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.     

植物分类学在化石珊瑚藻(珊瑚藻目,红藻门)中的应用

最近有人认为将化石藻类的分类归入现生藻类分类单元有利于珊瑚藻作为古环境的标志,便于理解该类群的演化。然而,这样分类可能很难,因为并不是所有现生藻类分类特征都能在化石种中保存下来。Sporolithacea科的钙化部分(独立或者聚集的孢子囊群)的出现,可以把它们与这个类群的另一个现生科Corallinaceae区别开,这个科在生殖窠中产生孢子囊。节片的有无,丝间细胞的联系类型,生殖窠中孢子囊释放的数目都是用来划分Coral1inaceae科的亚科的标准,在化石样品中也可以用合适的条件进行观察。在大多数情况下,对现生珊瑚藻类属的划分特征可以在化石藻类中鉴别出来,但在几种现生珊瑚藻没有钙化的生殖结构或发育特征。因此,它们生殖结构无法与相应的化石藻类进行对比,也不能进行化石藻类的分类。近年来的趋势认为生殖结构和发育特征是对现生珊瑚藻进行分类的优先鉴定标准,然而,某些特征的稳定性在属的划分上仍然存在争论。在许多情况下,现生藻类的分类标准特征都不能在化石中保存,对古生物化石的分类标准的最佳选择是在化石藻类中选择辅助的,并且可以识别的其它鉴定特征,或者应用非正式的比现生藻类代表定义更宽的属名。     

RE-ASSESSMENT OF THE TYPE COLLECTIONS OF CORALLINALEAN GENERA (CORALLINALES, RHODOPHYTA) DESCRIBED BY V. P. MASLOV

Abstract:  From 1935 to 1962 Maslov published two monographs and several other papers on the taxonomy of fossil calcareous algae of diverse ages from the large geographical area of the former USSR. Among many other taxa, he described five new genera ( Solenophyllum , Palaeophyllum , Mesolithon , Bicorium and Tomilithon , as a subgenus of Parachaetetes Deninger) which he attributed to the Corallinaceae, and the new genus Karpathia , which he included in the Squamariaceae. Type material of these taxa, except for Mesolithon , is housed in the Geological Institute of the Russian Academy of Sciences, Moscow. Re-examination of the genus types from a modern perspective of coralline algal taxonomy shows that Karpathia is a validly published genus of corallinaceans (subfamily Mastophoroideae) to which several Cenozoic fossil species can be assigned. In contrast, the holotype of P. elegans , the type species of Palaeophyllum , cannot be assigned to any subfamily within the Corallinaceae and its preserved features are inadequate for confidently delimiting a separate genus. Solenophyllum is a valid genus in which Palaeozoic representatives of Corallinales formerly attributed to Parachaetetes Deninger can be included. Tomilithon is considered a younger heterotypic synonym of Solenophyllum . The algal nature of the bioclasts in the type of Bicorium is uncertain.     

PARASITIC INTERACTIONS AND VEGETATIVE ULTRASTRUCTURE OF CHOREOAEMA THURETII (CORALLINALES,RHODOPHYTA)1

The monotypic coralline red alga, Choreonema thuretii (Bornet) Schmitz (Choreonematoideae), grows endophytically within three geniculate genera of the Corallinoideae. Although the thallus of Choreonema is reduced, lacks differentiated plastids, and is endophytic except for its conceptacles, its status as a parasite has been questioned because cellular connections to the host had not been ob served. Transmission electron microscopy, however, disclosed a previously undescribed type of parasitic interaction in which Choreonema interacts with its host through specialized cells known as lenticular cells. These small, lens-shaped cells are produced from the single file of host-penetrating vegetative cells. Pit plug morphology between vegetative and lenticular cells is polarized. Plug caps facing the vegetative cell have normal coralline morphology, while those facing the lenticular cell are composed of three layers. Regions of lenticular cells near host cells protrude toward the host cell; upon encountering the host cell wall, the prolrusion produces numerous finger-like fimbriate processes that make cellular connections with the host cell. Lenticular cells may extend several protrusions toward a host cell or penetrate more than one host cell; two or more lenticular cells may also penetrate the same host cell. The lack of secondary pit connections, cell fusions, and passage of parasitic nuclei suggest that this parasitic relationship may be evolutionarily older than previously reported cases of parasitism in red algae.     

LITHIUM CHLORIDE EXTRACTION OF DNA FROM THE SEAWEED PORPHYRA PERFORATA (RHODOPHYTA)1

Lithium chloride facilitates the softening of cell walls resulting in a simple, quick (2 h) method for DNA extraction from the red seaweed Porphyra perforata J. Agardh. A 5-min treatment of tissues in Lid at 55°C extracts DNA that is relatively free of the viscous polysaccharides and proteins that are usually coextracted in large amounts from cell walls and cytoplasm. This protocol does not require grinding of tissues, hydroxyapatite binding, cetyl trimethyl ammonium bromide treatments, enzymatic treatments, phenol extraction, or CsCl-gradient centrifugation. The resulting DNA is of sufficient quality to be used as a template for polymerase chain reaction amplification.     

PATTERNS OF REPRODUCTION,GENETIC DIVERSITY,AND GENETIC DIFFERENTIATION IN CALIFORNIA POPULATIONS OF THE GENICULATE CORALLINE ALGA LITHOTHRIX ASPERGILLUM (RHODOPHYTA)1

The reproductive composition and genetic diversity of populations of the red seaweed Lithothrix aspergillum Gray (O. Corallinales) were studied at three southern California sites (Shaw's Cove and Treasure Island, Laguna Beach; Indian Rock, Santa Catalina Island) and at a fourth site (Bodega Bay) located in northern California. Sexually reproducing populations were confined to southern California. Diploid individuals were numerically dominant over haploid (gametophytic) individuals at all sites. Intertidal and subtidal subpopulations from Shaw's Cove differed in their reproductive profiles. Most intertidal specimens found on emersed surfaces were densely branched, turf-forming, and bore tetrasporangial (68.6%), carposporangial (11.4%), or spermatangial (5.7%) conceptacles, reflecting a sexual life history; none produced asexual bispores. In contrast, 74.3% of the larger, loosely branched subtidal specimens bore bisporangial conceptacles indicative of asexual reproduction. Nearly 70% of the Indian Rock thalli showed no evidence of conceptacle formation. Only asexual, diploid bispore-producing thalli were obtained from the Bodega Bay site. Genetic diversity (mean number of alleles per locus, percent of polymorphic loci, and average expected heterozygosity) of diploid L. aspergillum populations varied with life-history characteristics and geographic location. A total of 30 alleles was inferred from zymograms of 16 loci examined by starch-gel electrophoresis; of these loci, 11 were polymorphic. The genetic diversity of sexual, diploid populations of L. aspergillum (alleles per locus [A/L] = 1.4-1.5; percent polymorphic loci [%P] = 37.5-50.0) was relatively high compared with other red seaweeds. Lowest diversity (A/L = 1.0; %P = 0.0) occurred in the exclusively asexual Bodega Bay population which consisted of genetic clones. All sexual L. aspergillum populations deviated significantly from Hardy-Wein-berg expectations due to lower than expected heterozygosity. Genetic differentiation (Wright's F_statistic [F_ST]; Nei's Genetic Distance [D]) among sexually reproducing southern California populations was low (F_ST= 0.030) on a local scale (ca. 5 km), suggesting high levels of gene flow, but high genetic differention (F_ST= 0.390 and 0.406) occurred among southern California populations separated by ca. 70 km. Very high genetic differentiation (F_ST= 0.583–0.683) was obtained between northern and southern California populations separated by 700–760 km. Our genetic and reproductive data suggest that the L. aspergillum population from Bodega Bay is sustained by perennation, vegetative propagation, or asexual reproduction by bispores and may represent an isolated remnant or a population established by a founder event.     

REVISION OF THE MASTOPHOROIDEAE (CORALLINALES,RHODOPHYTA) AND POLYPHYLY IN NONGENICULATE SPECIES WIDELY DISTRIBUTED ON PACIFIC CORAL REEFS1

The subfamily Mastophoroideae (Corallinaceae, Rhodophyta) is characterized by species possessing nongeniculate, uniporate tetrasporangial conceptacles without apical plugs, the presence of cell fusions, and the absence of secondary pit connections. However, molecular phylogenetic studies not including the type genus Mastophora indicated that the Mastophoroideae was polyphyletic. Our molecular phylogenetic analysis of the subfamily including the type genus using DNA sequences of SSU rDNA and plastid‐encoded gene of PSII reaction center protein D1 (psbA) revealed that Mastophora formed a robust clade only with Metamastophora. The other mastophoroid genera were divided into six lineages within the family Corallinaceae. Five supported lineages—(i) Pneophyllum; (ii) Hydrolithon gardineri (Foslie) Verheij et Prud’homme, Hydrolithon onkodes (Heydr.) Penrose et Woelk., and Hydrolithon pachydermum (Foslie) J. C. Bailey, J. E. Gabel et Freshwater; (iii) Hydrolithon reinboldii (Weber Bosse et Foslie) Foslie; (iv) Spongites; and (v) Neogoniolithon—were clearly distinguished by the combination of characters including the presence or absence of palisade cells and trichocytes in large, tightly packed horizontal fields and features of tetrasporangial and spermatangial conceptacles. Therefore, we amend the Mastophoroideae to be limited to Mastophora and Metamastophora with a thin thallus with basal filaments comprised of palisade cells, tetrasporangial conceptacles formed by filaments peripheral to fertile areas, and spermatangia derived only from the floor of male conceptacles. This emendation supports Setchell’s (1943) original definition of the Mastophoroideae as having thin thalli. We also propose the establishment of three new subfamilies, Hydrolithoideae subfam. nov. including Hydrolithon, Porolithoideae subfam. nov. including the resurrected genus Porolithon, and Neogoniolithoideae subfam. nov. including Neogoniolithon. Taxonomic revisions of Pneophyllum and Spongites were not made because we did not examine their type species.     

MESOPHYLLUM SPHAERICUM SP. NOV. (CORALLINALES,RHODOPHYTA): A NEW MAËRL‐FORMING SPECIES FROM THE NORTHEAST ATLANTIC1

Mesophyllum sphaericum sp. nov. is described based on spherical maërl individuals (up to 10 cm) collected in a shallow subtidal maërl bed in Galicia (NW Spain). The thalli of these specimens are radially organized, composed of arching tiers of compact medullary filaments. Epithallial cells have flattened to rounded outermost walls, and they occur in a single layer. Subepithallial initials are as long as, or longer than the daughter cells that subtend them. Cell fusions are abundant. Multiporate asexual conceptacles are protruding, mound‐like with a flattened pore plate, lacking a peripheral raised rim. Filaments lining the pore canal and the conceptacle roof are composed of five to six cells with straight elongate and narrow cells at their base. Carposporangial conceptacles are uniporate, protruding, and conical. Spermatangial conceptacles were not observed. Molecular results placed M. sphaericum near to M. erubescens, but M. sphaericum is anatomically close to M. canariense. The examination of the holotype and herbarium specimens of M. canariense indicated that both species have pore canal filaments with elongate basal cells, but they differ in number of cells (five to six in M. sphaericum vs. four in M. canariense). Based on the character of pore canal filaments, M. canariense shows similarities with M. erubescens (three to five celled). The outermost walls of epithallial cells of M. canariense are flared compared to the round to flattened ones of M. erubescens, the latter being widely accepted for the genus Mesophyllum. The addition of M. sphaericum as new maërl‐forming species suggests that European maërl beds are more biodiverse than previously understood.     

LIFE HISTORY AND GROWTH OF LAURENCIA BRACHYCLADOS (RHODOPHYTA,CERAMIALES)1

Laurencia brachyclados Pilger from Hawai'i completed a “Polysiphonia-type” tri-phasic life history in 21 weeks in laboratory culture. Tetraspores developed into gametophytes in a nearly 1:1:1 ratio of females: males: non-reproductive. Carpospores were released as early as 21 days after mixing virgin female and male gametophytes. Cultured thalli showed a “guerilla type” growth form. Other Hawaiian Laurencia species in culture had longer maturation times or remained non-reproductive. Variation in life history schedules may influence Laurencia species coexistence and algal community structure.     

FRACTIONATION OF NUCLEAR,CHLOROPLAST, AND MITOCHONDRIAL DNA FROM POLYSIPHONIA BOLDII (RHODOPHYTA) USING A RAPID AND SIMPLE METHOD FOR THE SIMULTANEOUS ISOLATION OF RNA AND DNA1

Extraction of nucleic acids from red algae is complicated by the presence of phycocolloids. For this reason, methods used for nucleic acid isolation from other organisms are not always amenable to use with red algal preparations; modifications in some cases lead to protocols that are time consuming and complicated, often requiring large amounts of algal tissue for starting material. Here we describe the isolation of both RNA and DNA followed by fractionation and identification of nuclear, chloroplast, and mitochondrial DNAs from a single preparation of Polysiphonia boldii Wynne and Edwards using a simple method that yielded approximately 100 μg of total RNA and 20 μg of total DNA from 1 g of frozen powdered algae. The potent protein denaturant guanidinium thiocyanate and the detergent sarkosyl were used to gently lyse the cells and organelles and immediately inhibit nuclease activity in the extract. The nucleic acids were isolated by ultracentrifugation into a dense solution of CsCl; the RNA was recovered as a pellet and the DNA as a band within the CsCl solution. Agarose gel electrophoresis of the total RNA showed discrete ribosomal RNA bands, indicating little nonspecific degradation. The nuclear, chloroplast, and mitochondrial DNAs were fractionated by density gradient ultracentrifugation in the presence of the DNA binding dye, bisbenzimide H (Hoechst 33258), which binds preferentially to DNA with a high A + T:G + C ratio, thus altering its density to a greater degree than it does that of DNA with a lower nucleotide ratio. The three fractions were identified by Southern blot analysis using heterologous gene probes specific for the different genomes. The protocol should be applicable to different types of algae. The simple nucleic acid isolation step can be performed on multiple samples simultaneously without subsequent fractionation of DNA, allowing comparison of DNA from different individuals, populations, or species.     

Brief communication: ancient DNA prospects from Sri Lankan highland dry caves support an emerging global pattern

Recovery of ancient DNA has become an increasingly important tool in elucidating the origins of past populations and their relationships. Unfortunately, many human skeletal remains do not contain original DNA amplifiable by polymerase chain reaction (PCR). Amino-acid racemization has proven to be a useful predictor of ancient DNA results. We analyzed the relative levels of amino-acid preservation and racemization of human samples from two highland dry-cave sites in Sri Lanka, and found that amino-acid enantiomer ratios were inconsistent with successful authentic DNA recovery. A review of the literature reveals that these results are consistent with a global pattern of poor DNA preservation in the tropics.     

A METHOD FOR EXTRACTION OF HIGH MOLECULAR WEIGHT DNA FROM THE MACROALGA PORPHYRA YEZOENSIS (RHODOPHYTA)1,2

We developed a method for extraction of DNA from the red alga Porphyra yezoensis Ueda. The method consists of three preparation steps that include CsCl-gradient ultracentrifugation, cetyl trimethyl ammonium bromide treatment, and a final RNase step. The amount of DNA extracted from 1.5 g of starting material averaged 17.7 μg. The resulting DNA had a high molecular weight, was 25-166 kb in length, was digested with five common restriction enzymes, and showed no nuclease activity. It was of sufficient quality for construction of genomic libraries.     

MOLECULAR ANALYSIS REVEALS CRYPTIC DIVERSITY IN PORPHYRA (RHODOPHYTA)1

The small subunit ribosomal RNA (SSU rRNA) gene was amplified from 15 species of the red alga Porphyra and digested with restriction enzymes to generate data for species identification. The subset of species selected for phylogenetic analysis was P. cuneiforms (Setchell et Hus) Krishnamurthy, P. nereocystis anderson, P. schizophylla Hollenberg et Abbott, P. thuretii Setchell et Dawson and Porphyra 1674. Bangia sp. was used as an out-group. Restriction sites were mapped and used as characters in parsimony and maximum likelihood analysis. The phylogenetic hypotheses generated were compared statistically to possible alternative phylogenies based on traditional morphological taxonomic characters. The results indicate that the current subgenera in Porphyra do not represent monophyletic groups and that traditional morphological and ecological taxonomic characters alone may not be adequate for definitive species identification and cannot be relied on as an indication of Porphyra have large insertions in the SSU gene that are apparently splicesd from the final SSU rRNA molecule. The possible character, distribution and potential significance of these putative introns are discussed.     

MOLECULAR SYSTEMATICS OF CERAMIUM AND CENTROCERAS (CERAMIACEAE,RHODOPHYTA) FROM BRAZIL1

Morphological investigations identified 11 Ceramium Roth species, of the 18 previously reported from Brazil. Phylogenetic analyses of sequences of the chloroplast‐encoded rbcL gene confirmed the presence of seven of these species. Three other species are reported from Brazil for the first time. Ceramium affine Setchell & Gardner and C. filicula Harvey ex Womersley were previously known only from the Pacific Ocean (Mexico and Australia, respectively). A new species, C. fujianum Barros‐Barreto et Maggs sp. nov., is described here. Its general habit is similar to that of C. strictum sensu Harvey from Europe but it has one less periaxial cell than C. strictum; its cortical filament arrangement is closest to C. deslongchampsii Chauvin ex Duby, also from Europe, but whorled tetrasporangia partially covered by cortical cells differ strikingly from the naked protruding tetrasporangia of C. deslongchampsii. Ceramium species in which each periaxial cell cuts off transversely only a single basipetal cell formed a robust clade. The genus Ceramium as represented in Brazil is not monophyletic with respect to Centroceras Kützing and Corallophila Weber‐van Bosse; Ceramium nitens, which has axial cells completely covered by rounded cortical cells formed by acropetal and basipetal filaments, did not group with any Ceramium clade but was weakly allied to a species of Corallophila. All three Brazilian Centroceras sequences were attributed to a single species, C. clavulatum.     

CHIHARAEA AND YAMADAIA (CORALLINALES,RHODOPHYTA) REPRESENT REDUCED AND RECENTLY DERIVED ARTICULATED CORALLINE MORPHOLOGIES1

Phycologists have hypothesized that the diminutive fronds produced by species in the genera Chiharaea and Yamadaia, which are composed of comparatively few genicula and intergenicula, represent morphological intermediates in the evolution of articulated corallines from crustose ancestors. We test this “intermediate frond hypothesis” by comparing rbcL sequences from the generitype species Chiharaea bodegensis and Yamadaia melobesioides to sequences from other coralline genera. We demonstrate that Chiharaea includes two other NE Pacific species, Arthrocardia silvae and Yamadaia americana. Chiharaea species are characterized morphologically by inflated intergenicula and axial conceptacles with apical or acentric pores. Although relationships among the three species are unresolved, Chiharaea bodegensis, C. americana comb. nov., and C. silvae comb. nov. are distinguished from one another by DNA sequences, morphology, habitat, and biogeography. Chiharaea occurs together with Alatocladia, Bossiella, Calliarthron, and Serraticardia macmillanii in a strongly supported clade of nearly endemic north Pacific articulated coralline genera and species that have evolved relatively recently compared to other articulated corallines. In contrast, NW Pacific Yamadaia melobesioides belongs in a clade with Corallina officinalis, the generitype species of Corallina, and therefore we reduce Yamadia to a synonym of Corallina and propose Corallina melobesioides comb. nov. We reject the ‘intermediate frond hypothesis’ and conclude that Chiharaea and Yamadaia are recently derived taxa that evolved from articulated coralline ancestors and represent a reduction in the number of genicula and intergenicula.     

TWO NEW SPECIES OF TAYLORIELLA (RHODOMELACEAE,RHODOPHYTA) FROM THE NORTHEASTERN NORTH PACIFIC1

Two new species are recognized in the rhodomelacean genus Tayloriella Kylin: T. divaricata sp. nov. and T. abyssalis sp. nov. These two taxa are distributed in the northeastern North Pacific, the former ranging from Amchitka Island in the Aleutians through southcentral Alaska to northern British Columbia, and the latter ranging also from Amchitka Island through southcentral Alaska and British Columbia into northern Washington. A characteristic of these two species shared with the type of Tayloriella is that the abaxial lateral always overtops the monopodially developed axes in every order of branching. The laterals have little congenital fusion with the parent axes. A common feature in these two species is that the laterals are terminated in a relatively long monosiphonous portion (usually 6 or 7 cells). The relationship of Tayloriella to Pterosiphonia and Pterosiphoniella is discussed.     

ULTRASTRUCTURE OF MEIOSIS IN DASYA BAILLOUVIANA (RHODOPHYTA). I. PROPHASE I1

This first of two papers on ultrastructural observations of meiosis in the red alga Dasya baillouviana (Gmelin) Montague describes stages of prophase I of meiosis. Although the five stages of prophase were originally derived from light microscopic studies, the same stages were utilized for this study based on the developmental sequence of the synaptonemal complex, which has the same morphology and mode of development as those reported for other red algae. The cytoplasm in early prophase sporocytes was typically less electron dense than either vegetative cells or sporocytes in later stages of meiosis. The reduction in density suggests clearing of ribosomes and other cytoplasmic components prior to conversion from sporophyte to gametophyte control. Leptotene cells often had an amorphous, chromatin-free area, function unknown, which was not obviously associated with any specific nuclear region. Diplotene cells were characterized by nuclei containing prominent ring-shaped nucleoli composed of a dark staining ring of material surrounding an electron-translucent “vacuole.” Packets of electron-dense, fibrillar material were often noted in the cytoplasm of late prophase cells. These packets are thought to he “nuage,” a term applied to large cytoplasmic aggregations of RNA in germ cells of several other phyla. It is suggested that nuage may represent a new infusion of ribosomal and messenger RNA for post-meiotic development. The division pales are established by late prophase and a single polar ring is found within each large “exclusion zone” in close association with a pore-free area of nuclear envelope. Both annulate lamellae and small, numerous vesicles are located in the exclusion zones. The significance of the various aspects of prophase I is discussed with the overall observation that this phase of meiosis in red algae is very similar to the process in higher plant and animal cells.