in homobasidiomycetes.     

Molecular phylogenetics of the Gloeophyllales and relative ages of clades of Agaricomycotina producing a brown rot

The Gloeophyllales is a recently described order of Agaricomycotina containing a morphologically diverse array of polypores (Gloeophyllum), agarics (Neolentinus, Heliocybe) and resupinate fungi (Veluti-Veluticeps, Boreostereum, Chaetodermella), most of which have been demonstrated to produce a brown-rot mode of wood decay and are found preferentially on coniferous substrates. Multiple phylogenetic studies have included taxa of Gloeophyllales, but none have sampled the order thoroughly, and so far only ribosomal RNA genes have been used. Consequently the limits and higher level placement of the Gloeophyllales are obscure. We obtained sequence data for three protein-coding genes (rpb2, atp6, tef1) and three rRNA regions (nuc-ssu, nuc-lsu, 5.8S) in 19 species of Gloeophyllales representing seven genera and analyzed them together with a diverse set of Agaricomycotina, emphasizing Polyporales. Boreostereum, which is suspected to produce a white rot, is the sister group of the rest of the Gloeophyllales, all of which produce a brown rot. Gloeophyllum contains at least two independent clades, one of which might correspond to the genus Osmoporus. White rot and resupinate fruiting bodies appear to be plesiomorphic in Gloeophyllales. Relaxed molecular clock analyses suggest that the Gloeophyllales arose in the Cretaceous, after the origin of Pinaceae.     

Trends in morphological evolution in homobasidiomycetes inferred using maximum likelihood: a comparison of binary and multistate approaches

The homobasidiomycetes is a diverse group of macrofungi that includes mushrooms, puffballs, coral fungi, and other forms. This study used maximum likelihood methods to determine if there are general trends (evolutionary tendencies) in the evolution of fruiting body forms in homobasidiomycetes, and to estimate the ancestral forms of the homobasidiomycetes and euagarics clade. Character evolution was modeled using a published 481-species phylogeny under two character-coding regimes: additive binary coding, using DISCRETE, and multistate (five-state) coding, using MULTISTATE. Inferences regarding trends in character evolution made under binary coding were often in conflict with those made under multistate coding, suggesting that the additive binary coding approach cannot serve as a surrogate for multistate methods. MULTISTATE was used to develop a"minimal"model of fruiting body evolution, in which the 20 parameters that specify rates of transformations among character states were grouped into the fewest possible rate categories. The minimal model required only four rate categories, one of which is approaching zero, and suggests the following conclusions regarding trends in evolution of homobasidiomycete fruiting bodies: (1) there is an active trend favoring the evolution of pileate-stipitate forms (those with a cap and stalk); (2) the hypothesis that the evolution of gasteroid forms (those with internal spore production, such as puffballs) is irreversible cannot be rejected; and (3) crustlike resupinate forms are not a particularly labile morphology. The latter finding contradicts the conclusions of a previous study that used binary character coding. Ancestral state reconstructions under binary coding suggest that the ancestor of the homobasidiomycetes was resupinate and the ancestor of the euagarics clade was pileate-stipitate, but ancestral state reconstructions under multistate coding did not resolve the ancestral form of either node. The results of this study illustrate the potential sensitivity of comparative analyses to character state definitions.     

Fruiting body and soil rDNA sampling detects complementary assemblage of Agaricomycotina (Basidiomycota, Fungi) in a hemlock-dominated forest plot in southern Ontario

This is the first study to assess the diversity and community structure of the Agaricomycotina in an ectotrophic forest using above-ground fruiting body surveys as well as soil rDNA sampling. We recovered 132 molecular operational taxonomic units, or 'species', from fruiting bodies and 66 from soil, with little overlap. Fruiting body sampling primarily recovered fungi from the Agaricales, Russulales, Boletales and Cantharellales. Many of these species are ectomycorrhizal and form large fruiting bodies. Soil rDNA sampling recovered fungi from these groups in addition to taxa overlooked during the fruiting body survey from the Atheliales, Trechisporales and Sebacinales. Species from these groups form inconspicuous, resupinate and corticioid fruiting bodies. Soil sampling also detected fungi from the Hysterangiales that form fruiting bodies underground. Generally, fruiting body and soil rDNA samples recover a largely different assemblage of fungi at the species level; however, both methods identify the same dominant fungi at the genus-order level and ectomycorrhizal fungi as the prevailing type. Richness, abundance, and phylogenetic diversity (PD) identify the Agaricales as the dominant fungal group above- and below-ground; however, we find that molecularly highly divergent lineages may account for a greater proportion of total diversity using the PD measure compared with richness and abundance. Unless an exhaustive inventory is required, the rapidity and versatility of DNA-based sampling may be sufficient for a first assessment of the dominant taxonomic and ecological groups of fungi in forest soil.     

After the gold rush,or before the flood? Evolutionary morphology of mushroom-forming fungi (Agaricomycetes) in the early 21st century

Mushroom-forming fungi (Agaricomycetes, approx. syn.: Homobasidiomycetes) produce a diverse array of fruiting bodies, ranging from simple crust-like forms to complex, developmentally integrated forms, such as stinkhorns and veiled agarics. The 19th century Friesian system divided the mushroom-forming fungi according to macromorphology. The Friesian taxonomy has long been regarded as artificial, but it continues to influence the language of mycology and perceptions of fungal diversity. Throughout the 20th century, the phylogenetic significance of anatomical features was elucidated, and classifications that departed strongly from the Friesian system were proposed. However, the anatomical studies left many questions and controversies unresolved, due in part to the paucity of characters, as well as the general absence of explicit phylogenetic analyses. Problems in fruiting body evolution were among the first to be addressed when molecular characters became readily accessible in the late 1980s. Today, GenBank contains about 108,000 nucleotide sequences of ‘homobasidiomycetes’, filed under 7300 unique names. Analyses of these data are providing an increasingly detailed and robust view of the phylogeny and the distribution of different fruiting body forms across the 14 major clades that make up the agaricomycetes. However, it would be wrong to suggest that all the important questions about fruiting body evolution have been resolved. Recent studies focusing on resupinate forms suggest that there may still be undetected major clades of agaricomycetes, which could have a significant impact on our estimates of the ancestral forms in this morphologically diverse group. Modern approaches, including comparative phylogenetic analyses and developmental studies, have the potential to yield novel insights into both the macroevolutionary processes and cellular mechanisms of fungal morphological evolution.     

Molecular systematics and biological diversification of Boletales

Historical patterns of morphological evolution and ecology in the Boletales are largely unresolved but appear to involve extensive convergence. We studied phylogenetic relationships of Boletales based on two datasets. The nuc-lsu dataset is broadly sampled and includes roughly 30% of the described species of Boletales and 51 outgroup taxa across the Hymenomycetes. The multigene dataset (nuc-ssu, nuc-lsu, 5.8S, mt-lsu, atp6) sampled 42 key species of Boletales in a framework of 14 representative Hymenomycetes. The Boletales are strongly supported as monophyletic in our analyses on both datasets with parsimony, maximum likelihood and Bayesian approaches. Six major lineages of Boletales that currently are recognized on subordinal level, Boletineae, Paxillineae, Sclerodermatineae, Suillineae, Tapinellineae, Coniophorineae, received varied support. The backbone of the Boletales was moderately resolved in the analyses with the nuc-lsu dataset, but support was strong for most major groups. Nevertheless, most brown-rot producing forms were placed as a paraphyletic grade at the base of the Boletales. Analyses on the multigene dataset confirm sister group relationships among Boletales, Agaricales and Atheliales. Boletineae and Suillineae received the highest support values; Paxillineae and Sclerodermatineae were not consistently resolved as monophyletic groups. The Coniophorineae were not monophyletic in any analyses. The Tapinellineae consisting of morphologically diverse brown-rotting fungi forms the basal group in the Boletales. We performed ancestral state reconstruction with BayesMultiState, which suggested that the ancestor of the Boletales was a resupinate or polyporoid saprotrophic fungus, producing a brown-rot.     

Analysis of character correlations among wood decay mechanisms,mating systems,and substrate ranges in homobasidiomycetes

Homobasidiomycetes include the majority of wood-decaying fungi. Two basic forms of wood decay are known in homobasidiomycetes: white rot, in which lignin and cellulose are degraded, and brown rot, in which lignin is not appreciably degraded. An apparent correlation has been noted between production of a brown rot, decay of conifer substrates, and possession of a bipolar mating system (which has a single mating-type locus, in contrast to tetrapolar systems, which have two mating-type loci). The goals of this study were to infer the historical pattern of transformations in decay mode, mating type, and substrate range characters, and to determine if a causal relationship exists among them. Using nuclear and mitochondrial rDNA sequences, we performed a phylogenetic analysis of 130 species of homobasidiomycetes and performed ancestral state reconstructions by using parsimony on a range of trees, with various loss:gain cost ratios. We evaluated pairwise character correlations by using the concentrated changes test (CCT) of Maddison and the maximum likelihood (ML) method of Pagel. White rot, tetrapolar mating systems, and the ability to decay conifers and hardwoods appear to be plesiomorphic in homobasidiomycetes, whereas brown rot, bipolar mating systems, and exclusive decay of conifers appear to have evolved repeatedly. The only significant correlation among characters was that between brown rot (as the independent character) and exclusive decay of conifer substrates (P < 0.03). This correlation was supported by the CCT on a range of plausible trees, although not with every reconstruction of ancestral states, and by the ML test. Our findings suggest that the evolution of brown rot has promoted repeated shifts to specialization for confier substrates.     

Phylogenetic study of Baylisascaris schroederi isolated from Qinling subspecies of giant panda in China based on combined nuclear 5.8S and the second internal transcribed spacer (ITS-2) ribosomal DNA sequences

The nuclear ribosomal DNA (rDNA) region spanning 5.8S rDNA and the second internal transcribed spacer (ITS-2) of Baylisascaris schroederi isolated from the Qinling subspecies of giant panda in Shaanxi Province, China were amplified and sequenced. Sequence variations in the two rDNA regions within B. schroederi and among species in the family Ascarididae were examined. The lengths of B. schroederi 5.8S and ITS-2 rDNA sequences were 156 bp and 327 bp, respectively, and no nucleotide variation was found in these two rDNA regions among the 20 B. schroederi samples examined, and these ITS-2 sequences were identical to that of B. schroederi isolated from giant panda in Sichuan province, China. The inter-species differences in 5.8S and ITS-2 rDNA sequences among members of the family Ascarididae were 0-1.3% and 0-17.7%, respectively. Phylogenetic relationships among species in the Ascarididae were re-constructed by Bayesian inference (Bayes), maximum parsimony (MP), and maximum likelihood (ML) analyses, based on combined sequences of 5.8S and ITS-2 rDNA. All B. schroederi samples clustered together and sistered to B. transfuga with high posterior probabilities/bootstrap values, which further confirmed that nematodes isolated from the Qinling subspecies of giant panda in Shaanxi Province, China represent B. schroederi. Because of the large number of ambiguously aligned sequence positions (difficulty of inferring homology by positions), ITS-2 sequence alone is likely unsuitable for phylogenetic analyses at the family level, but the combined 5.8S and ITS-2 rDNA sequences provide alternative genetic markers for the identification of B. schroederi and for phylogenetic analysis of parasites in the family Ascarididae.     

Phylogenetic diversity of lichen-associated homobasidiomycetes

The vast majority of lichenicolous fungi are relatively host-specific, nonvirulent ascomycetes and heterobasidiomycetes. A few known lichenicolous homobasidiomycetes (mushroom-forming fungi) generally exhibit broad host ecologies and in some cases, high virulence. Many produce conspicuous sclerotia or bulbils, thought to be adaptive in dispersal and survival. To more clearly understand the evolution of these atypical behaviors in lichenicolous basidiomycetes, we isolated or acquired specimens or cultures of 23 lichenicolous homobasidiomycetes and their relatives, from which we obtained mainly nuclear and some mitochondrial rDNA sequences. Phylogenetic analyses in this study indicate that a lichenicolous habit arose in four major clades. In two of these clades the habit represents a major evolutionary theme linked to the origin of well-known basidiolichens. The phylogenetic diversity of these fungi indicates that the lichenicolous habit arose recently and independently in the mushroom-forming fungi.     

Berkleasmium crunisia sp. nov. and its phylogenetic affinities to the Pleosporales based on 18S and 28S rDNA sequence analyses

Berkleasmium crunisia sp. nov. is described from a decaying rachis of Calamus sp. (Arecaceae) from Khuan Ka Long, Satun Province, Thailand. This Berkleasmium species differs morphologically from other species in possessing subtending cells and larger conidia. The phylogenetic relationship of the genus Berkleasmium among sexual ascomycetes also was examined. Sequence analyses from 18S, 28S and ITS-5.8S rDNA were analyzed phylogenetically under maximum parsimony, Bayesian and neighbor joining criteria. Phylogenies revealed that Berkleasmium is not monophyletic. Berkleasmium micronesicum and B. nigroapicale are related to Westerdykella cylindrica and Sporormia australis, which are members of the family Sporormiaceae (Pleosporales). Other species, including our new taxon, appear to share phylogenetic affinities with other anamorphic fungi, whose classification within the Pleosporales is still obscure. Analyses of 18S, 28S, ITS (+5.8S) rDNA and combined (18S+28S) gene sequences fail to give sufficient phylogenetic resolution within the Pleosporales.     

Reconstructing the evolution of agarics from nuclear gene sequences and basidiospore ultrastructure

Traditional classifications of agaric fungi involve gross morphology of their fruit bodies and meiospore print-colour. However, the phylogeny of these fungi and the evolution of their morphological and ecological traits are poorly understood. Phylogenetic analyses have already demonstrated that characters used in traditional classifications of basidiomycetes may be heavily affected by homoplasy, and that non-gilled taxa evolved within the agarics several times. By integrating molecular phylogenetic analyses including domains D1–D3 and D7–D8 of nucLSU rDNA and domains A–C of the RPB1 gene with morphological and chemical data from representative species of 88 genera, we were able to resolve higher groups of agarics. We found that the species with thick-walled and pigmented basidiospores constitute a derived group, and hypothesize that this specific combination of characters represents an evolutionary advantage by increasing the tolerance of the basidiospores to dehydration and solar radiation and so opened up new ecological niches, e.g. the colonization of dung substrates by enabling basidiospores to survive gut passages through herbivores. Our results confirm the validity of basidiospore morphology as a phylogenetic marker in the agarics.     

Nucleotide sequence diversity of rDNA internal transcribed spacers extracted from conidia and cleistothecia of several powdery mildew fungi

An improved protocol, including DNA extraction with Chelex, two amplifications with a nested primer set, and DNA purification by electrophoresis, made it possible to analyze nuclear rDNA sequences of powdery mildew fungi using at most several hundred conidia or 20 cleistothecia. Nucleotide sequence diversity of the nuclear rDNA region containing the two internal transcribed spacers (ITS1 and ITS2) and 5.8S rRNA gene derived from conidia and cleistothecia was investigated for four kinds of powdery mildew fungi including two isolates of the same species. The results showed that the nucleotide sequences of the nuclear rDNA region were highly conserved between the teleomorph and the anamorph. Thus, the nucleotide sequence data obtained from either developmental stage can be used for phylogenetic studies of powdery mildew fungi. The nucleotide sequences of the 5.8S rRNA genes of the four species were highly conserved, but those of their ITS regions were variable. This suggests that the nuclear rDNA region is not suitable for phylogenetic studies of distantly related powdery mildew fungi, because too much sequence diversity exists, within the ITS, and too little phylogenetic information is contained within the 5.8S rRNA gene. However, the ITS region will be useful for phylogenetic comparison of closely related species or intraspecies. Contribution No. 132 from the Laboratory of Plant Pathology, Mie University.     

A new poroid species of Resupinatus from Puerto Rico, with a reassessment of the cyphelloid genus Stigmatolemma

A fungus with gelatinous poroid fruiting bodies was found in Puerto Rico and determined by macro- and micromorphology to be most similar to members of the lamellate agaric genus Resupinatus. This species is described as a new species, Resupinatus porosus. Phylogenetic analyses of ribosomal DNA sequences support the inclusion of this fungus in the clade containing Resupinatus, and indicate that this monophyletic group also includes members of Asterotus and the cyphelloid genus Stigmatolemma. Resupinatus porosus is another example of tropical poroid representatives of lamellate agaric genera. Resupinatus is emended to include species with poroid (R. porosus) or merulioid (R. merulioides) hymenophore as well as those with laterally stipitate (Asterotus) or cyphelloid (Stigmatolemma) fruiting bodies. Seven new combinations in Resupinatus are proposed to accommodate well-known species of Stigmatolemma.     

Detection and taxonomic placement of endophytic fungi within frond tissues of Livistona chinensis based on rDNA sequences.

The 5.8S gene and flanking internal transcribed spacers (ITS1 and ITS2) of the rDNA were amplified from total DNA extracted from frond tissues of Livistona chinensis with universal and fungal-specific primers. These amplified fragments were cloned and sequenced. Phylogenetic analysis based on the 5.8S gene sequences indicated that the six clone sequences obtained were of different origins. Five sequences, P1-9, P2-6, P4-4, P4-5, and P4-7, belonged to the fungi and one sequence, P3-2, belonged to the plants. P1-9 was inferred to belong to the Basidiomycota based on the phylogenetic analysis of the 5.8S gene sequences but could not be identified to lower taxonomic levels. Further identification of the other four fungal clones to lower taxonomic levels was attempted based on phylogenetic analysis and sequence comparison of both the conserved 5.8S gene and the variable ITS regions. The origin of P2-6 was identified to be Glomerella and its anamorph Colletotrichum, the origins of P4-5 and P4-7 were Mycosphaerella and its anamorph Cladosporium, and the origin of P4-4 was the Herpotrichiellaceae. The direct approach to detection and taxonomic placement of endophytic fungi within host tissue without the need for conventional in vitro culturing is discussed.     

Phylogenetic relationships of Malaysian monkeys, Cercopithecidae, based on mitochondrial cytochrome c sequences

Mitochondrial DNA cytochrome c oxidase II (COII) gene sequences of Malaysian Cercopithecidae were examined to ascertain their phylogenetic relationships. Colobinae were represented by the genera Presbytis, Trachypithecus and Nasalis, while the genus Macaca represented Cercopithecinae. DNA amplification and sequencing of the COII gene was performed on 16 samples. Symphalangus syndactylus (Hylobatidae) was used as the outgroup. Data were analyzed using both character (maximum parsimony) and distance (neighbor-joining) methods. Tree topologies indicated that Colobinae and Cercopithecinae have their own distinct monophyletic clade. This result was well supported by bootstrap values and genetic distances derived from the Kimura-2-parameter algorithm. Separation of Macaca nemestrina from M. fascicularis was also well supported by bootstrap values. In addition, tree topologies indicate a good resolution of the Colobinae phylogenetic relationships at the intergeneric level, but with low bootstrap support. The position of Nasalis remained problematic in both trees. Overall, COII is a good gene candidate for portraying the phylogenetic relationships of Malaysian primates at the inter- and intra-subfamily levels.     

赤潮棕囊藻(Phaeocystis globosa)rDNA ITS区序列变异与二级结构分析

测定了南海球形棕囊藻香港株P1、P2和湛江株ZhJ1的rDNAITS区序列(含5.8srDNA),结合Gen Bank的13条同源序列,比对长度为904bp,变异位点271个,简约信息位点221个,平均(A+T)(34.5%)<(G+C)(65.4%).藻株P1、P2和ZhJ1序列存在变异位点20个,序列间相似性为97.9%～98.5%.ITS序列在种间和种内的解析度高于18srDNA和28srDNA基因;构建的NJ树、MP树、贝叶斯推断系统树的结构是一致的,不同种类的棕囊藻单独聚类,不同地理来源的球形棕囊藻混杂分布但相同地理来源的藻株多聚类在一起.RNA二级结构显示,不同藻种间5.8srDNA区结构基本一致,表现出属的特异性;ITS1、2区结构表现较大的种间差异,表明ITS区RNA二级结构可为棕囊藻分类鉴定提供有用的分子结构信息.     

Phylogenetic evidence for horizontal transmission of group I introns in the nuclear ribosomal DNA of mushroom-forming fungi

Group I introns were discovered inserted at the same position in the nuclear small-subunit ribosomal DNA (nuc-ssu-rDNA) in several species of homobasidiomycetes (mushroom-forming fungi). Based on conserved intron sequences, a pair of intron-specific primers was designed for PCR amplification and sequencing of intron-containing rDNA repeats. Using the intron-specific primers together with flanking rDNA primers, a PCR assay was conducted to determine presence or absence of introns in 39 species of homobasidiomycetes. Introns were confined to the genera Panellus, Clavicorona, and Lentinellus. Phylogenetic analyses of nuc-ssu-rDNA and mitochondrial ssu-rDNA sequences suggest that Clavicorona and Lentinellus are closely related, but that Panellus is not closely related to these. The simplest explanation for the distribution of the introns is that they have been twice independently gained via horizontal transmission, once on the lineage leading to Panellus, and once on the lineage leading to Lentinellus and Clavicorona. BLAST searches using the introns from Panellus and Lentinellus as query sequences retrieved 16 other similar group I introns of nuc-ssu-rDNA and nuclear large-subunit rDNA (nuc-lsu-rDNA) from fungal and green algal hosts. Phylogenetic analyses of intron sequences suggest that the mushroom introns are monophyletic, and are nested within a clade that contains four other introns that insert at the same position as the mushroom introns, two from different groups of fungi and two from green algae. The distribution of host lineages and insertion sites among the introns suggests that horizontal and vertical transmission, homing, and transposition have been factors in intron evolution. As distinctive, heritable features of nuclear rDNAs in certain lineages, group I introns have promise as phylogenetic markers. Nevertheless, the possibility of horizontal transmission and homing also suggest that their use poses certain pitfalls.      

Implications of complete nuclear large subunit ribosomal RNA molecules from the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) and relatives

The D1/D2 domains of large subunit (LSU) rDNA have commonly been used for phylogenetic analyses of dinoflagellates; however, their properties have not been evaluated in relation to other D domains due to a deficiency of complete sequences. This study reports the complete LSU rRNA gene sequence in the causative unarmored dinoflagellate Cochlodinium polykrikoides, a member of the order Gymnodiniales, and evaluated the segmented domains and secondary structures when compared with its relatives. Putative LSU rRNA coding regions were recorded to be 3433 bp in length (49.0% GC content). A secondary structure predicted from the LSU and 5.8S rRNAs and parsimony analyses showed that most variation in the LSU rDNA was found in the 12 divergent (D) domains. In particular, the D2 domain was the most informative in terms of recent evolutional and taxonomic aspects, when compared with both the phylogenetic tree topologies and molecular distance (approximately 10 times higher) of the core LSU. Phylogenetic analysis was performed with a matrix of LSU DNA sequences selected from domains D2 to D4 and their flanking core sequences, which showed that C. polykrikoides was placed on the same branch with Akashiwo sanguinea in the “GPP” complex, which is referred to the gymnodinioid, peridinioid and prorocentroid groups. A broad phylogeny showed that armored and unarmored dinoflagellates were never clustered together; instead, they were clearly divided into two groups: the GPP complex and Gonyaulacales. The members of Gymnodiniales were always interspersed with peridinioid, prorocentroid and dinophysoid forms. This supports previous findings showing that the Gymnodiniales are polyphyletic. This study highlights the proper selection of LSU rDNA molecules for molecular phylogeny and signatures.