CHARACTERIZATION AND MOLECULAR PHYLOGENYOF A PROTEIN KINASE cDNA FROM THE DINOFLAGELLATE GONYAULAX (DINOPHYCEAE)1

Degenerate primers corresponding to conserved protein kinase motifs were used to amplify potential kinase DNA fragments from a Gonyaulax polyedra Stein cDNA library using PCR. One PCR fragment, potentially encoding a CAMP-dependent protein kinase, was used as a probe to isolate a near full-length cDNA from the library. The nucleic acid sequence of the entire cDNA clone had a high homology to the catalytic subunit of cAMP-dependent protein kinase (cAPK subfamily and affiliated members. Northern blot analysis showed that the corresponding mRNA had a size (about 1.4 kb) and a relative high abundance consistent with a cAPK homologue. Southern blot analysis showed that while there are roughly 30 copies of the kinase gene per genome, the pattern of restriction fragments is inconsistent with the hypothesis of a large gene family. Phylogenetic analyses comparing the deduced amino acid sequence from the Gonyaulax cDNA with other cAPK sequences place Gonyaulax close to the slime mold Dictyostelium discoideum. This is the first phylogenetic analysis of dinoflagellates based on protein sequence, and the results are in agreement with similar analyses based on rRNA sequences.     

BIOGEOGRAPHYOF BATRACHOSPERMUM GELATINOSUM (BATRACHOSPERMALES,RHODOPHYTA) IN NORTH AMERICA BASED ON MOLECULAR AND MORPHOLOGICAL DATA1

Fifteen populations of the widespread fieshwater red alga Batrachospermum gelatinosum (L.) De Candolle were sampled throughout the geographic range in North America from central Alabama, U.S.A. (33° N), to Ellesmere Island, Northwest Territories (NWT), Canada (80° N). Analysis of ribosomal DNA internal transcribed spacer (ITS) 1 and 2 sequences yielded a parsimony tree with a large polytomy consisting of most populations plus a branch with one Nova Scotia and two NWT populations. The nucleotide variation, both within the polytomy and within the branch, was small (< 1%). The sequence divergence between the branch and polytomy was 3%. The lengths of the ITS 1 and 2 sequences of B. gelatinosum, 216–229 and 448–458 base pairs, respectively, fall within the very broad ranges reported for other red algae. The cluster analysis of 11 morphometric characteristics revealed three groupings of populations, partly based on geographic distribution. All tundra, eastern boreal forest, and mid-western hemlock-hardwood populations were in one grouping, whereas the deciduous forest, coastal plain, and eastern hemlock-hardwood populations were in a second. How ever, one deciduous forest population from Rhode Island, U.S.A. was unassociated. There was considerable overlap in morphometric characteristics among the three groupings. Based on this fact and the relatively small nucleotade variation in ITS sequences, we conclude that B. gelatinosum is a morphologically variable and geographically widespread species that is a valid taxonomic entity.     

EXTENSIVE INTRASPECIFIC MORPHOLOGICAL VARIATION IN ENTEROMORPHA MUSCOIDES (CHLOROPHYTA) REVEALED BY MOLECULAR ANALYSIS

Enteromorpha muscoides (Clemente y Rubio) Cremades and E. clathrata Roth (Greville) are morphologically variable species that can easily be distinguished from other Enteromorpha species but not from each other. The key morphological character separating the two species is the presence or absence of spine-like branches: E. muscoides has small spine-like branches throughout the thallus, whereas E. clathrata lacks spines. The spiny branches in E. muscoides are not as obvious in summer as in winter, so summer samples may be difficult to distinguish from those of E. clathrata. In this study, molecular data were used to investigate whether these two species, as defined by morphological characters, might be conspecific. The sequences of the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene differed by 0%–0.6% between all samples of both E. muscoides and E. clathrata. Phylogenetic analysis of these sequences in an alignment with 13 other representatives of both Enteromorpha and Ulva showed that this highly supported monophyletic E. muscoides / E. clathrata clade is separated by long branch lengths from other Enteromorpha and Ulva clades. Based on these results, we suggest that Enteromorpha muscoides (Clemente y Rubio) Cremades and Enteromorpha clathrata Roth (Greville) are conspecific, with the older name E. muscoides taking priority.     

UNUSUAL DIHYDROXYSTEROLS AS CHEMOTAXONOMIC MARKERS FOR MICROALGAE FROM THE ORDER PAVLOVALES (HAPTOPHYCEAE)1

Recent studies have led to the identification of an unusual class of dihydroxysterols (steroidal diols termed “pavlovols”)in a few species of microalgae from the genus Pavlova (family Pavlovaceae, class Haptophyceae = Prymnesiophyceae). These compounds have an additional hydroxyl group at G-4 in the sterol A ring, which appears to be very rare in sterol biosynthetic pathways. The sterol compositions of many other haptophytes from different orders have been analyzed, but to date all have lacked pavlovols. We now report the occurrence of these compounds in Diacronema vlkianum Prauser and two strains of Pavlova pinguis Green. This is the first report of the lipid composition of these species. Both microalgae contained “24-methylpavlovol” (4α, 24-dimethyl-5α-cholestan-3β, 4β-diol), P. pinguis also contained “24-ethylpavlovol” (4α-methyl-24-ethyl-5α-cholestan-3β, 4β-diol), and D. vlkianum contained a diol identified from its mass spectrum as 4α, 24β-dimethyl-5α-cholest-22E-en-3β,4β-diol. Both species contained structurally analogous 4-desmethyl sterols and 4-methyl sterols, although there were major differences in the proportions in each series. The major 4-desmethyl sterol in both species was 24-ethylcholesta-5, 22E-dien-3β-ol and the major 4-methyl sterol was 4α-methyl-24-ethyl-5α-cholest-22E-en-3β-ol. The presence of pavlovols in P. pinguis, combined with earlier data, suggests that all Pavlova species might have this distinguishing lipid feature. However, their identtjication in D. vlkianum extends the occurrence of these compounds to another genus and shows that they are not unique to the genus Pavlova. However, they are probably restricted to species from the order Pavlov ales. The modes of biosynthesis and functions of pavlovols remain unknown.     

CHARACTERIZATION OF MORPHOSPECIES AND STRAINS OF MICROCYSTIS (CYANOBACTERIA) FROM NATURAL POPULATIONS AND LABORATORY CLONES USING CELL PROBES (LECTINS AND ANTIBODIES)1

The genus Microcystis (cyanobacteria) includes toxic and bloom-forming morphotypes which are usually arranged into species based on morphological features. Immunofluorescence assays using polyclonal and preadsorbed antibodies, as well as FITC-labebd lectins were used to characterize three morphospecies of Microcystis (M. viridis, M. wesenbergii, and M. aeruginosa) from natural populations (several lakes/reservoirs in Denmark and Spain) and laboratory clones. The cell probes used were unaffected by the different phases of the cell division cycle, growth phase, or environmental factors, such as culture medium, light, or temperature. Anhbody and lectin binding patterns were specific to each clone. In nature, the cell probes were useful tools to characterize Microcystis populations. Antibodies and lectins revealed geographic differentiation within the same morphospecies. Differentiation was moderate among nearby locales and intensified among areas distant from one another. Microcystis aeruginosa from Spain has very different cell surface antigens and lectin binding sites than M. aeruginosa from Denmark. A taxonomy of Microcystis based on cell probes reveals some discrepancies with classical morphospecies. The binding affinities were more closely related to the geographic origin of the tested material than to the morphospecies identification. Different morphospecies from the same lake in some cases were more similar than the same morphospecies from different lakes. Microcystis viridis and M. aeruginosa from Danish lakes appeared to be closely related species, whereas M. wesenbergii emerged as a different species.     

FEEDING IN PERANEMA TRICHOPHORUM REVISITED (EUGLENOPHYTA)1

Feeding in Peranema trichophorum (Ehrenberg 1838) Stein 1878 was first observed over a century ago, yet there is still contention over how the feeding apparatus is used in feeding. Using video microscopy and scanning microscopy, this study documents two types of feeding. Peranema may engulf prey cells whole. Immotile cells are preferred, but moving cells occasionally are engulfed. Details of the early stages of engulfment are presented using scanning electron microscopy. A second method of feeding begins with the attachment of Peranema to a prey cell. The rods of the feeding apparatus then are repeatedly scraped over the surface of the prey until a tear occurs in the cell. The feeding apparatus is inserted into the opening and the internal cell contents are sucked out. The anterior flagellum is inserted into the prey to help remove the cell's contents.     

INFECTIOUS DISEASES OF MAZZAELLA LAMINARIOIDES (RHODOPHYTA): CHANGES IN INFECTION PREVALENCE AND DISEASE EXPRESSION ASSOCIATED WITH SEASON,LOCALITY, AND WITHIN-SITE LOCATION1

This study addresses the issues of infection prevalence and disease expression in two wild populations of the red algal host Mazzaella laminarioides and their variability associated with locality, season, and spatial location of the host in the intertidal zone. Our results demonstrated that Endophyton ramosum is the most frequent infective pathogen affecting M. laminarioides in Matanzas and Pucatrihue. This situation prevailed through the year and across the high-to-low intertidal gradient. Although there was a general trend for lower levels of infection in late winter and early spring, only in a few, cases was well-defined seasonality detected. Furthermore, clear seasonal patterns, as displayed by deformative disease in the high intertidal zone of Pucatrihue, were attenuated in the middle and lower intertidal zones. Differences in levels of infection in M. laminarioides between the high intertidal zones of Matanzas and Pucatrihue diminished toward the low intertidal zone. Thus, effects of seasonality and locality on infection prevalence may be influenced, at least in part, by the position of the hosts an the intertidal zone. Spatial distribution of the diseased individuals also varied along the beach. This pattern was consistent between the two sites and seemed related to wave exposure and the specific pathogen. Comparisons of the size distribution of noninfected fronds with their infected counterparts showed that infections by Endophyton ramosum and Pleurocapsa sp. more frequently affected medium-and large-sized fronds. This pattern was consistent temporally and similar in the two localities. Finally, a clear association between maturity and prevalence of infection was detected. This association resulted in most fronds of the noninfected segment of the host population being immature, whereas most mature fronds were infected. In conclusion, infectious diseases affecting the red alga Mazzaella laminarioides are a persistent phenomenon in wild populations of the host, although only a small segment of the infected populations displays the full expression of the disease. In spite of the suggested role of factors such as season, latitude, and spatial location of the host on disease prevalence and expression, additional studies are needed to understand fully the dynamics of infectious diseases in wild populations of algal hosts.     

VARIATION IN THE OCCURRENCE OF EXTERNAL CARBONIC ANHYDRASE AMONG STRAINS OF THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Eleven different strains of Phaeodactylum tricornutum Bohlin were obtained from three culture collections and were examined for the presence of external and internal carbonic anhydrase (CA). Cells of all strains, grown in standing culture at alkaline pH and low, dissolved inorganic carbon had internal CA, but only eight were found to have external CA. External CA activity was reduced when cultures were bubbled with air and was completely repressed when they were grown on 5% CO₂. Expression of external CA activity appears to be regulated by CO₂ concentration in the growth medium, but within one species, there appears to be a variation in occurrence of external CA and consequently in the mode of inorganic carbon acquisition.     

GENETIC VARIABILITY AND SPATIAL SEPARATION IN THE SEA PALM KELP POSTELSIA PALMAEFORMIS (PHAEOPHYCEAE) AS ASSESSED WITH M13 FINGERPRINTS AND RAPDS1

Postelsia palmaeformis Ruprecht is an annual species, occuring from southern California to Vancouver Island, Canada, in upper intertidal sites exposed to extreme wave shock. Because of its limited spore dispersal, discrete and inbred populations are likely on the local scale, yet dispersal of drifting and fertile thalli raises the possibility of outbred populations on a regional scale. M13 minisatellite DNA fingerprinting and random amplified polymorphic DNA (RAPD) marks were used in a complementary fashion to investigate genetic variability among 24 individuals on scales of clusters (= coalesced holdfasts). < 1 m, 10 m, 25 m, 16 km, and 250 km. Based on M13 fingerprinting, genetic relatedness within clusters was extremely high. Three of six clusters had at hast two identical individuals, and similarity values within five clusters were ≧0.90. Similarities between two of three clusters separated by < 1 m were significantly higher than between cluster pairs separated by 25 m and 250 km: however, the similarity between two clusters separated by 25 m was equivalent to the similarity between two clusters separated by 250 km. Thus, genetic relatedness as determined by M13 fingerprinting generally decreased as distance increased to 25 m. Conversely, RAPD data easily discriminated populations separated by 16 and 250 km but were not useful in discriminating individuals from < 1 to 25 m. Results from the complementary data sets suggest that most dispersal occurs over distances of 1–5 m, individuals within a cluster are siblings, and distinguishable biogeographic populations are present along the coast.     

PHYLOGENYOF CHLOROMONAS (CHLOROPHYCEAE): A STUDY OF 185 RIBOSOMAL RNA GENE SEQUENCES1

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.     

RECENT APPEARANCE OF CYLINDROSPEMOPSIS (CYANOBACTEIUA) IN FIVE HYPEREUTROPHIC FLORIDA LAKES1

Observations of historical and recent phytoplankton samples from five hypereutrophic Florida lakes indicate that nitrogen-fixing cyanobacteria in the genus Cylindrospermopsis Seenayya et Subba Raju have entered these water-bodies sometime in the last 30 years. Cylindrospermopsis forms a year-round bloom in one of the five lakes, dominates seasonally in another, and is at least at times an important component of the phytoplankton community in the remaining three. The increase in abundance of Cylindrospermopsis in Florida lakes could have implications for water management.     

DNA EXTRACTION AND RAPD MARKERS TO ASSESS THE GENETIC SIMILARITY AMONG GELIDIUM SESQUIPEDALE (RHODOPHYTA) POPULATIONS1

A simple method developed for genomic DNA isolation from fungus was tested on the red alga, Gelidium sesquipedale (Clem.) Born et Thur., which is commercially exploited for its high sulfated polysaccharide (agar) content. This method is faster, cheaper, and less toxic than conventional phenol/chloroform methods. Random amplified polymorphic DNA (RAPD) amplifications were performed successfully without the necessity of purifying the DNA. RAPD markers were used to investigate the genetic similarity among three natural populations of G. sesquipedale from southern Portugal. Bulked-genomic DNA samples of 15 different individuals were made in each population. These can be conceived of as a sample of the population DNA. Of the 62 primers screened, 41 produced bands and 22 revealed polymorphisms. Genetic similarities among populations were high. Populations that are further away from each other have the lowest similarity coefficients, whereas the intermediate Ingrina population, located on the south coast, showed higher genetic similarity with the Odeceixe population located on the southwest coast, than with the São Rafael southern population. This suggests a higher genetic flow between Odeceixe and Ingrina or the result may be a founder effect in the sense that the species has propagated from the east coast to the south coast of Portugal. We conclude that the use of this isolation method with RAPD analysis is appropriate to characterize the genetic variability of this commercial species along its geographical distribution. Large sample sizes can be screened at a relatively low cost. Finding genetic markers for commercial populations of G. sesquipedale may be of industrial interest.     

PHYLOGENETIC AFFINITIES OF THE SARCINOCHRYSIDALES AND CHRYSOMERIDALES (HETEROKONTA) BASED ON ANALYSES OF MOLECULAR AND COMBINED DATA1

Small-subunit ribosomal RNA nucleotide sequences were inferred for Giraudyopsis stellifera Dangeard (Chrysomeridales), as well as for Pulvinaria sp. and Sarcinochrysis marina Geitler (Sarcinochrysidales,). Phylogenetic analyses of the molecular data indicate that the former is weakly related to the Phaeophyceae/Xanthophyceae clade, whereas the latter two have affinities to the Pelagophyceae, and the Sarcinochrysidales sensu stricto is transferred to this class. A recent study proposed that the Pelagophyceae belongs to a larger assemblage of chromophytic species characterized by reduced flagellar apparatuses. Although the flagellar apparatus characterizing the Sarcinochrysidales is reduced relative to the Chysomeridaels and some other chromophytes, it is the most complicated to be associated with “the reduced flagellar apparatus” lineage. Cladistic analyses of a traditional data set (largely ultrastructural features of the flagellar apparatus) and a combined traditional/molecular data set were used to assess the evolutionary trends of reduction in the flagellar apparatus within the heterokont chromophytes.     

PHYLOGENETIC RELATIONSHIPS OF SELECTED EUGLENOID GENERA BASED ON MORPHOLOGICAL AND MOLECULAR DATA1

The small subunit rRNA (SSU rRNA) coding regions sequenced from the euglenoids Petalomonas cantuscygni, Peranema trichophorum, and Khawkinea quartana were used to assess the phylogenetic relationships of these genera within the Euglenozoa. Phylogenies derived from distance, parsimony, and maximum likelihood methods infer that the euglenoids and kinetoplastids form sister clades within a monophyletic assemblage. Distances representative of closely related lineages separate the genera within the Kinetoplastida, whereas larger distance values separate genera within the euglenoid assemblage. The results of the morphological and molecular studies suggest that phagotrophy arose early in the euglenozoan lineage with the subsequent acquisition of phototrophy, osmotrophy, and parasitism. Phagotrophic euglenoids with a pellicle composed of longitudinal strips appear to have diverged prior to genera with helically arranged strips. This study suggests that the hypothetical ancestor to the Euglenozoa was a phagotroph with two flagella, both containing paraxonemal rods. Furthermore, its basal bodies contained proximal cartwheels, were connected by a prominent fiber, and were anchored with three asymmetrically arranged flagellar roots.     

PHYLOGENETIC ANALYSIS OF YAMAGISHLELLA AND PLATYDORINA (VOLVOCACEAE,CHLOROPHYTA) BASED ON rbcL GENE SEQUENCES1

Yamagishiella, based on Pandorina unicocca Rayburn et Starr, is distinguished from Eudorina by its isogamous sexual reproduction, whereas Platydorina exhibits anisogamous sexual reproduction. In the present study, we sequenced the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) genes from five Japanese and North American strains of Y. unicocca (Rayburn et Starr) Nozaki, two Platydorina caudata Kofoid strains, and two strains of Eudorina unicocca G. M. Smith, as well as eight related colonial and unicellular species. Phylogenetic trees were constructed based on these sequence data and on previously published rbcL gene sequences from 23 volvocalean species in order to deduce phylogenetic relationships within the colonial Volvocales, with particular regard to the phylogenetic positions and status of the genera Yamagishiella and Platydorina. Two robust monophyletic groups of the anisogamous/oogamous volvocacean species were resolved in the maximum-parsimony tree as well as in the neighbor-joining distance tree. One of the two groups comprises three species of Volvox section Volvox, whereas the other is composed of other sections of Volvox as well as of all the species of Eudorina and Pleodorina. Platydorina, however, was positioned outside these two monopliyletic groups. Therefore, derivation of the Platydorina lineage may be earlier than that of such anisogamous/oogamous groups, or orgin of “anisogamy with sperm packets” in Platydorina may be independent of sperm packet evolution in Eudorina, Pleodorina, and Volvox. It was also resolved with high bootstrap values that all of the Y. unicocca strains form a monophyletic group positioned outside the large monophyletic group including Eudorina and Pleodorina. These reject the possibility of the reverse evolution of isogamy from anisogamy to give rise to Yamagishiella within the lineage of Eudorina.     

STUDIES ON TRANSPARENT EXOPOLYMER PARTICLES (TEP) PRODUCED IN THE ROSS SEA (ANTARCTICA) AND BY PHAEOCYSTIS ANTARCTICA (PRYMNESIOPHYCEAE)1

The distribution and production of transparent exopolymer particles (TEPs) were studied quantitatively both in cultures of Phaeocystis antarctica Karsten (Prymnesiophyceae) and in natural phytoplankton assemblages in the Ross Sea, Antarctica. TEP production in culture was a function of growth rate and photosynthetic activity and was strongly influenced by photon flux density. The concentrations of TEP measured during a bloom, dominated by P. antarctica, were higher than those produced by coastal diatom blooms and were correlated with chlorophyll a (Chl a), being low at Chl a levels below 3 μgL^?1 but increasing rapidly at greater Chl a concentrations. Because higher chlorophyll hek are dominated 4 larger P. antarctica colonies, this relationship suggests that TEP was produced primarily by sloughing and disintegration of the colonial matrix. TEP concentrations (both absolute and relative to Chl a) increased as the bloom's biomass increased. Vertical distributions of TEP and Chl a showed TEP: chlorophyll maxima at the bottom of the water column at most stations. Because TEP and floc formation are tightly coupled, we suggest that mucous flocs derived from TEP, rather than intact P. antarctica colonies, are the dominant component of aggregates and subsequent organic carbon vertical flux.     

GENETIC DIVERSITY WITHIN BALTIC SEA POPULATIONS OF NODULARIA (CYANOBACTERIA)1

The determination of the genetic structure of microbial populations has, until recently, required the establishment of many independent clonal cultures for genotypic analysis. In such studies it has been necessary to assume that isolates able to grow in laboratory culture are representative of the full range of diversity within the natural population. In order to test this assumption we used the polymerase chain reaction (PCR) to amplify the intergenic spacer region of the Phycocyanin operon (PC-IGS) from filaments of Nodularia taken both from clonal cultures and from natural populations in the Baltic Sea. Analysis of the nucleotide sequences revealed more variation among 16 cultured isolates than within 23 single filaments sampled from a natural population. As a means of rapidly determining population genetic structure we designed and used mixtures of allele-specific amplification primers in diagnostic PCRs to identify which PC-IGS allele was present in single filaments from natural cyanobacterial assemblages. Using this method, we determined the PC-IGS genotype of 156 filaments from 9 sampling stations throughout the central basin of the Baltic Sea in July 1996. Our results show that two distinct genotypes of Nodularia are present in the population at all stations. Although the two types were present in approximately equal numbers, they were not distributed uniformly.     

FIVE NEW GOMPHONEMA SPECIES (BACILLARIOPHYCEAE) FROM RIVERS IN SOUTH AFRICA AND SWAZILAND1

Diatom material from South Africa and Swaziland was examined with light and scanning electron microscopy. Five new taxa are proposed: Gomphonema crocodilei, G. quasicrocodilei, G. venusta, G. latistigmata, and G. cholnokyi. Gomphonema venusta has been previously misidentified as Gomphonema clevei Fricke and G. cholnokyi as G. subclavatum (Grunow) Grunow. All of the species possess biseriate striae, areolae with external vela, a true stigma, and marginal laminae underlying the valve margin and mantle. Compared to the other members of gomphonemoid lineage, the five new species show greatest similarity to Gomphoneis mesta Passy-Tolar & Lowe, G. magna Kociolek & Stoermer, and G. rhombica (Fricke) Merino, García, Hernández-Mariné, & Fernández and to the Herculeana lineage of Gomphoneis sensu Kociolek and Stoermer. The most recent phylogenetic analysis of the genus Gomphoneis showed that the two lineages of the genus. Herculeana and Elegans, are not monophyletic. The Herculeana lineage is more closely allied to Gomphonema than to the Elegans lineage. Since the Elegans lineage contains the generitype of Gomphoneis (G. elegans (Grunow) Cheve), and the species described here are more closely related to the Herculeana lineage, we have assigned them to Gomphonema.     

STEROLS OF PORPHYRIDIUM(RHODOPHYTA)1,2

Species of the unicellular Porphyridium have been examined for their sterol content. Clones of 4 species maintained in axenic, chemically-defined culture were analyzed—these included P. sordidum Geitler, P. purpureum (Bory) Ross, P. aerugineum Geitler and P. violaceum Kormnann(P. griseum Geitler was not available to use for examination). The major sterol was 22-dehydrocholesterol in all except P. aerugineum in which there was a mixture of this sterol, cholesterol and higher sterols. Traces of C₂₈ and C₂₉ sterols were detected in most instances as well.     

INTERSPECIFIC AND INTRASPECIFIC MORPHOLOGICAL VARIATION IN MANATEES (SIRENIA: TRICHECHUS)

The three living species of Trichechus are clearly defined and well exemplify the degree of variability and taxonomic value of morphological characters in a well-understood mammalian genus. Statistical analysis of the largest sample of manatee skulls yet studied has allowed us to identify small suites of characters that effectively distinguish these species. The two subspecies of T. manatus proposed by Hatt (1934) can likewise be distinguished, and their use as taxonomic categories seems justified. This suggests that the cool winters of the northern Gulf Coast, on the one hand, and the deep water and strong currents of the Straits of Florida, on the other, are effective barriers to gene flow between Florida and Antillean manatees. Alleged taxonomic distinctions within T. senegalensis , however, have no demonstrated basis. No significant sexual dimorphism was detected in skulls of any of the species. Many interspecific differences can be correlated with feeding ecology, but others remain unexplained. Features of tooth crown morphology are among the most constant characters examined, but some osteological characters are equally good. T. manatas and T. senegalensis (which are phenetically the most similar) also seem to share a more recent common ancestor than either does with T. inunguis. However, the three species probably separated from each other at nearly the same time. T. inunguis has since become the most derived species, while T. senegalensis has changed the least.