178 Molecular Systematics of the Ulvellaceae (Ulvales, Ulvophyceae) Inferred from Nuclear and Chloroplast DNA Sequences

The traditional use of thallus morphology to define lineages within the Ulvophyceae proved problematic because parallelism and convergence obscured natural relationships. The advent of transmission electron microscopy led to a re-evaluation of groups on the basis of zoid characters. Within TEM-defined lineages, the Ulotrichales and Ulvales were considered closely related orders, and molecular evidence has substantiated this hypothesis. In our molecular systematic studies of ulvophyceous green algae, we have broadened taxon sampling among these two orders. We show that the Ulvales and Ulotrichales are indeed monophyletic orders. However, the phylogenetic placement of species in the genera Bolbocoleon, Ctenocladus, Phaeophila, Pseudendoclonium and Acroblaste indicates that one or more additional lineages, placed between Ulvales and Ulotrichales in gene sequence trees, must be recognized. These species may constitute a separate order (Ctenocladales) consisting of multiple families. Our findings will be compared with taxonomic concepts based on morphological criteria.     

The Distribution of Elongation Factor-1 Alpha (EF-1α), Elongation Factor-Like (EFL), and a Non-Canonical Genetic Code in the Ulvophyceae: Discrete Genetic Characters Support a Consistent Phylogenetic Framework

ABSTRACT. The systematics of the green algal class Ulvophyceae have been difficult to resolve with ultrastructural and molecular phylogenetic analyses. Therefore, we investigated relationships among ulvophycean orders by determining the distribution of two discrete genetic characters previously identified only in the order Dasycladales. First, Acetabularia acetabulum uses the core translation GTPase Elongation Factor 1α (EF-1α) while most Chlorophyta instead possess the related GTPase Elongation Factor-Like (EFL). Second, the nuclear genomes of dasycladaleans A. acetabulum and Batophora oerstedii use a rare non-canonical genetic code in which the canonical termination codons TAA and TAG instead encode glutamine. Representatives of Ulvales and Ulotrichales were found to encode EFL, while Caulerpales, Dasycladales, Siphonocladales, and Ignatius tetrasporus were found to encode EF-1α, in congruence with the two major lineages previously proposed for the Ulvophyceae. The EF-1α of I. tetrasporus supports its relationship with Caulerpales/Dasycladales/Siphonocladales, in agreement with ultrastructural evidence, but contrary to certain small subunit rRNA analyses that place it with Ulvales/Ulotrichales. The same non-canonical genetic code previously described in A. acetabulum was observed in EF-1α sequences from Parvocaulis pusillus (Dasycladales), Chaetomorpha coliformis , and Cladophora cf. crinalis (Siphonocladales), whereas Caulerpales use the universal code. This supports a sister relationship between Siphonocladales and Dasycladales and further refines our understanding of ulvophycean phylogeny.     

PHYLOGENETIC POSITION OF BOLBOCOLEON PILIFERUM (ULVOPHYCEAE,CHLOROPHYTA): EVIDENCE FROM REPRODUCTION,ZOOSPORE AND GAMETE ULTRASTRUCTURE,AND SMALL SUBUNIT RRNA GENE SEQUENCES1

Aspects of the reproduction of Bolbocoleon piliferum N. Pringsheim, a common, small, filamentous, endophytic marine green alga, were examined by LM and TEM. These observations were combined with phylogenetic analysis of nuclear‐encoded small subunit rRNA gene sequences to assess the phylogenetic position of B. piliferum. Quadriflagellate zoospores and planozygotes derived from fusion of isogametes yielded plants with identical morphology. Zoosporangia and gametangia divided by sequential cleavages. Plugs at the apices of zoosporangia and gametangia formed during development; tubes were found at zoosporangial and gametangial apices after swarmer release. Flagellar apparatuses of zoospores and gametes were similar to those of algae in the Ulvales (Ulvophyceae), except that terminal caps were entire rather than bilobed and rhizoplasts and “stacked” microtubular root configurations were absent. Structures associated with planozygotes were identical to those observed in other algae currently assigned to Ulotrichales and Ulvales. Molecular phylogenetic analyses placed B. piliferum within the Ulvophyceae, at the base of a clade that contains representatives of the families Ulvaceae, Ulvellaceae, and Kornmanniaceae. The results support an earlier hypothesis that B. piliferum constitutes a distinct lineage. Analyses including Kornmanniaceae recover monophyletic Ulotrichales and Ulvales, whereas analyses omitting the Kornmanniaceae indicate that Ulotrichales is paraphyletic. The structures associated with gamete fusion are conserved within Ulotrichales and Ulvales and perhaps more widely within Chlorophyta.     

Flip‐flop organization in the chloroplast genome of Capsosiphon fulvescens (Ulvophyceae,Chlorophyta)

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561 bp in length with 31.3% GC content that contained 108 genes including 77 protein‐coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip‐flop organization. The flip‐flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C. fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C. fulvescens genome, we found three major conflicting signals for C. fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70‐gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data‐rich approach (i.e., organelle genome data) from broader taxon sampling.     

The emergence of a new chlorophytan system,and Dr. Kornmann's contribution thereto

In traditional chlorophytan systems the organizational level was the primary character for the distinction of main groups (classes and orders). For instance, in Fott (1971), the flagellate level corresponds with the Volvocales, the coccoid level with the Chlorococcales, the filamentous level with the Ulotrichales, the siphonocladous level with the Siphonocladales, and the siphonous level with the Bryopsidales. The new system presented here is an elaboration and emendation of recently proposed taxonomies and their underlying phylogenetic hypotheses, and it is mainly based on ultrastructural features which have become available over the last 15 years. The following criteria are used for the distinction of classes and orders: (1) architecture of the flagellate cell (flagellate cells are considered as the depositories of primitive characters); (2) type of mitosis-cytokinesis; (3) place of meiosis in the life history and, consequently, the sexual life history type; (4) organizational level and thallus architecture; (5) habitat type (marine versus feshwater and terrestrial); (6) chloroplast type. The following classes are presented: Prasinophyceae, Chlamydophyceae, Ulvophyceae (orders Codiolales, Ulvales, Cladophorales, Bryopsidales, Dasycladales), Pleurastrophyceae (?), Chlorophyceae s.s. (orders Cylindrocapsales, Oedogoniales, Chaetophorales), Zygnematophyceae, Trentepohliophyceae, Charophyceae (orders Klebsormidiales, Coleochaetales, Charales). The new system no longer reflects the traditional hypothesis of a stepwise evolutionary progression of organizational levels in which the flagellate level represents the most primitive lineage, the coccoid and sarcinoid levels lineages of intermediate derivation, and the filamentous, siphonocladous and siphonous levels the most derived lineages. Instead, it is now hypothesized that these levels have arisen over and over again in different chlorophytan lineages which are primarily characterized by their type of flagellate cell. The flagellate green algal classes Prasinophyceae (with organic body scales) and Chlamydophyceae probably represent bundles of highly conservative lineages that diverged very long ago. Consequently, extant genera and species in these classes can be expected to have emerged long ago. Fossil evidence points to a minimum age of 600 Ma of certain extant Prasinophycean genera, and molecular evidence to a minimum age of 400–500 Ma of a fewChlamydomonas species. On the contrary, the most derived “green algal” lineage, the Angiosperms, can be expected to consist of, on average, much younger genera and species. Fossil evidence points to a minimum age of genera of 5–60 Ma. Lineages of intermediate evolutionary derivation (Ulvophyceae, Chlorophyceae, Charophyceae) can be expected to encompass genera and species of intermediate age. Fossil and (limited) molecular evidence point to a minimum age of 230–70 Ma of extant genera in Bryopsidales, Dasycladales and Cladophorales (Ulvophyceae) and of 250–80 Ma of extant genera in Charales (Charophyceae).     

MOTILE CELL ULTRASTRUCTURE AND THE CIRCUMSCRIPTION OF THE ORDERS ULOTRICHALES AND ULVALES (ULVOPHYCEAE,CHLOROPHYTA)

We have examined the motile cell ultrastructural features of several green algal species having filamentous or foliose thallus morphology and probable affinities with the Ulvophyceae, and compared them with the structural, reproductive, and life history features known for these taxa. We separate the algae studied into the orders Ulotrichales and Ulvales on the basis of consistent variations in terminal cap and proximal sheath structure that correlate well with life history patterns and certain features of sporangial and gametangial structure and development. Body scales are present only in certain members of the Ulotrichales. Both orders encompass a variety of thallus forms, demonstrating parallel evolution of thallus morphology. Flagellar apparatus features common to all the motile cells examined include 180° rotational symmetry, counterclockwise absolute orientation, the positioning of the basal bodies in an apical papilla, and the presence of one or more sets of striated bands associated with the X rootlets. Additional features that are usually present include basal body overlap and orientation roughly perpendicular to the long axis of the cell during forward swimming, striated distal fibers, and a single, striated, microtuble-associated component underlying each two-membered rootlet. These similarities indicate to us that the two groups are closely related members of the Ulvophyceae. We suggest that the Ulotrichales is the most primitive ulvophyceous assemblage known, but that all groups studied have advanced features relative to those supposed to have been present in the ancestral members of the Ulvophyceae.     

PHYLOGENETIC SYSTEMATICS OF THE ULVACEAE (ULVALES,ULVOPHYCEAE) USING CHLOROPLAST AND NUCLEAR DNA SEQUENCES1

Systematic hypotheses for the Ulvaceae were tested using phylogenetic analysis of sequences for the gene encoding the large subunit of RUBISCO, small subunit rDNA and a combined data matrix. Representatives of eight putative ulvaceous genera and twelve additional taxa from the Ulvophyceae and Trebouxiophyceae were included in analyses using maximum parsimony and maximum likelihood criteria. Molecular data supported hypotheses for the Ulvaceae that are based on the early development of vegetative thalli and motile cell ultrastructure. Ulvaceae sensu Floyd and O'Kelly, including Percursaria Bory de Saint‐Vincent, Ulvaria Ruprecht and a complex of closely related species of Chloropelta Tanner, Enteromorpha Link and Ulva L. was supported; however, monophyly of Enteromorpha and Ulva was not supported. The Ulvales and Ulotrichales sensu Floyd and O'Kelly were monophyletic. Blidingia Kylin and Kornmannia Bliding were allied with the former and Capsosiphon Gobi with the latter, although relationships among these and other taxa in these orders remain uncertain. The Ulvales are characterized by an isomorphic life history pattern, gametangia and sporangia that are identical in structure and development, motile cells with bilobed terminal caps and proximal sheaths consisting of two equal subunits. Method of motile cell release and the gross morphology of vegetative thalli are not systematically reliable characters.     

PHYLOGENY OF THE ULVOPHYCEAE (CHLOROPHYTA): CLADISTIC ANALYSIS OF NUCLEAR-ENCODED rRNA SEQUENCE DATA1

Cladistic analysis of nuclear-encoded rRNA sequence data provided us with the basis for some new hypotheses of relationships within the green algal class Ulvophyceae. The orders Ulotrichales and Ulvales are separated from the clade formed by the remaining orders of siphonous and siphonocladous Ulvophyceae (Caulerpales, Siphonocladales /Cladophorales [S/C] complex, and the Dasycladales), by the Chlorophyceae and Pleurastrophyceae. Our results suggest that the Ulvophyceae is not a monophyletic group. Examination of inter- and intra-ordinal relationships within the siphonous and siphonocladous ulvophycean algae revealed that Cladophora, Chaetomorpha, Anadyomene, Microdictyon, Cladophoropsis and Dictyosphaeria form a clade. Thus the hypothesis, based on ultrastructural features, that the Siphonocladales and Cladophorales are closely related is supported. Also, the Caulerpales is a monophyletic group with two lineages; Caulerpa, Halimeda, and Udotea comprise one, and Bryopsis and Codium comprise the other. The Dasycladales (Cymopolia and Batophora) also forms a clade, but this clade is not inferred to be the sister group to the S/C complex as has been proposed. Instead, it is either the sister taxon to the Caulerpales or basal to the Caulerpales and S/C clade The Trentepohliales is also included at the base of the siphonous and siphonocladous ulvophycean clade. The Pleurastrophyceae, which, like the Ulvophyceae, posses a counter-clockwise arrangement of flagellar basal bodies, are more closely related to the Chlorophyceae than to the Ulvophyceae based on rRNA sequences. Thus, the arrangement of basal bodies does not diagnose a monophyletic group. Previously reported hypotheses of phylogenetic relationships of ulvophycean algae were tested. In each case, additional evolutionary steps were required to obtain the proposed relationships. Relationships of ulvophycean algae to other classes of green algae are discussed.     

Molecular phylogenetic analyses of the Japanese Ulva and Enteromorpha (Ulvales, Ulvophyceae), with special reference to the free-floating Ulva

In order to elucidate the species composition of free‐floating Ulva that cause green tide in several bays in Japan, and to clarify the generic status of Ulva and Enteromorpha (Ulvales, Ulvophyceae), the nuclear encoded internal transcribed spacer (ITS) region including the 5.8S gene and the plastid encoded large subunit of ribulose‐1, 5‐bisphosphate carboxylase/ oxgenase (rbcL) gene sequences for 15 species were determined. Both ITS and rbcL analyses indicate that free‐floating Ulva samples are divided into four different lineages that correspond to Ulva lactuca Linnaeus, U. pertusa Kjellman, U. armoricana Dion etal. and U. fasciata Delile. These four species are distinguished by cell morphology including the arrangement of cells, the shape and size of cells and the position of chloroplasts. Molecular data also indicated that Ulva and Enteromorpha are not separated as respective monophyletic groups within a large monophyletic clade and congeneric as shown by previous molecular studies using the ITS sequences alone. This strongly suggests that these genera are congeneric and Enteromorpha should be reduced to the synonym of Ulva.     

MOLECULAR PHYLOGENY OF THE UPRIGHT ERYTHROPELTIDALES (COMPSOPOGONOPHYCEAE,RHODOPHYTA): MULTIPLE CRYPTIC LINEAGES OF ERYTHROTRICHIA CARNEA1

The phylogeny of morphologically simple algae is problematic due to insufficient morphological characters to aid in distinguishing species and relationships. The problem is further compounded because multiple evolutionary lineages of morphologically similar species occur in most well‐sampled biogeographic locations; therefore, location cannot be used as a proxy for species. The phylogeny of the upright members of the Erythropeltidales is partially clarified by combining molecular data, unialgal culture observations, and worldwide sampling. Our results show that there are several well‐supported lineages within the Erythropeltidales with only two morphologically recognizable taxa at present. The first is the genus Porphyrostromium, with a well‐developed basal crust, which includes two Erythrotrichia species (Porphyrostromium ligulatum comb. nov. and Porphyrostromium pulvinatum comb. nov.). The second is the branched species Erythrotrichia welwitschii (Rupr.) Batters. There are also six strongly supported Erythrotrichia carnea–like lineages. While not completely satisfactory, we propose that one lineage (lineage 2) with samples close to the type locality be designated as E. carnea with a specific isolate as an epitype. The lack of morphology to differentiate the other lineages leads to a taxonomy based solely on gene sequencing and molecular phylogeny, with rbcL sequences differentiating the lineages proposed. We hold off on proposing more species and genera until more data and samples can be gathered.     

Phylogenetic studies of marsupials based on phosphoglycerate kinase DNA sequences

Phosphoglycerate kinase sequences were obtained for 313 aligned bases of 41 individuals from 39 marsupial species. In contrast to previous molecular analyses, the relationships suggested by these data show a high level of congruence with morphologically defined orders and families. Four main monophyletic lineages are recognizable. These are the monogeneric orders Microbiotheria (Dromiciops australis) and Notoryctemorphia (Notoryctes typhlops), a grouping of the American orders Didelphimorphia and Paucituberculata, and the Australasian species other than N. typhlops. Within the Australasian lineage, there are again four main monophyletic groups; the Dasyuridae, two peramelemorph (bandicoot) lineages (one comprised of pseudogene sequences) and the Diprotodontia. This topology is not greatly affected by the exclusion of pseudogenes except that a clade of syndactylous species (Peramelemorphia plus Diprotodontia) is recovered. Two other peramelemorph pseudogenes have inserts of about 1 kb with high levels of similarity to LINE 1 elements. The Diprotodontia is notable for its relative lack of intersequence variation in comparison to the Dasyuromorphia.     

BOOM AND BUST: ANCIENT AND RECENT DIVERSIFICATION IN BICHIRS (POLYPTERIDAE: ACTINOPTERYGII), A RELICTUAL LINEAGE OF RAY‐FINNED FISHES

Understanding the history that underlies patterns of species richness across the Tree of Life requires an investigation of the mechanisms that not only generate young species‐rich clades, but also those that maintain species‐poor lineages over long stretches of evolutionary time. However, diversification dynamics that underlie ancient species‐poor lineages are often hidden due to a lack of fossil evidence. Using information from the fossil record and time calibrated molecular phylogenies, we investigate the history of lineage diversification in Polypteridae, which is the sister lineage of all other ray‐finned fishes (Actinopterygii). Despite originating at least 390 million years (Myr) ago, molecular timetrees support a Neogene origin for the living polypterid species. Our analyses demonstrate polypterids are exceptionally species depauperate with a stem lineage duration that exceeds 380 million years (Ma) and is significantly longer than the stem lineage durations observed in other ray‐finned fish lineages. Analyses of the fossil record show an early Late Cretaceous (100.5–83.6 Ma) peak in polypterid genus richness, followed by 60 Ma of low richness. The Neogene species radiation and evidence for high‐diversity intervals in the geological past suggest a “boom and bust” pattern of diversification that contrasts with common perceptions of relative evolutionary stasis in so‐called “living fossils.”     

Simulating and detecting autocorrelation of molecular evolutionary rates among lineages

Evolutionary timescales can be estimated from genetic data using phylogenetic methods based on the molecular clock. To account for molecular rate variation among lineages, a number of relaxed‐clock models have been developed. Some of these models assume that rates vary among lineages in an autocorrelated manner, so that closely related species share similar rates. In contrast, uncorrelated relaxed clocks allow all of the branch‐specific rates to be drawn from a single distribution, without assuming any correlation between rates along neighbouring branches. There is uncertainty about which of these two classes of relaxed‐clock models are more appropriate for biological data. We present an R package, NELSI, that allows the evolution of DNA sequences to be simulated according to a range of clock models. Using data generated by this package, we assessed the ability of two Bayesian phylogenetic methods to distinguish among different relaxed‐clock models and to quantify rate variation among lineages. The results of our analyses show that rate autocorrelation is typically difficult to detect, even when there is complete taxon sampling. This provides a potential explanation for past failures to detect rate autocorrelation in a range of data sets.     

Mitochondrial rate variation among lineages of passerine birds

The order Passeriformes comprises the majority of extant avian species. Analyses of molecular data have provided important insights into the evolution of this diverse order. However, molecular estimates of the evolutionary and demographic timescales of passerine species have been hindered by a lack of reliable calibrations. This has led to a reliance on the application of standard substitution rates to mitochondrial DNA data, particularly rates estimated from analyses of the gene encoding cytochrome b (CYTB). To investigate patterns of rate variation across passerine lineages, we used a Bayesian phylogenetic approach to analyse the protein‐coding genes of 183 mitochondrial genomes. We found that the most commonly used mitochondrial marker, CYTB, has low variation in rates across passerine lineages. This lends support to its widespread use as a molecular clock in birds. However, we also found that the patterns of among‐lineage rate variation in CYTB are only weakly related to the evolutionary rate of the mitochondrial genome as a whole. Our analyses confirmed the presence of mutational saturation at third codon positions across the protein‐coding genes of the mitochondrial genome, reinforcing the view that these sites should be excluded in studies of deep passerine relationships. The results of our analyses have provided information that will be useful for molecular‐clock studies of passerine evolution.     

Calibrating the molecular clock beyond cytochrome b: assessing the evolutionary rate of COI in birds

Estimating the age of species or their component lineages based on sequence data is crucial for many studies in avian evolutionary biology. Although calibrations of the molecular clock in birds have been performed almost exclusively using cytochrome b (cyt b), they are commonly extrapolated to other mitochondrial genes. The existence of a large, standardized cytochrome c oxidase subunit I (COI) library generated as a result of the DNA barcoding initiative provides the opportunity to obtain a calibration for this mitochondrial gene in birds. In this study we compare the evolutionary rate of COI relative to cyt b across ten different avian orders. We obtained divergence estimates for both genes from nearly 300 phylogenetically independent pairs of species through the analysis of almost 5000 public sequences. For each pair of species we calculated the difference in divergence between COI and cyt b. Our results indicate that COI evolves on average 14% slower than cyt b, but also reveal considerable variation both among and within avian orders, precluding the use of this value as a standard adjustment for the COI molecular clock for birds. Our findings suggest that this variation is partially explained by a clear negative relationship between the difference in divergence in these genes and the age of species. Distances for cyt b are higher than those for COI for closely related species, but the values become similar as the divergence between the species increases. This appears to be the result of a stronger pattern of negative time‐dependency in the rate of cyt b than in that of COI, a difference that could be related to lower functional constraints on a small number of sites in cyt b that allow it to initially accumulate mutations more rapidly than COI.     

A trans-Amazonian screening of mtDNA reveals deep intraspecific divergence in forest birds and suggests a vast underestimation of species diversity

The Amazonian avifauna remains severely understudied relative to that of the temperate zone, and its species richness is thought to be underestimated by current taxonomy. Recent molecular systematic studies using mtDNA sequence reveal that traditionally accepted species-level taxa often conceal genetically divergent subspecific lineages found to represent new species upon close taxonomic scrutiny, suggesting that intraspecific mtDNA variation could be useful in species discovery. Surveys of mtDNA variation in Holarctic species have revealed patterns of variation that are largely congruent with species boundaries. However, little information exists on intraspecific divergence in most Amazonian species. Here we screen intraspecific mtDNA genetic variation in 41 Amazonian forest understory species belonging to 36 genera and 17 families in 6 orders, using 758 individual samples from Ecuador and French Guiana. For 13 of these species, we also analyzed trans-Andean populations from the Ecuadorian Chocó. A consistent pattern of deep intraspecific divergence among trans-Amazonian haplogroups was found for 33 of the 41 taxa, and genetic differentiation and genetic diversity among them was highly variable, suggesting a complex range of evolutionary histories. Mean sequence divergence within families was the same as that found in North American birds (13%), yet mean intraspecific divergence in Neotropical species was an order of magnitude larger (2.13% vs. 0.23%), with mean distance between intraspecific lineages reaching 3.56%. We found no clear relationship between genetic distances and differentiation in plumage color. Our results identify numerous genetically and phenotypically divergent lineages which may result in new species-level designations upon closer taxonomic scrutiny and thorough sampling, although lineages in the tropical region could be older than those in the temperate zone without necessarily representing separate species. In-depth phylogeographic surveys are urgently needed to avoid underestimating tropical diversity, and the use of mtDNA markers can be instrumental in identifying and prioritizing taxa for species discovery.     

Diversity of the lichenized fungi in King George Island, Antarctica, revealed by phylogenetic analysis of partial large subunit rDNA sequences

Lichens are predominant and important components of flora in the terrestrial ecosystem of Antarctica. However, relatively few researches on the phylogenetic position of Antarctic lichen-forming fungi have been accomplished. In this study, partial sequences of nuclear large subunit rDNAs from 50 Antarctic specimens were obtained and the phylogeny was reconstructed. Antarctic lichen species were distributed among 4 orders, including the monophyletic order Agyrales, paraphyletic orders Pertusariales and Teloschistales, and polyphyletic order Lecanorales. Species diversity was highest in the order Lecanorales, followed by Teloschistales and Pertusariales. Based on the phylogeny and sequence similarity analyses, it is proposed that the taxonomy of Stereocaulon alpinum, Physcia caesia, Usnea aurantiacoatra, and Cladonia species should be revised by careful examination of their phenotypic and molecular characteristics. Six species known to be endemic to Antarctica, Catillaria corymbosa, Himantormia lugubris, Leptogium puberulum, Pertusaria pertusa, Rhizoplaca aspidophora, and Umbilicaria antarctica, formed unique lineages, implying independent origins in the Antarctic area.