Galls on the marine red alga Prionitislanceolata (Halymeniaceae): specific induction and subsequentdevelopment of an algal-bacterial symbiosis

Gall formation in Prionitis lanceolata is associated with aspecific eubacterium (Proteobacteria [alphasubclass], Rhodobacter grouping), which, typical ofbacterial symbionts, has not yet been cultivated or isolated in pureculture. This investigation tested the hypothesis that P.lanceolata gall formation was caused by the associated eubacteriumusing a species-specific rDNA probe (S-S-P.l.sym-0949-a-A-25) toidentify and assay for symbiont presence during consecutive laboratoryinduction trials. Gall induction was quantified and whole-cell in situhybridization used to determine the relative percentage of symbioticeubacteria in inoculation homogenates. In situ hybridization ofsymbionts in sections allowed localization and monitoring of thismicrobe during gall development. Induction trial results indicate asignificant correlation between bacterial symbiont presence and gallinitiation (P = 0.00005). The gall bacterium comprisedthe majority of the eubacteria hybridized in laboratory inductionhomogenates (85-97%), in galls induced in the laboratoryand in three algal populations in nature. The evidence presented heredemonstrates the causative role of the identified eubacterium in gallinduction and formation. This investigation is significant in theapplication of molecular methods towards understanding the roles ofnoncultivable marine bacteria in marine algal-microbeinteractions.     

MOLECULAR IDENTIFICATION OF A BACTERIUM ASSOCIATED WITH GALL FORMATION IN THE MARINE RED ALGA PRIONITIS LANCEOLATA1

The polymerase chain reaction (PCR) was used to amplify eubacterial small-subunit (16S) ribosomal DNA (rDNA) genes from galls of the marine red alga Prionitis lanceolata Harvey (Gigartinales). These tumors consist of hypertrophied algal cells containing large numbers of intercellular bacteria that remain uncultivable. PCR-amplified 16S rDNAs from surface-sterilized gall tissue plugs were cloned, sequenced, and analyzed by alignment to available small-subunit rRNA sequences (University of Illinois Ribosomal Database Project). Variable regions were identified and used to construct a fluorescently labeled, species-specific oligodeoxynucleotide probe for whole cell in situ hybridization to the gall symbiont. Probe 949 (PLANC.949) localized the P. lanceolata bacterial symbiont in preparations from mature gall tissue. This probe did not hybridize to the rDNA of closely related bacteria included as controls in the same hybridization reactions, In situ hybridization revealed the presence of the same bacterium in association with P. lanceolata gall formation from three central California localities. Distance and parsimony analyses suggest that this organism is a member of the Proteobacteria (alpha subdivision; Rhodobacter group) and is most closely related to Roseobacter denitrificans .     

Some new species and new combinations of marine red algae from the central Pacific

Six new species in five genera of Rhodophyta are described. A new combination for Polyopes hakalauensis is also proposed. These taxa were encountered while preparing a manual of marine red algae of the Hawaiian Islands. Among the Halymeniales, two blade-like species of Halymenia, H. cromwellii sp. nov. and H. stipi-tata sp. nov., are distinguished from other species by habit and anatomical differences, including the possession of a stipe by the second. Halymenia hakalauensis Tilden 1902 is transferred to Polyopes as P. hakalauensis (Tilden) Abbott, replacing P. hawaiiensis Kajimura which is considered a synonym. A new species of Prionitis, P. corymbifera sp. nov., shows a corymbose branching pattern different from the mostly pinnate to subdichotomous branching of Japanese and Californian species. In the Gigartinales, Chondracanthus okamurae Abbott is named for a specimen which Okamura included in his understanding of Gigartina (now Chondracanthus tenellus), but which has terete axes, differing from C. tenellus (Harvey) Hommersand which is com-planate throughout. Chondracanthus intermedius (Sur-ingar) Hommersand is placed in synonymy with C. tenellus. The occurrence of C. tenellus in Hawai'i is a new distribution record from Asia. Three new species are added to the Ceramiales, Ceramium tranquillum Meneses (Ceramiaceae), Dasya kristeniae sp. nov. and Dasya murrayana Abbott & Millar (Dasyaceae). Distinctive features of Ceramium tranquillum are internodes that are three to five times longer than the height of nodes, and nodes that never project beyond the diameter of the internodes, giving straight margins except when fertile. Dasya kristeniae is a diminutive epiphyte which is nearly ecorticate, with tetrasporangial stichidia that are rectangular having apices that are curved or nodding, and cystocarps that have flaring ostioles above a constricted pericarp. Dasya murrayana was earlier misidentified with Dasya iyengarii Børgesen, and re-examination of those plants show continuous cortication throughout, where cortication is almost completely lacking in D. iyengarii except near the base. Dasya murrayana has a tufted habit, with indeterminate branches about the same sizes as the main axes, the plants growing on rock, whereas plants of D. iyengarii are not tufted, with indeterminate branches attenuate, and an epiphytic habit.     

THE RED ALGAL EPIPHYTES MICROCLADIA COULTERI AND M. CALIFORNICA (RHODOPHYCEAE,CERAMIACEAE). II. BASIPHYTE SPECIFICITY1

The phenomenon of basiphyte specificity in the settlement and growth of the red algal epiphytes Microcladia californica Farl. and M. coulteri Harv. was examined by studying the interface with their respective basiphytes and by cross-inoculation experiments. Microcladia californica attaches only to the surface of its single basiphyte Egregia menziesii (Aresch.) Turn. whereas M. coulteri penetrates the tissue of a wide range of basiphytes. The pattern of primary rhizoid development in both epiphytes determines the mode of attachment and may influence the range of basiphytes possible for each epiphyte. Cross-inoculation experiments show that Microcladia californica is not able to colonize the basiphytes of M. coulteri, Iridaea and Prionitis, or Ulva. The mechanisms by which these algae restrict the growth of epiphytes include short life-span, “cuticle peeling” and chemical defense. Microcladia coulteri, which naturally colonizes Iridaea and Prionitis, has evolved mechanisms to counteract the antifouling effects of those basiphytes. The question of why Egregia is the exclusive substratum for M. californica remains undetermined. However, Egregia may provide the appropriate ecological conditions and a surface topography conductive to M. californica spore settlement and growth.     

GC-SIM-MS DETECTION AND QUANTIFICATION OF FREE INDOLE-3-ACETIC ACID IN BACTERIAL GALLS ON THE MARINE ALGA PRIONITIS LANCEOLATA (RHODOPHYTA)

Indole-3-acetic acid (IAA), a plant hormone necessary for terrestrial plant growth and development, was detected and quantified in the marine red alga Prionitis lanceolata Harvey (Halymeniaceae, Gigartinales, Rhodophyta) using gas chromatography–selective ion-monitoring mass spectrometry (GC-SIM-MS). This allowed comparison of free IAA levels between the algal thallus and eubacterially induced galls on this alga characterized by abnormal algal growth and cell division and extensive, intercellular microbial proliferation. The levels of free IAA in the P. lanceolata thallus averaged 2.5 (±1.1) ng·g⁻¹ fresh wt. Free IAA levels in galls were more variable, ranging from ca. 4 to 39 (8.3 ± 10.9) ng·g⁻¹ fresh wt, but were significantly higher overall ( P = 0.0022). The identity of the IAA in this marine florideophycean alga was confirmed by full scan GC-MS analysis of both galls and thalli. The levels of free IAA in P. lanceolata were two to three orders of magnitude higher than those observed previously in the Rhodophyta. The origin of elevated IAA levels in P. lanceolata galls is unknown because it is possible that this compound is produced by either the gall-inducing bacterial symbiont or the host alga.     

A morphological and molecular assessment of the genus Prionitis J. Agardh (Halymeniaceae, Rhodophyta)

A critical reassessment of the morphological features of two closely related red algal genera, Grateloupia C. Agardh and Prionitis J. Agardh (Halymeniaceae), shows that members of the two genera share very similar reproductive (including the Grateloupia‐type auxiliary‐cell ampullae) and vegetative characters. Diagnostic features hitherto used for distinguishing these two genera, the texture of blades (lubricous to leathery in Grateloupia vs cartilaginous in Prionitis) and the position of reproductive structures (scattered over the entire blade in Grateloupia vs confined to particular portions of the blade in Prionitis), are continuous across some 75 species of both genera, thus making it difficult to draw a clear‐cut distinction between the two genera. In ribulose‐1,5‐bisphosphate carboxylase/oxygenase gene (rbcL) sequence analyses, the species of Grateloupia and Prionitis, including the two generitypes, constitute a large monophyletic clade in the Halymeniaceae. It is therefore proposed that Prionitis be included in the synonymy under Grateloupia and the appropriate combinations are proposed.     

On the absence of previously reported Japanese and Peruvian species of Prionitis (Halymeniaceae, Rhodophyta) in the northeast Pacific

Partial rbc L sequences of type specimens, historically significant specimens and recently collected comparative specimens are used to demonstrate that Prionitis decipiens (Montagne) J. Agardh (type locality: Paita, Peru), Prionitis angusta (Harvey) Okamura (type locality: Shimoda, Japan, currently known as Grateloupia angusta (Harvey) Kawaguchi et Wang) and Prionitis cornea (Okamura) E. Y. Dawson (type locality: Shizuoka, Japan, currently known as Grateloupia cornea Okamura), all previously reported from the northeast (NE) Pacific (California, USA and Baja California, Mexico), were not, and currently are not, present. Historically significant specimens from the NE Pacific specifically annotated by E. Y. Dawson as belonging to P. cornea or specifically annotated by I. A. Abbott as belonging to P. angusta were sequenced and shown to belong to either Prionitis linearis Kylin (type locality: La Jolla, California, USA) or to Prionitis filiformis Kylin (type locality: San Francisco, California, USA). The lectotype of Gelidium decipiens Montagne (basionym of Prionitis decipiens ) is narrowed to a single specimen illustrated by Montagne, and a lectotype is designated for P. linearis . The utility of partial rbc L sequences (less than 200 base pairs) for determining the identity of specimens is discussed, as is the importance of sequencing type and historically significant specimens.