PHYLOGENETIC ANALYSIS OF THE GENUS EUGLENA (EUGLENOPHYCEAE) WITH PARTICULAR REFERENCE TO THE TYPE SPECIES EUGLENA VIRIDIS1

Euglena viridis (subgenus Euglena) serves as the type species for the genus Euglena. In this study, molecular phylogenetic analyses using a small subunit (SSU) and a combined SSU–partial large subunit rDNA data set for members of the genus Euglena showed that strains identified as E. viridis on the basis of morphology are distributed between two separate nonsister clades. Although all the E. viridis strains examined were morphologically indistinguishable and possessed spherical mucocysts and stellate chloroplasts with one paramylon center, there was a high degree of sequence divergence between the E. viridis strains in different clades, making this a cryptic species. Like E. viridis, all taxa from the subgenus Euglena are characterized by having one or more stellate chloroplasts with paramylon grains clustered around the center of the chloroplast. These additional taxa were divided into four clades in all the molecular analyses. Strains of Euglena stellata formed two nonsister clades whose members had a single aggregate chloroplast with paramylon center and spindle‐shaped mucocysts. A geniculata clade included species with one or two stellate chloroplasts with paramylon centers and spherical mucocysts, and the cantabrica clade had members with one stellate chloroplast with paramylon center and spherical mucocysts often arranged in spiral rows. Interspersed among these were three additional clades bearing taxa from the subgenus Calliglena that contains members with discoid plastids and pyrenoids that may or may not be capped with paramylon. These taxa formed a laciniata clade, mutabilis clade, and gracilis clade. This study demonstrates that E. viridis and E. stellata are cryptic species that can only be distinguished at the molecular level. Because E. viridis is the designated type species for the genus Euglena, we designated an epitype for E. viridis.     

VARIABILITY IN THE PRIMARY SITE OF PHOTOSYNTHETIC DAMAGE IN SYMBIODINIUM SP. (DINOPHYCEAE) EXPOSED TO THERMAL STRESS1

Exposure to elevated temperature is known to cause photosynthetic inhibition in the coral symbiont Symbiodinium sp. Through the use of the artificial electron acceptor, methyl viologen, this study identified how reduced photosynthetic capacity occurs as a result of inhibition up‐ and/or downstream of ferredoxin in Symbiodinium sp. in hospite and in culture. Heterogeneity between coral species and symbiont clades was identified in the thermal sensitivity of photosynthesis in the symbionts of the scleractinian corals Stylophora pistillata and Pocillopora damicornis, as well as among Symbiodinium cultures of clades A, B, and C. The in hospite symbionts of S. pistillata and the cultured clade C Symbiodinium both exhibited similar patterns in that their primary site of thermal inhibition occurred downstream of ferredoxin at 32°C. In contrast, the primary site of thermal inhibition occurred upstream of ferredoxin in clades A and B at 32°C, while at 34°C, all samples showed combined up‐ and downstream inhibition. Although clade C is common to both P. damicornis and S. pistillata, the manner of thermal inhibition was not consistent when observed in hospite. Results showed that there is heterogeneity in the primal site of thermal damage in Symbiodinium among coral species and symbiont clades.     

EVOLUTION OF MORPHOLOGICAL NOVELTY: A PHYLOGENETIC ANALYSIS OF GROWTH PATTERNS IN STREPTOCARPUS (GESNERIACEAE)

Abstract.— Streptocarpus shows great variation in vegetative architecture. In some species a normal shoot apical meristem never forms and the entire vegetative plant body may consist of a single giant cotyledon, which may measure up to 0.75 m (the unifoliate type) or with further leaves arising from this structure (the rosulate type). A molecular phylogeny of 87 taxa (77 Streptocarpus species, seven related species, and three outgroup species) using the internal transcribed spacers and 5.8S region of nuclear ribosomal DNA suggests that Streptocarpus can be divided into two major clades. One of these broadly corresponds to the caulescent group (with conventional shoot architecture) classified as subgenus Streptocarpella, whereas the other is mainly composed of acaulescent species with unusual architecture (subgenus Streptocarpus). Some caulescent species (such as S. papangae) are anomalously placed with the acaulescent clade. Available cytological data are, however, completely congruent with the two major clades: the caulescent clade is x = 15 and the acaulescent clade (including the caulescent S. papangae) is x = 16 (or polyploid multiples of 16). The genera Linnaeopsis, Saintpaulia, and Schizoboea are nested within Streptocarpus. The sequenced region has evolved, on average, 2.44 times faster in the caulescent clade than in the acaulescent clade and this is associated with the more rapid life cycle of the caulescents. Morphological variation in plant architecture within the acaulescent clade is homoplastic and does not appear to have arisen by unique abrupt changes. Instead, rosulate and unifoliate growth forms have evolved several times, reversals have occurred, and intermediate architectures are found. An underlying developmental plasticity seems to be a characteristic of the acaulescent clade and is reflected in a great lability of form.     

Allopolyploidy in the Wintergreen Group of tribe Gaultherieae (Ericaceae) inferred from low‐copy nuclear genes

DNA sequence data from the low‐copy nuclear genes waxy (GBSSI) and leafy were compared with plastid and ITS sequence data from prior studies to reconstruct phylogenetic relationships in the Wintergreen Group of tribe Gaultherieae (Ericaceae). We conducted phylogenetic analysis with 102 species that includes representatives of all 15 major clades previously identified within the Wintergreen Group and that together span its circum‐Pacific distribution. Results yielded two distinct homeologous copies of waxy for two of the clades, each in widely separated parts of the tree. It also yielded two copies of leafy for one of the clades; only one copy of leafy was found for the other clade, but it was placed in the same major clade as its waxy counterpart and well away from its placement in a prior plastid analysis. A combined four‐locus (waxy, leafy, ITS and plastid data) phylogenetic analysis of all available relevant data placed the copies of each of the clades in two distinct positions in the phylogeny with strong overall statistical support. In combination with evidence from morphology, reproductive biology and cytology, the results suggest that these clades arose through allopolyploid hybridization between lineages deep in the phylogeny but relatively close geographically. This finding confirms previous assumptions that hybridization has played an important role in the evolution of the Gaultherieae.     

GENETIC VARIABILITY AND MOLECULAR PHYLOGENY OF DINOPHYSIS SPECIES (DINOPHYCEAE) FROM NORWEGIAN WATERS INFERRED FROM SINGLE CELL ANALYSES OF rDNA1

The objectives of this study were to determine rDNA sequences of the most common Dinophysis species in Scandinavian waters and to resolve their phylogenetic relationships within the genus and to other dinoflagellates. A third aim was to examine the intraspecific variation in D. acuminata and D. norvegica, because these two species are highly variable in both morphology and toxicity. We obtained nucleotide sequences of coding (small subunit [SSU], partial large subunit [LSU], 5.8S) and noncoding (internal transcribed spacer [ITS]1, ITS2) parts of the rRNA operon by PCR amplification of one or two Dinophysis cells isolated from natural water samples. The three photosynthetic species D. acuminata, D. acuta, and D. norvegica differed in only 5 to 8 of 1802 base pairs (bp) within the SSU rRNA gene. The nonphotosynthetic D. rotundata (synonym Phalacroma rotundatum[Claparède et Lachmann] Kofoid et Michener), however, differed in approximately 55 bp compared with the three photosynthetic species. In the D1 and D2 domains of LSU rDNA, the phototrophic species differed among themselves by 3 to 12 of 733 bp, whereas they differed from D. rotundata by more than 100 bp. This supports the distinction between Dinophysis and Phalacroma. In the phylogenetic analyses based on SSU rDNA, all Dinophysis species were grouped into a common clade in which D. rotundata diverged first. The results indicate an early divergence of Dinophysis within the Dinophyta. The LSU phylogenetic analyses, including 4 new and 11 Dinophysis sequences from EMBL, identified two major clades within the phototrophic species. Little or no intraspecific genetic variation was found in the ITS1–ITS2 region of single cells of D. norvegica and D. acuminata from Norway, but the delineation between these two species was not always clear.     

MULTIGENE ANALYSES OF PHOTOSYNTHETIC EUGLENOIDS AND NEW FAMILY,PHACACEAE (EUGLENALES)1

Bayesian and maximum‐likelihood (ML) analyses of the combined multigene data (nuclear SSU rDNA, and plastid SSU and LSU rDNA) were conducted to evaluate the phylogeny of photosynthetic euglenoids. The combined data set consisted of 108 strains of photosynthetic euglenoids including a colorless sister taxon. Bayesian and ML analyses recovered trees of almost identical topology. The results indicated that photosynthetic euglenoids were divided into two major clades, the Euglenaceae clade (Euglena, Euglenaria, Trachelomonas, Strombomonas, Monomorphina, Cryptoglena, Colacium) and the Phacaceae clade (Phacus, Lepocinclis, Discoplastis). The Euglenaceae clade was monophyletic with high support and subdivided into four main clades: the Colacium, the Strombomonas and Trachelomonas, the Cryptoglena and Monomorphina, and the Euglena and Euglenaria clades. The genus Colacium was positioned at the base of the Euglenaceae and was well supported as a monophyletic lineage. The loricate genera (Strombomonas and Trachelomonas) were located at the middle of the Euglenaceae clade and formed a robust monophyletic lineage. The genera Cryptoglena and Monomorphina also formed a well‐supported monophyletic clade. Euglena and the recently erected genus Euglenaria emerged as sister groups. However, Euglena proxima branched off at the base of the Euglenaceae. The Phacaceae clade was also a monophyletic group with high support values and subdivided into three clades, the Discoplastis, Phacus, and Lepocinclis clades. The genus Discoplastis branched first, and then Phacus and Lepocinclis emerged as sister groups. These genera shared a common characteristic, numerous small discoid chloroplasts without pyrenoids. These results clearly separated the Phacaceae clade from the Euglenaceae clade. Therefore, we propose to limit the family Euglenaceae to the members of the Euglena clade and erect a new family, the Phacaceae, to house the genera Phacus, Lepocinclis, and Discoplastis.     

GENE SEQUENCE DIVERSITY AND THE PHYLOGENETIC POSITION OF ALGAE ASSIGNED TO THE GENERA PHAEOPHILA AND OCHLOCHAETE (ULVOPHYCEAE,CHLOROPHYTA)1

The phylogenetic position of microfilamentous marine green algae assigned to the species Phaeophila dendroides, Entocladia tenuis (Phaeophila tenuis, and Ochlochaete hystrix was examined through phylogenetic analyses of nuclear‐encoded small subunit rDNA and chloroplast‐encoded tufA gene sequences. These analyses placed the P. dendroides strains within the Ulvophyceae, at the base of a clade that contains representatives of the families Ulvaceae, Ulvellaceae, and the species Bolbocoleon piliferum, supporting an earlier hypothesis that P. dendroides constitutes a distinct lineage. Substantial divergence in both nuclear and plastid DNA sequences exists among strains of P. dendroides from different geographic localities, but these isolated strains are morphologically indistinguishable. The lineage may have an accelerated rate of gene sequence evolution relative to other microfilamentous marine green algae. Entocladia tenuis and O. hystrix are placed neither in the P. dendroides clade nor in the Ulvellaceae as previous taxonomic schemes predicted but instead form a new clade or clades at the base of the Ulvaceae. Ruthnielsenia gen. nov. is proposed to accommodate Kylin's species, which cannot be placed in Entocladia (=Acrochaete), Phaeophila, or Ochlochaete. Ruthnielsenia tenuis (Kylin) comb. nov., previously known only from Atlantic coasts, is reported for the first time from the Pacific coast of North America (San Juan Island, WA, USA). Isolates of R. tenuis from the Atlantic and Pacific coasts of North America have identical small subunit rDNA and tufA gene sequences.     

PHYLOGENY OF THE EUGLENALES BASED UPON COMBINED SSU AND LSU RDNA SEQUENCE COMPARISONS AND DESCRIPTION OF DISCOPLASTIS GEN. NOV. (EUGLENOPHYTA)1

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.     

Craniid brachiopods: aspects of clade structure and distribution reflect continental drift (Brachiopoda: Craniiformea)

We present maximum likelihood and Bayesian inference relative time‐tree analyses of aligned gene sequences from a worldwide collection of craniiform brachiopods belonging to two genera, Novocrania and Neoancistrocrania. Sequences were obtained from one mitochondrial and three nuclear‐encoded ribosomal RNA genes from varying numbers of specimens. Data‐exploration by network (splits) analyses indicates that each gene identifies the same divergent clades and (with one minor exception) the same inter‐clade relationships. Neoancistrocrania specimens were found only in the Pacific Ocean, near Japan, on the Norfolk and Chesterfield Ridges, and near the Solomon Islands. The Novocrania clades, in approximate order of increasing distance from the root comprise 1. a ‘Northern’ clade of animals collected in the NE. Atlantic, W. Mediterranean and Adriatic; 2. a ‘Tethyan’ clade comprising animals from the E. Mediterranean, Cape Verde islands and the Caribbean (Belize and Jamaica); 3. a ‘NE. Pacific’ clade containing animals from Vancouver Island and from localities near Japan and south of Taiwan; 4. a ‘Southern’ clade that contains two widely separated subclades, one from New Zealand and the other with an extraordinarily wide distribution, ranging from near Japan in the north to the Chesterfield Ridge and Solomon Islands in the West, and in the East to the Galapagos Islands, the coast of South America (Chile) and Richardson seamount (off South Africa) in the South Atlantic. To the South, members of this clade were found in the Weddell, Scotia and Bellinghausen Antarctic Seas. The root of the extant craniid radiation was previously found (by relaxed‐clock analysis) to lie on the branch connecting the two genera so that, in effect, the one clade of Neoancistrocrania serves to polarise evolutionary relationships within the several clades of Novocrania. As previously suggested, all results confirm that Neoancistrocrania is sister to the ‘Northern’ Novocrania clade, and this leads to a proposal that Neoancistrocrania represents one extreme of a wide range of variation in ancestral ventral valve mineralisation, speciation (～90 Ma) resulting from competitive exclusion in rapidly‐growing reef environments. To the extent possible, the identified molecular clades are correlated with named species of Novocrania. The reproductive and population biology of craniid brachiopods is not well known, but from available evidence they are considered to have low‐dispersal potential and, except in enclosed localities such as cold‐water fjords, to have small effective population sizes, features which are consistent with the observed divergent populations in well‐separated localities. Exceptionally slow craniid molecular (rDNA) evolution is suggested by the short branch of Novocrania where it has been used as an outgroup for large‐scale analyses of metazoans. Slow molecular evolution is also indicated by the existence of a distinct Tethyan clade, reflecting restricted dispersal at former times, and by the uniform, short, genetic distances and exceptionally wide geographical distribution of the Southern clade. Thus, the geographical distribution and phylogenetic divergence of craniid brachiopods is an example of phylotectonics, in which relationships revealed by phylogenetic analyses reflect opportunities for dispersal and settlement that were created by tectonic plate movements associated, in this case, with opening and closure of Tethys and the breakup of Gondwana. Molecular dating of craniid divergences and radiochemical dating of tectonic events thus illuminate one another. © 2014 The Linnean Society of London     

Phylogeny of the families Pyuridae and Styelidae (Stolidobranchiata, Ascidiacea) inferred from mitochondrial and nuclear DNA sequences

The Order Stolidobranchiata comprises the families Pyuridae, Styelidae and Molgulidae. Early molecular data was consistent with monophyly of the Stolidobranchiata and also the Molgulidae. Internal phylogeny and relationships between Styelidae and Pyuridae were inconclusive however. In order to clarify these points we used mitochondrial and nuclear sequences from 31 species of Styelidae and 25 of Pyuridae. Phylogenetic trees recovered the Pyuridae as a monophyletic clade, and their genera appeared as monophyletic with the exception of Pyura. The Styelidae, on the other hand, appeared as a paraphyletic group split into several clades. One of them was formed by solitary oviparous species, of which the Pyuridae were a sister group. A second clade included the colonial genera Botryllus, Botrylloides and Symplegma. The remaining colonial and solitary genera formed several poorly resolved clades. One of the more species genus, Polycarpa, was shown to be polyphyletic, and the species Styela plicata grouped into two genetically distant clades suggesting the existence of two cryptic species. The internal phylogeny of Styelidae has bearings on the origin of coloniality in this family. We suggest to abandon the traditional division of colonial forms into social and compound species and use instead the categories of aggregated colonies that do not have common vascular systems, and integrated colonies, that do possess such systems. Our molecular results indicate that there have been several independent acquisitions of coloniality in the Styelidae, and that viviparity may be a pre-adaptation for a colonial life-style.     

Circumscription of Fulbrightiella gen. nov. and Sherwoodiella gen. nov., Two Novel Genera in the Calotrichaceae (Nostocales,Cyanobacteria)

Three novel strains in Calotrichaceae from tropical habitats were isolated and characterized with regard to their morphology, phylogenetic placement, and secondary structures of conserved domains in the 16S-23S internal transcribed spacer (ITS). The strains fell into two clades formerly identified as Calothrix from freshwater and brackish habitats. Based on both morphology and ecology, they differed from the type species of Calothrix, C. confervicola, which is marine, has wide trichomes with short cells, and narrows abruptly to a hyaline hair. The first clade grouped species with heteropolar filaments widened at the base and narrowed gradually toward the apex but not ending in a hair, with basal heterocytes that are formed in series as the apically placed heterocytes senesce; this clade is being named Fulbrightiella gen. nov., with two named species, F. bharadwajae sp. nov. and F. oahuensis sp. nov. The second clade was comprised of a single species with isopolar trichomes that are untapering as hormogonia, but which widen midfilament and taper toward both ends following growth. These trichomes develop pairs of heterocyte mid-filament, causing fragmentation into heteropolar trichomes with basal heterocytes and ends that taper, but not to a hair. This clade consists of a single species at present, Sherwoodiella mauiensis. With this action, four clades in the Calotrichaceae have been named: Macrochaete, Dulcicalothrix, Fulbrightiella, and Sherwoodiella. Calothrix sensu stricto is truly marine, morphologically distinct, and unsequenced; finding and sequencing the generitype for Calothrix remains as the most important and unfinished task in the revision of the Calotrichaceae.     

Phylogenetic Framework for Trema (Celtidaceae)

We used ITS and trnL sequence data, analyzed separately and combined by MP, to explore species relationships and concepts in Trema (Celtidaceae), a pantropical genus of pioneer trees. Whether Trema is monophyletic or includes Parasponia is still unresolved. Three clades within Trema received moderate to high support, one from the New World and two from the Old World, but their relationships were not resolved. In the New World, specimens of T. micrantha formed two groups consistent with endocarp morphology. Group I, with smaller brown endocarps, is a highly supported clade sister to T. lamarckiana. Group II, with larger black endocarps, is poorly resolved with several subclades, including the highly supported T. integerrima clade. Both Old World clades contain Asian and African species, with three or more species in each region. Trema orientalis is not monophyletic: specimens from Africa formed a highly supported clade sister to T. africana, while those from Asia were sister to T. aspera from Australia.     

POLYOL PATTERNS IN BIOFILM‐FORMING AEROTERRESTRIAL GREEN ALGAE (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

The distribution of polyols was examined for the first time in 34 green algal strains from four different clades belonging to the class Trebouxiophyceae, which dominate aeroterrestrial biofilms of many regions. Sorbitol was detected in representatives of the Prasiola clade, while ribitol was present in the Elliptochloris and Watanabea clades. Apatococcus lobatus (Chodat) J. B. Petersen contained erythritol in addition to ribitol. Polyols are considered as effective stress metabolites exerting multiple protective functions in metabolism and hence mainly occur in organisms colonizing extreme environments. In contrast, members of the Chlorella clade, which mainly occur in aquatic habitats, did not contain polyols. Thus, the constitutive presence of specific polyols facilitates a differentiation between species of the Prasiola clade from the Elliptochloris and Watanabea clades, respectively, and further allows differentiation of morphologically converging taxa.     

PHYLOGENETIC ANALYSIS OF 32 STRAINS OF VAUCHERIA (XANTHOPHYCEAE) USING THE rbcL GENE AND ITS TWO FLANKING SPACER REGIONS1

The complete rbcL gene was sequenced for 21 species and 32 strains of Vaucheria and for five other Xanthophyceae (Asterosiphon dichotomus (Kützing) Rieth, Botrydium becharianum Vischer, B. cystosum Vischer, B. stoloniferum Mitra, Tribonema intermixtum Pascher). The psbA‐rbcL spacer, upstream of the rbcL gene, and the RUBISCO spacer between the rbcL and rbcS genes were also completely sequenced for the Vaucheria strains and Asterosiphon. The psbA‐rbcL spacer was the most variable region that was sequenced, and only the 3′ end of the spacer could be aligned. Phylogenetic analyses (maximum parsimony, neighbor joining, and maximum likelihood) were conducted using the DNA sequence and the amino acid sequence for the rbcL gene, and a second analysis was conducted using a portion of the psbA‐rbcL spacer +rbcL gene + RUBISCO spacer. All analyses showed that Vaucheria species formed monophyletic clades that corresponded with morphologically based subgeneric sections, including the section Racemosae. Species producing a gametophore (= fruiting branch, bearing both an antheridium and oogonium) formed a monophyletic clade in all analyses. The nongametophore species sometimes formed a monophyletic clade but other times formed a basal grade. Pair‐wise comparisons of nucleotides and amino acids showed that for some species, numerous nucleotide changes resulted in relatively few amino acid changes. Consequently, phylogenetic analysis of the amino acids produced numerous trees, which in a strict consensus tree resulted in numerous polychotomies. An original strain of V. terrestris that was deposited in two culture collections over 25 years ago had identical sequences, suggesting no rapid change was occurring in the sequenced regions. Two strains of V. prona, isolated from Europe and North America, had identical sequences. Other species, for which two or more strains were examined, had different sequences. These results suggest that cryptic species complexes exist within Vaucheria because the rbcL gene is a conservative gene that is identical in other protists.     

PHYLOGEOGRAPHIC STUDIES IN THE TROPICAL SEAWEED CLADOPHOROPSIS MEMBRANACEA (CHLOROPHYTA,ULVOPHYCEAE) REVEAL A CRYPTIC SPECIES COMPLEX1

The seaweed Cladophoropsis membranacea (Hofman Bang ex. C. Agardh) Børgesen is a widely distributed species on coral reefs and along rocky coastlines throughout the tropics and subtropics. In a recent population‐level survey openface>1600 individuals with eight microsatellite loci, a number of isolates from biogeographically disjunct locations could not be amplified for any of the loci. Nonamplifiable and amplifiable isolates co‐occurred within the Canary Islands, Cape Verde Islands, and in the Caribbean. These unexpected results led to question whether or not C. membranacea is a single species. Phylogenetic relationships were evaluated using rDNA ITS1 and ITS2 sequence comparisons from 42 isolates sampled from a subset of 30 of the 66 locations. Four well‐supported clades were identified. Sequence divergence within clades was <1%, whereas between‐clade divergence was 2%–3%. Intraindividual variation was extremely low with no effects on the analysis. A strong, but imperfect, correspondence was found between ITS clades and amplifiable microsatellite loci. It is concluded that C. membranacea consists of three cryptic species. Using Pacific isolates as an outgroup, the most basal clade included the Central Canary Islands, Cape Verde, and Bonaire (Caribbean) isolates and thus spanned the widest latitude. Two derived sister clades consisted of a southern transtropical group stretching across the SE Caribbean to the Cape Verde Islands and African coast (but not the Canary Islands) and a NE‐Canary Island‐Mediterranean clade that also included the Red Sea. The detection of overlapping biogeographic distributions highlights the importance of ecotypic differentiation with respect to temperature and the importance of shifting sea surface isotherms that have driven periodic extinctions and recolonizations of the Canary Islands—a crossroads of marine floral exchange—since the last glacial maximum.     

Morphology evolution and molecular phylogeny of Pestalotiopsis (Coelomycetes) based on ITS2 secondary structure

The internal transcribed spacer 2 (ITS2) located between the 5.8S and 28S genes of the nuclear ribosomal gene cistron is conserved at the level of secondary structure rather than primary sequence. Within the fungal genus Pestalotiopsis, there were two types of ITS2 sequence patterns, and hence secondary structures, which were supported by high bootstrap values in phylogenies based on the ProfDist distance and Profile neighbor-joining algorithms. Pestalotiopsis consists of two groups that differ in color intensity of the spore as measured by optical density (OD) in three median cells of conidia comprised of five cells with one basal and two to four apical appendages. OD was quantified using a novel method with the publicly available software, Image J. OD values of species within one clade were high (dark OD >0.6), while OD values of species in the other clade were low (pale OD <0.6). However, knobbed-tipped appendages, which have been used to classify species of Pestalotiopsis, were observed in both clades. In the dark clade, knobbed-tipped appendage strains aggregated in one subclade, but in the pale group, these strains did not aggregate.     

A COMBINED 18S rDNA AND rbcL PHYLOGENETIC ANALYSIS OF CHLOROMONAS AND CHLAMYDOMONAS (CHLOROPHYCEAE,VOLVOCALES) EMPHASIZING SNOW AND OTHER COLD‐TEMPERATURE HABITATS1

An extensive phylogenetic analysis of the biflagellate genera, Chlamydomonas Ehrenberg and Chloromonas Gobi emend. Wille, was undertaken using 18S rDNA and rbcL gene sequence analysis. Emphasis was placed on 21 cold‐tolerant taxa of which 10 are from snow. These taxa occurred in four distinct clades each in the 18S rDNA and rbcL phylogenies, and when taken together suggest at least five distinct origins in cold habitats. Most of these taxa occur in a single clade (A), and all snow species occurred in this clade. In the rbcL and combined rbcL–18S rDNA analyses, the snow taxa fell into three groups. Two groups occurred in subclade 1: Chlamydomonas augustae Skuja CU, Chlamydomonas augustae UTEX, and Chlamydomonas sp.‐A and Chloromonas clathrata Korshikov, Chloromonas rosae Ettl CU, and Chloromonas rosae v. psychrophila var. nov. The third snow group, subclade 2, included three species with unique cell divisions, Chloromonas brevispina (Fritsch) Hoham, Roemer et Mullet, Chloromonas pichinchae (Lagerheim) Wille, and Chloromonas sp.‐D, and the basal Chloromonas nivalis (Chodat) Hoham et Mullet with normal cell divisions. This suggests that the snow habitat has been colonized at least twice and possibly three times in the history of these biflagellates. In the 18S rDNA tree, one cold‐tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa (Pascher et Jahoda) Gerloff et Ettl. In the rbcL tree, three cold‐tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa, Chloromonas sp.‐ANT1, and Chloromonas sp.‐ANT3. These results support previous findings that pyrenoids have been gained and lost several times within this complex.     

INTRAGENOMIC NUCLEOTIDE POLYMORPHISM AMONG SMALL SUBUNIT (18S) RDNA PARALOGS IN THE DIATOM GENUS SKELETONEMA (BACILLARIOPHYTA)1

Morphological features of the siliceous cell wall traditionally have been used to diagnose and classify species of diatoms, though an increasing number of studies distinguish new species, in part, by phylogenetic analysis of rDNA sequences. Intragenomic sequence variation is common among the hundreds to thousands of rDNA cistrons present within a genome, and this variation has strong potential to obscure species boundaries based on rDNA sequences. We screened six Skeletonema culture strains for intragenomic nucleotide polymorphisms in the small subunit (SSU) rDNA gene and found that all strains had polymorphic sites, with proportions ranging from 0.57% to 1.81%. In all cases, transitions accounted for more than 70% of nucleotide differences at polymorphic sites. Polymorphic sites were split nearly evenly in the SSU rRNA molecule between the base‐paired regions of helices (52%) and the unpaired regions of loops and bulges (48%). Phylogenetic analysis showed that SSU rDNA genotypes were monophyletic for two of the six culture strains examined. Genotypes from the other four culture strains either showed little or no phylogenetic structure compared with genotypes of other conspecific culture strains or had phylogenetic structure that was incongruent with existing species boundaries. Moderate to strong support for monophyly was recovered for four of the seven species included in the analysis. Phylogenetic results combined with the low sequence divergence of SSU rDNA genotypes within species suggest that concerted evolution has not proceeded to completion in these species and/or that the rate at which variation is being generated exceeds the rate at which concerted evolution is expunging variation.