MONOPHYLY OF THE GENUS CLOSTERIUM AND THE ORDER DESMIDIALES (CHAROPHYCEAE, CHLOROPHYTA) INFERRED FROM NUCLEAR SMALL SUBUNIT rDNA DATA

We newly sequenced the nuclear-encoded small subunit (SSU) rDNA coding region for 21 taxa of the genus Closterium. The new sequences were integrated into an alignment with 13 known sequences of conjugating green algae representing six traditional families (i.e. Zygnemataceae, Mesotaeniaceae, Gonatozygaceae, Peniaceae, Closteriaceae, and Desmidiaceae) and five known charophycean sequences as outgroups. Both maximum likelihood and maximum parsimony analyses supported with high bootstrap values one large clade containing all placoderm desmids (Desmidiales). All the Closterium taxa formed one clade with 100% bootstrap support, indicating their monophyly, but not paraphyly, as suggested earlier. As to the taxa within the genus Closterium , we found two clades of morphologically closely related taxa in both maximum likelihood and maximum parsimony trees. They corresponded to the C. calosporum species complex and the C. moniliferum-ehrenbergii species complex. It is of particular interest that the homothallic entity of C. moniliferum v. moniliferum was distinguished from and ancestral to all other entities of the C. moniliferum-ehrenbergii species complex. Superimposing all 50 charophycean sequences on the higher order SSU rRNA structure model of Closterium , we investigated degrees of nucleotide conservation at a given position in the nucleotide sequence. A characteristic "signature" structure to the genus Closterium was found as an additional helix at the tip of V1 region. In addition, eight base deletions at the tip of helix 10 were found to be characteristic of the C. calosporum species complex, C. gracile , C. incurvum , C. pleurodermatum , and C. pusillum v. maius. These taxa formed one clade with an 82% bootstrap value in maximum parsimony analysis.     

SEXUAL REPRODUCTION IN CLOSTERIUM MONILIFERUM AND CLOSTERIUM EHRENBERGII1

Sexual reproduction in C. moniliferum is described for the first time. The morphology of conjugation is quite like that of C. ehrenbergii. Homothallic strains of both species usually produce single zygospores between daughter cells that have just divided. However, 2 homothallic clones of C. moniliferum form twin zygospores between conjugants which have paired before division and conjugation. This has not been observed in C. ehrenbergii. Heterothallic strains of both species form twin zygospores in the same manner. Heterotfiallisrn seems a well-established feature in both species. Germination and the survival of 2 products of meiosis arc typical of other desmids which have been investigated.     

Phylogeny of the Macaronesian endemic Crambe section Dendrocrambe (Brassicaceae) based on internal transcribed spacer sequences of nuclear ribosomal DNA

The 14 species of Crambe L. sect. Dendrocrambe DC. (Brassicaceae) form a monophyletic group endemic to the Canary and Madeira archipelagos. Both parsimony and maximum likelihood analyses of sequence data from the two internal transcribed spacer regions of nuclear ribosomal DNA were used to estimate phylogenetic relationships within this section. These analyses support the monophyly of three major clades. No clade is restricted to a single island, and therefore it appears that inter-island colonization has been the main avenue for speciation in these two archipelagos. The two species endemic to Fuerteventura (C. sventenii) and Madeira (C. fruticosa) comprise a clade, providing the first evidence for a floristic link between the Eastern Canary Islands and the archipelago of Madeira. Both maximum likelihood and weighted parsimony analyses show that this clade is sister to the two other clades, although bootstrap support for this relationship is weak. Parsimony optimizations of ecological zones and island distribution suggest a colonization route from the low-altitude areas of the lowland scrub toward the high-elevation areas of the laurel and pine forests. In addition, Tenerife is likely the ancestral island for species endemic to the five westernmost islands of Gran Canaria, La Gomera, El Hierro, La Palma, and Tenerife.     

TIME-LAPSE ANALYSES OF SEXUAL ISOLATION BETWEEN TWO CLOSELY RELATED MATING GROUPS OF THE CLOSTERIUM EHRENBERG SPECIES COMPLEX (CHLOROPHYTA)1

Sexual isolation between Groups A and B of Closterium ehrenbergii, two closely related species, was studied by a multiple-choice mating method, as well as the nochoice mating method which has been used in previous work on microalgae. Time lapse photomicrographs and the difference in cell shape and size between the two mating groups allowed identification of a given cell in the mixture as either Group A or B, even when certain morphological changes occurred during the several day culture required for sexual induction. When plus and minus mating types of Group A were mixed with those of Group B (multiplechoice mating), no intergroup hybrid zygospores were formed. However, many intragroup zygospores of either Group A or B were formed. When one plus strain of Group A was mixed with one minus strain of Group B or when one plus strain of Group B was mixed with one minus strain of Group A (no-choice mating), intergroup sexual interactions took place resulting in a small number of hybrid zygospores; however, the process took much longer than intragroup sexual interactions. It was also shown that cell size difference itself hardly affects sexual interactions between haploid and autodiploid strains of Group A. It is suggested that sexual isolation between Groups A and B would be complete in nature, although they may interact sexually in the laboratory.     

Natural populations of the Closterium ehrenbergii (Desmidiales, Chlorophyta) species complex in Nepal

Several natural populations of the Closterium ehrenbergii Meneghini ex Ralfs species complex were collected in Nepal, in October–December 1982. Water temperature and pH were also recorded. Clonal isolates from these populations were identified to one of four mating groups (H, I, J and M) by test crossing with standard mating-type strains of known mating groups. Groups H and M have smooth walled zygospores, while Groups I and J have scrobiculated zygospore walls. Several undetermined isolates were found in some population samples. In contrast to the previously reported population samples from Nepal, especially from dried soil samples, some of these populations appeared to be rather heavily loaded with mutations that are deleterious to the sexual cycle (i.e. sexual compatibility, zygospore formation and germination). By genetic analysis, a zygote maturation-defective mutation (zym) was detected. One reason for such a heavy genetic load was suggested to be that most population samples had been maintained exclusively by asexual reproduction for a long period in large lakes and nearby ponds, or left-over vegetative populations in paddy fields after other members entered into dormancy through sexual reproduction. The significance of studying such mutations at sexual gene loci is discussed in the light of speciation problems in microalgae.     

Phylogenetic relationships in the Gesnerioideae (Gesneriaceae) based on nrDNA ITS and cpDNA trnL-F and trnE-T spacer region sequences

The Gesnerioideae includes most of the New World members of the Gesneriaceae family and is currently considered to include five tribes: Beslerieae, Episcieae, Gesnerieae, Gloxinieae, and Napeantheae. This study presents maximum parsimony and maximum likelihood phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer regions (ITS), and the chloroplast DNA trnL intron, trnL-trnF intergenic spacer region, and trnE-trnT intergenic spacer region sequences. The ITS and cpDNA data sets strongly support the monophyly of a Beslerieae/Napeantheae clade; an Episcieae clade; a Gesnerieae clade; a Gloxinieae clade minus Sinningia, Sinningia relatives, and Gloxinia sarmentiana; and a Sinningia/Paliavana/Vanhouttea clade. This is the first study to provide strong statistical support for these tribes/clades. These analyses suggest that Sinningia and relatives should be considered as a separate tribe. Additionally, generic relationships are explored, including the apparent polyphyly of Gloxinia. Chromosome number changes are minimized on the proposed phylogeny, with the exception of the n = 11 taxa of the Gloxinieae. Scaly rhizomes appear to have been derived once in the Gloxinieae sensu stricto. The number of derivations of the inferior ovary is unclear: either there was one derivation with a reversal to a superior ovary in the Episcieae, or there were multiple independent derivations of the inferior ovary.     

Molecular phylogenetics of Hypoxidaceae--evidence from plastid DNA data and inferences on morphology and biogeography

Phylogenetic relationships of the monocot family Hypoxidaceae (Asparagales), which occurs mainly in the Southern Hemisphere, were reconstructed using four plastid DNA regions (rbcL, trnL intron, trnL-F intergenic spacer, and trnS-G intergenic spacer) for 56 ingroup taxa including all currently accepted genera and seven species of the closely related families Asteliaceae, Blandfordiaceae, and Lanariaceae. Data were analyzed by applying parsimony, maximum likelihood and Bayesian methods. The intergenic spacer trnS-G--only rarely used in monocot research--contributed a substantial number of potentially parsimony informative characters. Hypoxidaceae consist of three well-supported major clades, but their interrelationships remain unresolved. Our data indicate that in the Pauridia clade one long-distance dispersal event occurred from southern Africa to Australia. Long-distance dispersal scenarios may also be likely for the current distribution of Hypoxis, which occurs on four continents. In the Curculigo clade, the present distribution of Curculigo s.s. on four continents could support a Gondwanan origin, but the level of divergence is too low for this hypothesis to be likely. The main clades correspond well with some floral characters, habit and palynological data, whereas chromosomal data exhibit plasticity and probably result from polyploidization and subsequent dysploidy and/or aneuploidy. Evolutionary flexibility is also suggested by the number of reported pollination syndromes: melittophily, myophily, sapromyophily, and cantharophily. Based on our phylogenetic results, we suggest cautious nomenclatural reorganization to generate monophyly at the generic level.     

Cryptic goldfields: a molecular phylogenetic reinvestigation of Lasthenia californica sensu lato and close relatives (Compositae: Heliantheae sensu lato)

Maximum parsimony analysis of DNA sequence data from the internal and external transcribed spacer (ITS and ETS) regions of 18S-26S nuclear ribosomal DNA and the 3' trnK intron of chloroplast DNA from over 60 populations of Lasthenia sect. Amphiachaenia yielded a well-supported tree showing that the most common species of Lasthenia, L. californica sensu lato (s.l.), is not monophyletic. Members of Lasthenia californica s.l. belong to two well-supported but morphologically cryptic clades. One clade includes members of L. macrantha; the other represents a basally divergent lineage in L. sect. Amphiachaenia. Members of each clade can be diagnosed by pappus morphology and by geographic distribution, except for epappose plants that occur in a broad region of sympatry in central California. Overall diversification in the clade corresponding to L. sect. Amphiachaenia has been accompanied by minimal morphological divergence, which has resulted in previously underappreciated cryptic diversity.     

Polyphyly of Limoniastrum (Plumbaginaceae): evidence from DNA sequences of plastid rbcL, trnL intron and trnL-F intergene spacer

Phylogenetic relationships of Limoniastrum and other genera of subfamily Staticoideae (Plumb-aginaceae) were studied using parsimony analysis of the plastid gene rbc L, the intron of trn L and the intergene spacer of trnL-trn F. Our analysis showed that Limoniastrum was polyphyletic. Limoniastrum ifniense , in both rbc L and combined data analyses, is sister to Armeria and Psylliostachys , whereas in the trn L-F (intron and spacer combined) analysis it is sister to a clade composed of Acantholimon, Dictyolimon and the remaining species of Limoniastrum . In all analyses, the five remaining species of Limoniastrum (excluding Limoniatrum ifniense ) formed a clade with two groups of species: L. monopetalum+L. guyonianum and those sometimes considered as the segregate genus Bubania ( L.feei, L. weygandiorum and L. rechingeri ). Levels of sequence divergence among these three groups of Limoniatrum were greater than for other well supported genera in the family and, in combination with morphological differences and paucity of synapomorphies, led us to conclude that separate generic status for each of the three clades is warranted.     

Recognition of two major clades and early diverged groups within the subfamily Cyperoideae (Cyperaceae) including Korean sedges

We aim to present phylogenetic major groups within the subfamily Cyperoideae (Cyperaceae) on the basis of three molecular data sets; nuclear ribosomal internal transcribed spacer and 5.8S ribosomal RNA region, the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit gene, and trnL intron and trnL-F intergenic spacer. Three molecular data and two combined data sets were used to obtain robust and detailed phylogenetic trees by using maximum parsimony and Bayesian inference, respectively. We analyzed 81 genera and 426 species of Cyperaceae, including Korean species. We suggest one early diverged group (EDGs), and two major clades (FAEC and SDC) within the subfamily Cyperoideae. And the clade EDGs comprises six tribes (Schoeneae, Bisboeckelereae, Sclerieae, Cryptangieae, Trilepideae, and Rhynchosporeae) at the basal nodes of Cyperoideae. The FAEC clade (posterior probability [PP]/bootstrap value [BS] = 1.00/85) comprises four tribes (Fuireneae, Abildgaardieae, Eleocharideae, Cypereae), and the SDC clade (PP/BS = 1.00/86) comprises three tribes (Scirpeae, Dulichieae, Cariceae). These three clades used for phylogenetic groups in our study will be useful for establishing the major lineage of the sedge family. The phylogeny of Korean sedges was also investigated within the whole phylogeny of Cyperaceae. The 20 genera of Korean sedges were placed in 10 tribes forming 14 clades.     

MOLECULAR PHYLOGENY OF STAURASTRUM MEYEN EX RALFS AND RELATED GENERA (ZYGNEMATOPHYCEAE,STREPTOPHYTA) BASED ON CODING AND NONCODING RDNA SEQUENCE COMPARISONS1

Nuclear‐encoded small subunit rDNA, 1506 group I intron, and internal transcribed spacer sequences were obtained from 39 strains representing five core desmid genera, Staurastrum, Staurodesmus Teil., Cosmarium Corda ex Ralfs, Xanthidium Ehr. ex Ralfs, and Euastrum Ehr. ex Ralfs (Desmidiaceae, Zygnematophyceae), and used individually and concatenated to assess phylogenetic relationships between putatively allied members of the family. To identify positional homology between divergent noncoding sequences, secondary structure models were generated and their reliability assessed by screening the alignment for compensating base changes. The phylogeny based on coding and noncoding sequence comparisons confidently resolved a monophyletic core of the genus Staurastrum but also revealed the artificial nature of the traditional genus. Twenty distinct species representing a wide range of morphotypes of Staurastrum formed a strongly supported generic clade that was further split into three well‐resolved lineages. The phylogenetic relationships revealed within Staurastrum were in conflict with all previous formal or informal classifications of the genus. The genera Staurodesmus and Cosmarium were shown to be highly polyphyletic, and some morphologically similar taxa displayed high sequence divergence that exceeded generic boundaries. Apparently, the taxonomic significance of some morphological characters in Staurastrum and other desmid genera has been greatly overestimated.     

Zygospore formation between homothallic and heterothallic strains of Closterium

Zygospore formation in different strains of the Closterium peracerosum-strigosum-littorale complex was examined in this unicellular isogamous charophycean alga to shed light on gametic mating strains in this taxon, which is believed to share a close phylogenetic relationship with land plants. Zygospores typically form as a result of conjugation between mating-type plus (mt⁺) and mating-type minus (mt⁻) cells during sexual reproduction in the heterothallic strain, similar to Chlamydomonas. However, within clonal cells, zygospores are formed within homothallic strains, and the majority of these zygospores originate as a result of conjugation of two recently divided sister gametangial cells derived from one vegetative cell. In this study, we analyzed conjugation of homothallic cells in the presence of phylogenetically closely related heterothallic cells to characterize the reproductive function of homothallic sister gametangial cells. The relative ratio of non-sister zygospores to sister zygospores increased in the presence of heterothallic mt⁺ cells, compared with that in the homothallic strain alone and in a coculture with mt⁻ cells. Heterothallic cells were surface labeled with calcofluor white, permitting fusions with homothallic cells to be identified and confirming the formation of hybrid zygospores between the homothallic cells and heterothallic mt⁺ cells. These results show that at least some of the homothallic gametangial cells possess heterothallic mt⁻-like characters. This finding supports speculation that division of one vegetative cell into two sister gametangial cells is a segregative process capable of producing complementary mating types.     

STABLE DIPLOIDS FROM INTRAGROUP ZYGOSPORES OF CLOSTERIUM EHRENBERGII MENEGH. (CONJUGATOPHYCEAE)1

Two pairs of stable diploid clones were obtained as aberrant forms among F₁ progeny of an intragroup (intraspecific) cross between R-11-4 (mating type +) and M-16-4b (mating type -) of Group A of Closterium ehrenbergii Menegh. Each pair was derived from the two germination products of a single zygospore, and both clones were mating type minus. The cell size range of these four diploid minus clones was considerably above that of normal (haploid) Group A clones. Chromosome counts at the second meiotic metaphase indicated that these clones were diploid with approximately 200 chromosomes, which was double the number for normal Group A clones. Diploid minus clones conjugated normally with any haploid Group A plus clones, and yielded many triploid zygospores. Triploid zygospores germinated normally as did intragroup diploid zygospores. In metaphase I preparations, only bivalents were observed except on a few occasions where some uni- and multivalents were also detected. Viability of F₁ progeny from triploid zygospores (55–74%) was somewhat lower than from diploid zygospores of Japanese Group A populations (65–90%), but higher than intergroup (interspecific) hybrid zygospores from Groups A, B and H (0–12%). In addition to lower viability, some F₁ progeny from triploid zygospores exhibited slow vegetative growth. Almost all pairs of F₁ clones from single triploid zygospores were of opposite mating type, similar to normal diploid zygospores of the intragroup cross. Morphological variability of F₁ progeny of triploid zygospores was great. The apparently normal meiosis of triploid zygospores and the high viability of F₁ progeny suggested that the genome of Group A contains several sets of chromosome complements with mechanisms by which bivalents are regularly formed in the first meiotic division.     

Species of the parasitic genus Duboscquella are members of the enigmatic Marine Alveolate Group I

Small subunit ribosomal RNA gene sequences of Duboscquella spp. infecting the tintinnid ciliate, Favella ehrenbergii, were determined. Two parasites were sampled from different localities. They are morphologically similar to each other and both resemble D. aspida. Nevertheless, two distinct sequences (7.6% divergence) were obtained from them. Phylogenetic trees inferred from maximum likelihood and maximum parsimony revealed that these two Duboscquella spp. sequences are enclosed in an environmental clade named Marine Alveolate Group I. This clade consists of a large number of picoplanktonic organisms known only from environmental samples from various parts of the ocean worldwide, and which therefore lack clear characterization and identification. Here, we provide morphological and genetic characterization of these two Duboscquella genotypes included in this enigmatic clade. Duboscquella spp. produce a large number of small flagellated spores as dispersal agents and the presence of such small cells partially explains why the organisms related to these parasites have been detected within environmental genetic libraries, built from picoplanktonic size fractions of environmental samples. The huge diversity of the Marine Alveolate Group I and the finding that parasites from different marine protists belong to this lineage suggest that parasitism is a widespread and ecologically relevant phenomenon in the marine environment.     

ITS rDNA sequence-based phylogenetic analysis of Tomentellopsis species from boreal and temperate forests, and the identification of pink-type ectomycorrhizas

Fungi in the genus Tomentellopsis were subjected to molecular phylogenetic analyses in order to clarify species-level relationships and mycorrhiza-forming ability with coniferous and deciduous trees. Fungal nucleotide sequence data from the internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) region were obtained from fruitbodies, ectomycorrhiza and pure cultures. Maximum parsimony (MP), distance (neighbor joining, NJ) and maximum likelihood (ML) analyses of aligned ITS sequences highlighted three clades designated T. echinospora, T. submollis and T. bresadoliana. Sporocarp tissue and ectomycorrhizas, or isolated mycelia, previously described as Pinirhiza rosea, Piceirhiza rosea or Pink were clearly identified in a strongly supported T. submollis clade. Host-linked population variation in this clade was also noted that may reflect active speciation activity. A fungus isolated from Beige-type mycorrhizas formed on Scots pine seedlings appeared in the paraphyletic T. echinospora clade which included sequences showing greater divergence. The data provide a primary classification of Tomentellopsis species that is urgently needed in assessment of the importance of resupinate thelephoroid fungi in mycorrhizal communities associated with trees in coniferous and deciduous forest ecosystems.     

Phylogenetics of Planipapillus, lawn-headed onychophorans of the Australian Alps, based on nuclear and mitochondrial gene sequences

We addressed phylogenetic relationships among species of Planipapillus, a clade of oviparous onychophorans from southeastern mainland Australia, to create a framework for understanding the evolution of the modified male head papillae used in mating in this clade. We sequenced fragments of two mitochondrial genes, COI and 12S rRNA, and a nuclear intron from the fushi tarazu gene, for individuals from 14 putative species of Planipapillus and six outgroups. We analyzed these data under both parsimony and likelihood criteria, incorporating heterogeneous parameter fitting guided by likelihood ratio tests. These analyses result in strong, congruent support for many clades. We infer multiple independent origins of spikes in Planipapillus male head structures.     

Evaluating the clade size effect in alternative measures of branch support

The clade size effect refers to a bias that causes middle‐sized clades to be less supported than small or large‐sized clades. This bias is present in resampling measures of support calculated under maximum likelihood and maximum parsimony and in Bayesian posterior probabilities. Previous analyses indicated that the clade size effect is worst in maximum parsimony, followed by maximum likelihood, while Bayesian inference is the least affected. Homoplasy was interpreted as the main cause of the effect. In this study, we explored the presence of the clade size effect in alternative measures of branch support under maximum parsimony: Bremer support and symmetric resampling, expressed as absolute frequencies and frequency differences. Analyses were performed using 50 molecular and morphological matrices. Symmetric resampling showed the same tendency that bootstrap and jackknife did for maximum parsimony and maximum likelihood. Few matrices showed a significant bias using Bremer support, presenting a better performance than resampling measures of support and comparable to Bayesian posterior probabilities. Our results indicate that the problem is not maximum parsimony, but resampling measures of support. We corroborated the role of homoplasy as a possible cause of the clade size effect, increasing the number of random trees during the resampling, which together with the higher chances that medium‐sized clades have of being contradicted generates the bias during the perturbation of the original matrix, making it stronger in resampling measures of support.     

TIME LAPSE ANALYSES OF SEXUAL REPRODUCTION IN CLOSTERIUM EHRENBERGII (CONJUGATOPHYCEAE)1

The process of sexual differentiation was studied using heterothallic clones of Closterium ehrenbergii Meneghini. The first visible sign of sexual reproduction was agglutination of two or more cells in a group and this was followed by gametangiogenic division and conjugation of gametangial cells. Movements of gametangial cells were carefully studied. Gametangial cells occasionally participated again in gametangiogenesis instead of proceeding directly to the formation of conjugation papilla. The whole process of sexual differentiation from vegetative cell to zygospore was considered to be basically similar in both of the two closely related mating groups, A and B, of C. ehrenbergii. Nevertheless, there were some differences between the two groups in patterns of the sexual differentiation. In Group A, vegetative cell division was completely suppressed by mixing the two complementary mating type clones together into the same medium with high light illumination. This suppression was not caused by the nitrogen depletion in the medium, but by the presence of cells of opposite mating type. In Group B, vegetative cell division and sexual reproduction occurred side by side repeatedly for several days.