Russell L. Chapman

Phylogenetic Relationships of the "Green Algae" and "Bryophytes"

Considerable progress has been made recently, based on classical morphological characters and newly described ultrastructural features, in understanding the phylogenetic relationships of the tracheophytes to the green algae and bryophytes. Recent technological advances in molecular biology, particularly the advent of the polymerase chain reaction (PCR), have allowed nucleotide sequence data relevant to such large-scale phylogenetic questions to accumulate, especially ribosomal RNA gene sequences (both the large and small subunits) from the nucleus and the chloroplast. We present synthetic cladistic analyses of the green plants that combine and compare available morphological and molecular data sets

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.

PHYLOGENETIC RELATIONSHIPS OF BRYOPHYTES INFERRED FROM NUCLEAR-ENCODED RRNA GENE SEQUENCES

We investigate phylogenetic relationships among hornworts, liverworts and mosses, and their relationships to other green plant groups, by analysis of nucleotide variation in complete 18s rRNA gene sequences of three green algae, two hornworts, seven liverworts, nine mosses, and six tracheophytes. Parsimony and maximum-likelihood analyses yield a single optimal tree in which the hornworts are resolved as the basal group among land plants, and the liverworts and mosses are sister taxa that together form the sister clade to the tracheophytes. This phylogeny is internally robust as indicated by decay indices and by comparison (using both parsimony and likelihood criteria) to topologies representing five alternative hypotheses of bryophyte relationships. We discuss some possible reasons for differences between the phylogeny inferred from the rRNA data and those inferred from other character sets.

PHYLOGENY OF CHLAMYDOMONAS (CHLOROPHYTA) BASED ON CLADISTIC ANALYSIS OF NUCLEAR 18S rRNA SEQUENCE DATA1

The genus Chlamydomonas Ehrenberg may contain as many as 450 described species. Morphological, physiological and molecular data show that variation among some Chlamydomonas species can he great, leading to speculation that multiple, generic‐level lineages exist within this genus. The most recent systematic studies of Chlamydomonas have led to proposals of nine distinct morphological and 15 distinct sporangial autolysin groups. Partial sequences from the nuclear small subunit rRNAs from 14 Chlamydomonas species representing 12 autolysin and four morphological groups, and from three flagellates thought to he related to Chlamydomonas were determined in a phylogenetic study of relationships among these algae. Sequence comparisons among some Chlamydomonas species revealed differences comparable to the sequence divergence between soybeans and cycads. Cladistic analysis of the sequence data suggests that multiple lineages exist among species of Chlamydomonas. Some of these lineages represent alliances of both Chlamydomonas and non‐Chlamydomonas taxa; thus, the current taxonomy does not reflect natural, or monophyletic, groups. Collectively, these lineages may represent distinct families or even orders.

THE PLEURASTROPHYCEAE AND MICROMONADOPHYCEAE: A CLADISTIC ANALYSIS OF NUCLEAR rRNA SEQUENCE DATA1

Partial sequences from the nuclear‐encoded 18S and 26S ribosomal RNA molecules from representatives of the five classes of Chlorophyta sensu Mattox and Stewart (1984) were analyzed cladistically in a study of the phylogenetic relationships among the Micromonadophyceae, Pleurastrophyceae, and other green algae. The sequence data indicate that the Micromonadophyceae (= Prasinophyceae) is not monophyletic but comprises at least three lineages occupying a basal position among the green algae. Though the Pleurastrophyceae and the Ulvophyceae both possess counter‐clockwise basal body orientations, the sequence data indicate that the Pleurastrophyceae is the sister group to the Chlorophyceae. The molecular data alone do not resolve the monophyly of the Pleurastrophyceae or the Ulvophyceae; however, a combined data set of molecular and non‐molecular characters support a monophyletic Pleurastrophyceae. Analyses with user‐defined tree topologies and the bootstrap method of character resampling indicate that the relationships shown in the most parasimonious cladograms are well supported by the character data.

A PHYLOGENETIC STUDY OF THE CORALLINALES (RHODOPHYTA) BASED ON NUCLEAR SMALL‐SUBUNIT rRNA GENE SEQUENCES

Conflicting classifications for the Corallinales were tested by analyzing partial sequences for the nuclear small‐subunit ribosomal RNA (SSU) gene of 35 species of coralline algae. Parsimony and likelihood analyses of these data yielded congruent hypotheses that are inconsistent with classifications for the group that include as many as eight subfamilies. Four major clades are resolved within the order, including the early‐diverging Sporolithaceae as well as the Melobesioideae and Corallinoideae. The fourth clade, which is supported robustly, includes both nongeniculate and geniculate species classified in the subfamilies Mastophoroideae, Metagoniolithoideae, Lithophylloideae, and Amphiroideae. Molecular and morphological data support the proposal that the latter two subfamilies are sister taxa. Although relationships among some genera are not resolved clearly, the order of branching of taxa among and within the four principal lineages is concordant with paleontological evidence for the group. Relationships inferred among genera within each of the clades is discussed. Seven morphological characters delimiting higher taxonomic groups within the order were combined with the sequence data, analyzed, and optimized onto the resulting tree(s). Except for the presence or absence of genicula, all other characters were found to be phylogenetically informative. Genicula are nonhomologous structures that evolved independently in the Amphiroideae, Corallinoideae, and Metagoniolithoideae. The phenetic practice of separating coralline algae into two categories solely on the basis of the presence or absence of genicula does not accurately reflect the evolutionary history of the group.

Phylogeny of the colonial green flagellates: a study of 18S and 26S rRNA sequence data.

A study of phylogenetic relationships of the colonial green algal flagellates based on nuclear 18S and 26S rRNA sequence data suggests that the colonial habit has had at least two independent origins. All colonial taxa included in the analysis, except Stephanosphaera, are allied in a clade with Chlamydomonas reinhardtii and other Chlamydomonas taxa ascribed to the Euchlamydomonas group by Ettl. In contrast, Stephanosphaera is allied with other unicellular flagellates including Haematococcus. Comparison of the 18S and 26S data shows that the two sets of data yield different results following cladistic analysis. The 18S data provide the principal signal that supports the more basal divergences, but the data do not unambiguously address relationships among taxa in the clade that includes most colonial flagellates and Chlamydomonas taxa representative of the Euchlamydomonas group (sensu Ettl). In contrast, the 26S data have fewer informative sites that support basal divergences than the 18S data, but provide much of the signal that supports resolution of taxa in the colonial flagellate clade in an analysis of the combined 18S and 26S rRNA sequence data. Additional sequence data from the 26S molecule and additional taxa may reduce the topological ambiguity inferred from the sequence data for the colonial flagellates. Alternatively, an ancient and rapid radiation of taxa in the colonial lineage could account for the topological ambiguity. Despite some unresolved questions of relationships, cladistic analysis of the combined data sets provides some robustly supported concepts of evolution in these flagellates.

Molecular Systematics of the Green Algae

Ranging from unicells to complex “plantlike” organisms that are adapted to habitats from subaerial or terrestrial to freshwater or marine, the green algae represent a diversity of life forms that offer a daunting challenge in the search for shared morphological characters. The ultrastuctural techniques that fueled the 1970s and early 1980s revolution in algal systematics revealed a suite of new morphological characters, but many were not global (i.e., present in all of the taxa). Controversy over the interpretation of the importance of ultrastructural features (e.g., of cell division versus flagellar apparatus) led to conflicting hypotheses. Also, different researchers studied different details of different taxa, and thus a data matrix reporting a complete set of morphological and ultrastructural characters over a wide range of algal taxa was not available. Thus, it is no wonder that many researchers interested in unraveling the mystery of green algal phylogeny embraced molecular systematics, hoping that its early promise of relatively simple access to ample global characters would lead, finally, to a “true” phylogeny. The extent to which this promise has been fulfilled, or is likely to be fulfilled, is the subject of this chapter.

PHYLOGENY OF CARTERIA (CHLOROPHYCEAE) INFERRED FROM MOLECULAR AND ORGANISMAL DATA1

Comparative ultrastructural data have shown that at least two distinct groups exist within Carteria. Similarly, interpretations of variation in gross morphological features have led to the discovery of morphologically distinct groups within the genus. Partial sequences from the nuclear‐encoded small‐ and large‐subunit ribosomal RNA molecules of selected Carteria taxa were studied as a means of 1) testing hypotheses that distinct groups of species exist within the genus and 2) assessing monophyly of the genus. Parsimony analysis of the sequence data suggests that three Carteria species, C. lunzensis, C. crucifera, and C. olivieri, form a monophyletic group that is the basal sister group to all other ingroup flagellate taxa (including species of Chlamydomonas, Haematococcus, Stephanosphaera, Volvox, and Eudorina). Two other Carteria taxa, C. radiosa and Carteria sp. (UTEX isolate LB 762), form a clade that is the sister group to a clade that includes Haematococcus spp., Chlamydomonas spp., and Stephanosphaera. Thus, the sequence data support the interpretations of ultrastructural evidence that described two distinct Carteria lineages. Moreover, the sequence data suggest that these two Carteria groups do not form a monophyletic assemblage. Parsimony analysis of a suite of organismal (morphological, ultra‐structural, life history, and biochemical) character data also suggest two distinct lineages among the five Carteria taxa; however, the organismal data are ambiguous regarding monophyly of these Carteria taxa. When the two independent data sets are pooled, monophyly of Carteria is not supported; therefore, the weight of available evidence, both molecular and organismal, fails to support the concept of Carteria as a natural genus.

Phylogeny of chloromonas (chlorophyceae): A study of 18S ribosomal RNA gene sequences

The unicellular, biflagellate genus Chloromonas differs from its ally, Chlamydomonas, primarily by the absence of pyrenoids in the vegetative stage of the former. As with most green flagellate genera, little is known about phylogenetic affinities within and among Chloromonas species. Phylogenetic analyses of nuclear‐encoded small‐subunit ribosomal RNA gene sequences demonstrate that a sampling of five Chloromonas taxa, obtained from major culture collections, do not form a monophyletic group. However, only three of these isolates, Chloromonas clathrata, Chloromonas serbinowi, and Chloromonas rosae, are diagnosable morphologically as Chloromonas species by the absence of a pyrenoid in the vegetative stage. The three diagnosable Chloromonas taxa form an alliance with two pyrenoid‐bearing chlamydomonads, Chlamydomonas augustae and Chlamydomonas macrostellata. With the exception of Chloromonas serbinowi, which represents the basal lineage within the clade, each of the diagnosable Chloromonas taxa and their pyrenoidbearing Chlamydomonas allies were isolated originally from mountain soils, snow, or cold peat. These observations suggest that habitat, independent of pyrenoid status, may be most closely linked to the natural history of this clade of chlamydomonad flagellates.