BIOGEOGRAPHY AND TAXONOMY OF BATRACHOSPERMUM HELMINTHOSUM (BATRACHOSPERMALES,RHODOPHYTA) IN JAPAN INFERRED FROM RBCL GENE SEQUENCES1

Twenty‐seven individuals of Batrachospermum helminthosum Bory were collected from various sites in Japan, from temperate Iwate Prefecture to subtropical Okinawa Prefecture. The chloroplast‐encoded rbcL gene was sequenced from each sample. Phylogenetic analyses were performed using these and previously published sequences of 12 samples from North America. There were five haplotypes among the Japanese samples. Haplotype 1 was collected throughout a large geographical area corresponding to most of the sampling range in this study and was represented by most individuals (21 individuals). The other haplotypes were represented by one to three individuals, respectively. These distributions of haplotypes suggest the occurrence of one large metapopulation and/or recent colonization over a wide geographical area in Japan. Phylogenetic analysis showed two well‐supported clades within B. helminthosum, one clade containing four haplotypes (1–4) from Japan and the other clade comprising five North American haplotypes. Such differences between Japanese and North American haplotypes might be a result of continental drift and subsequent isolation in the Mesozoic era. The position of haplotype 5 from Okinawa in Japan is poorly resolved, but it is not closely related to the other four Japanese haplotypes. It is suggested that several taxonomic characteristics (sexuality, point of origin of carpogonium‐bearing branches, and trichogynes with or without basal knobs or branching) is not fixed genetically but is affected by environmental conditions. Based on the results of the present study, B. coerulescens Sirodot and B. elegans Sirodot should be placed in synonymy with B. helminthosum.     

DISTRIBUTION AND SYSTEMATICS OF BATRACHOSPERMUM (BATRACHOSPERMALES,RHODOPHYTA) IN NORTH AMERICA. 3. SECTION SETACEA1

Six populations of Batirachospermum section Setacea from North America were compared to eight type specimens using multivariate morphometrics and image analysis. From this analysis, four species in this section were distinguished worldwide: B. atrum (Hudson) Hartley [syn. B. gallaei Sirodot]; B. orrthostichum Skuja, B. sertularina (Bory) Bory]; B. diatyches Entwisle; B. androinvolucrum sp. nov.; and B. puiggarianum Grunow in Wittrock et Nordstedt (syn. B. angolense Welwitsch ex West et West, B. nigrescens Welwitsch ex West et West). Two of these species were found in North America: B. atrum in California and Texas and B. androinvolucrum in British Columbia, Washington State, and Alabama. The new species, B. androinvolucrum, is distinguished by having spermatangia restricted to one-celled involucral bracts of the carpogonial branch.     

BIOGEOGRAPHY OF BATRACHOSPERMUM HELMENTOSUM (BATRACHOSPERMALES, RHODOPHYTA) IN NORTH AMERICA USING MOLECULAR AND MORPHOLOGICAL DATA

Over the course of 3 years (1997–1999), 72 stream sites were sampled for epilithic diatom communities. The analysis of these samples has led to the identification of over 325 species of diatoms. In addition to sampling the diatom community, selected physical and chemical parameters were recorded from each stream reach. These parameters included pH, specific conductance, current velocity, SRP, nitrate, silica, and total alkalinity. Canonical Correspondence Analysis (CCA) was used to identify influential environmental parameters and to assess the response of the diatom community to prominent anthropogenic inputs in the region (i.e. coal mine drainage, eutrophication). The initial analyses indicate that pH was the most influential environmental parameter along the first CCA axis. This shift was not unexpected, as acid mine drainage (AMD) in the region leads to a wide range of pH values (2.8–7.93). The highly acidic sites were characterized by species of the genus Eunotia (specifically E. exigua and E. steineckei ), Frustulia rhomboides , and Pinnularia subcapitata. Furthermore, Achnanthidium minutissimum was the most widely distributed of the diatom species encountered, being found at 94% of the sites sampled. Streams that fluctuated between acidic and circumneutral pH (termed "teeter-totter") had greater abundances of Brachysira vitrea than other streams in this survey. Further implications for the use of these diatom communities as biomonitoring tools and the distribution of assemblages within the Western Allegheny Plateau will be discussed.     

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.     

DISTRIBUTION AND SYSTEMATICS OF BATRACHOSPERMUM(BATRACHOSPERMALES,RHODOPHYTA) IN NORTH AMERICA. 6. SECTION TURFOSA1

Twenty-one populations of Batrachospermum section Turfosa from North America were compared to nine type and two historically important specimens using multivariate morphometrics and image analysis. The protologues of six other infrageneric taxa were also compared. From this analysis, six species are recognized worldwide: B, de-sikacharyi Sankaran, B. gombakense Kumano et Ratnasabapathy, B. keratophytum Bory de Saint-Vincent [syn. B. vagum var. keratophytum (Bory de Saint-Vincent) Sirodot, B. gulbenkianum Reis, and B. suevorum Kützing nom. Meg.], B. sinense Jao, B. turfosum Bory de Saint-Vincent [syn. B. vagum (Roth) C. Agardh and B. vagum var. undulato-pedicellatum Kumano et Watanabe], and B. vogesiacum F. G. Schultz ex Skuja [syn. B. vagum var. flagelliforme Sirodot, B. flagelliforme (Sirodot) Necchi], These species are distinguished on the basis of carposporophyte-bearing branch cortication, secondary fascicle development, monoecy or dioecy, presence of spermatangia on involucral filaments and monosporangia, and dimensions of trichogynes and carposporangia. Peripheral cortication has been previously used to separate species in this section, but we observed that this feature is quite widespread in the section. Presence of indeterminate gonimoblast filaments has been reported for some taxa in section Turfosa, but no such structures were seen in any of the specimens examined. Only B. keratophytum has been collected in North America, ranging from southwestern Greenland (64°N) to Louisiana (30° N).     

中国串珠藻属新记录

记述了我国串珠藻属的6个新记录种,即灌丛串珠藻(Batrachospermum lochmodes Skuja),内卷串珠藻(B. involutum Vis et Sheath),博雷串珠藻(B. boryanum Sirodot),下位串珠藻(B. hypogynum Kumano et Ratnasabapathy),阿比串珠藻(B. abilii Reis)和茶溪串珠藻(B. theaquum Skuja ex Entwisle et Foard)     

DISTRIBUTION AND SYSTEMATICS OF THE FRESHWATER GENUS SIRODOTIA(BATRACHOSPERMALES,RHODOPHYTA) IN NORTH AMERICA1

Multivariate morphometrics and image analysis were used to determine the number of well-delineated infrageneric taxa of Sirodotia in North America. Three groupings were distinguished from 25 populations examined from Newfoundland and Quebec in the north to central Mexico in the south. These groupings were statistically related to 10 type specimens, and the following species were recognized: Sirodotia huillensis (Welwitsch ex W. et G. S. West) Skuja (syn. S. ateleia Skuja), S. suecica Kylin (syn. S. acuminata Skuja ex Flint and S. fennica Skuja), and S. tenuissima (Collins) Skuja ex Flint. These species are differentiated on the basis of whorl shape and degree of separation at maturity (S. suecica, rounded and appressed; S. huillensis and S. tenuissima, truncated apex and separated), the density of spermatangia (S. huillensis, dense clusters; S. suecica and S. tenuissima, sparsely aggregated), and the mode of germination of the gonimoblast initial (S. suecica and S. tenuissima, from the nonprotuberant side of the fertilized carpogonium; S. huillensis from the protuberant side). Sirodotia huillensis was found only in the desert-chaparral, whereas S. suecica and S. tenuissima occurred from south-temperate to boreal regions in cool (temperature 8–18° C), low ion (specific conductance 10–99 μS · cm^?1), and mildly acidic to neutral (pH 5.7–7.3) waters.     

DISTRIBUTION AND SYSTEMATICS OF FRESHWATER CERAMIALES (RHODOPHYTA) IN NORTH AMERICA1

Twenty-five freshwater populations of Ceramiales were collected in North America, 24 of which were from the tropical rainforest region of Central America and the Caribbean. The streams tended to be moderate in mean current velocity (X?= 23.3 cm·S^?1) and maximum width (X?= 6.3 m) but high in temperature (X?= 23.1°C), pH (X?= 7.9), and specific conductance (X?= 320 μS·cm^?1). Three Bostrychia species were restricted to the Caribbean islands: B. moritziana (Sonder ex Kütz.) J. Ag. (syn. B. cornigera Mont. and B. radicans f. moliforme Post), with ecorticate indeterminate axes, monosiphonous ultimate branches, and cladohaptera; B. radicans (Mont.) Mont. (syn. B. leprieurii Mont and B. rivularis Harv.), with ecorticate and polysiphonous axes throughout and cladophaptera; and B. tenella (Lamour.) J. Ag., with corticate indeterminate axes, monosiphonous ultimate branches, and peripherohaptera. Ballia prieurii Kütz. was found in Belize and Costa Rica and was characterized by rebranched determinate laterals, opposite branching, and long apical cells (X?= 61 μm) and axial cells (X?= 43 μm). Caloglossa leprieurii (Mont.) J. Ag. was localized in Puerto Rico while. C. ogasawaerensis Okam. was collected only in Costa Rica. The two species were separated by site of branching (midrib vs. margin) and blade width (X?= 384 vs. 861 μm). Polysiphonia subtilissima Mont. from Florida and Jamaica had four pericentral cells, no cortication, rhizoids arising from pericentral cells, and branches initiated at trichoblast scars.     

GRACILARIA VERMICULOPHYLLA (RHODOPHYTA,GRACILARIALES) IN THE VIRGINIA COASTAL BAYS,USA: COX1 ANALYSIS REVEALS HIGH GENETIC RICHNESS OF AN INTRODUCED MACROALGA

Gracilaria vermiculophylla (Ohmi) Papenfuss is an invasive alga that is native to Southeast Asia and has invaded many estuaries in North America and Europe. It is difficult to differentiate G. vermiculophylla from native forms using morphology and therefore molecular techniques are needed. In this study, we used three molecular markers (rbcL, cox2‐cox3 spacer, cox1) to identify G. vermiculophylla at several locations in the western Atlantic. RbcL and cox2‐cox3 spacer markers confirmed the presence of G. vermiculophylla on the east coast of the USA from Massachusetts to South Carolina. We used a 507 base pair region of cox1 mtDNA to (i) verify the widespread distribution of G. vermiculophylla in the Virginia (VA) coastal bays and (ii) determine the intraspecific diversity of these algae. Cox1 haplotype richness in the VA coastal bays was much higher than that previously found in other invaded locations, as well as some native locations. This difference is likely attributed to the more intensive sampling design used in this study, which was able to detect richness created by multiple, diverse introductions. On the basis of our results, we recommend that future studies take differences in sampling design into account when comparing haplotype richness and diversity between native and non‐native studies in the literature.     

SYNTHESIS OF MOLECULAR RESEARCH ON BATRACHOSPERMUM HELMINTHOSUM (RHODOPHYTA) FROM STREAM REACHES IN EASTERN NORTH AMERICA

Vis, M. L., Hall, M. M., Machesky, N. J. & Miller, E. J. Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701 USA The freshwater red alga Batrachospermum helminthosum was collected from eleven streams throughout the species range in eastern North America as follows: three stream reaches from Ohio, and one each from Michigan, Indiana, Tennessee, Louisiana, North Carolina, Connecticut, Rhode Island and Massachusetts. The molecular marker technique of inter-simple sequence repeats (ISSR) and sequence data from the plastid encoded rubisco large subunit gene (rbcL), the mitochondrial COX2-COX3 gene spacer region, and the nuclear region of ITS1-5.8S rDNA-ITS2 were employed to examine biogeographic trends in this alga. Analysis of the rbcL sequence revealed 5 genotypes with one genotype representing individuals from seven stream reaches. Data from the ISSR molecular markers gave a distinct banding pattern for each of 165 individuals examined. ISSR results showed all individuals within a reach clustered together but did not provide well-defined groupings based on stream reach. The sequence data for the COX2-COX3 gene spacer was invariant among individuals from a stream reach. The individuals from Connecticut, Rhode Island and 2 Ohio stream reaches were identical and similarly the individuals from the North Carolina and another Ohio location did not vary in sequence so that seven genotypes were recorded among the individuals from the eleven stream reaches. Analysis of the ITS1-5.8S rDNA-ITS2 region showed sequence variation not only among individuals from different streams but also among individuals from the same reach. The utility and congruency of these data sets to answer biogeographic questions will be discussed.     

INCORPORATION OF FRESHWATER RHODOPHYTA INTO THE CASES OF CHIRONOMID LARVAE (CHIRONOMIDAE,DIPTERA) FROM NORTH AMERICA1

Chironomid larvae incorporate pieces of freshwater red algae into their cases from a wide geographic range in North America, extending from southern Canada to central Mexico. The Rhodophyta used in this process represent two orders (Acrochaetiales and Batrachospermales), five genera (Audouinella, Batrachospermum, Lemanea, Paralemanea, and Sirodotia), and 14 species from 21 locations. Three genera from the chironomid subfamily Orthocladiinae make these cases: Cardiocladius, Eukiefferiella, and Orthocladius. The Eukiefferiella claripennis group was the most frequently observed infrageneric taxon using red algae in its cases. The cases were tubular in shape with longitudinally oriented strips of algae held together by silken threads. Some of the cases constructed with Batrachospermum and Sirodotia also had several lateral branches of the alga radiating from the tube.     

PHYLOGENY OF THE BATRACHOSPERMALES (RHODOPHYTA) INFERRED FROM rbcL AND 18S RIBOSOMAL DNA GENE SEQUENCES

The sequence data from the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase ( rbc L) gene and 18S ribosomal DNA (small subunit) of taxa in the freshwater rhodophyte order Batrachospermales were used to construct phylogenetic hypotheses. Taxa examined in this study represent four families, eight genera, and six sections of the genus Batrachospermum . In addition, Rhododraparnaldia oregonica Sheath, Whittick et Cole, was included in the analysis because it shares particular ultrastructural, reproductive, and morphological characteristics with members of the Batrachospermales and Acrochaetiales. The trees generated from each gene, as well as a combined data set, were largely congruent. Rhododraparnaldia consistently occurs on an early branch within the Acrochaetiales – Palmariales clade and does not appear to be a member of the Batrachospermales. In addition, Thorea violacea Bory de St. Vincent was not closely related to the other taxa of the Batrachospermales in all trees and hence the family Thoreaceae does not appear to be a natural grouping within this order. All other taxa analyzed, which are presently classified within this order, formed a monophyletic clade in most analyses. Psilosiphon scoparium Entwisle was not closely allied with the taxa of the Lemaneaceae, lending support to the newly proposed family Psilosiphonaceae. Sequence data from the remaining taxa of the Lemaneaceae support the concept of a derived monophyletic clade. The genus Batrachospermum appears to comprise many morphologically similar but distantly related taxa, which will need further investigation to resolve their taxonomic status. Tuomeya, Sirodotia and Nothocladus are retained at the generic level until further data are obtained.     

MOLECULAR IDENTIFICATION OF TWO SIBLING SPECIES UNDER THE NAME GRACILARIA CHILENSIS (RHODOPHYTA,GRACILARIALES)1,3

Molecular markers belonging to three different genomes, mitochondrial (cox2‐3 spacer), plastid (RUBISCO spacer), and nuclear (internal transcribed spacer 1), were used to compare Gracilaria chilensis samples collected along the Chilean coast with samples ascribed to G. chilensis from the West Pacific Ocean (New Zealand and Australia). Our data are in agreement with previous studies suggesting two sibling species currently going under the name G. chilensis that co‐occur in New Zealand. One of these, a New Zealand sample previously examined by Bird and others in 1990, is conspecific with G. chilensis from Chile. Finally, our results demonstrate clearly that most of the sequences in GenBank reported as G. chilensis are based on misidentified material.     

PHYLOGEOGRAPHY OF CUTLERIA CYLINDRICA (CUTLERIALES,PHAEOPHYCEAE) IN NORTHEASTERN ASIA,AND THE IDENTITY OF AN INTRODUCED POPULATION IN CALIFORNIA1

Cutleria cylindrica Okamura was described from Japan in 1902 and has been reported only from northwestern Asia until its relatively recent discovery in California, USA, and Baja California, Mexico. To clarify the genetic relationships within and among the disjunct populations, we carried out a molecular phylogenetic study, as well as the examination of sex ratio and the life‐history patterns, of populations in Japan, Korea, and California. Based on the DNA sequences of mitochondrial genes cox2, cox3, the open reading frame (ORF) region, and the spacer between cox3 and ORF, a total of 23 haplotypes were detected in the 85 individuals from 20 localities in Japan, Korea, and California. All localities in Japan and Korea included multiple haplotypes, but only a single haplotype was found in California. There was a positive relationship between distance and genetic divergence in Japan and Korea. The single haplotype found in California was the same as one occurring in Japan (Aomori Pref. and Fukuoka Pref.) and Korea (Daedaepo, Pusan). Both male and female gametophytes were distributed in most northeastern Asian populations. Only female gametophytes, developing parthenogenetically from female gametes, were found in California and Aomori Pref., Japan. On the basis of these results, we conclude that the disjunct population of C. cylindrica in California originated from a relatively recent introduction from Japan and shares its origin with the parthenogenetic population in the Tsugaru Strait.     

PHYLOGEOGRAPHY AND WITHIN-STREAM GENETIC VARIATION OF BATRACHOSPERMUM HELMENTOSUM (RHODOPHYTA) USING INTER-SIMPLE SEQUENCE REPEATS

A relatively new molecular marker technique, inter-simple sequence repeats (ISSR), was used to assess the within-stream reach genetic variation and phylogeography of the freshwater red alga, Batrachospermum helmentosum. The within-stream reach study was undertaken in Southeastern Ohio, where 58 individuals were collected from an approximately 200 m stream length. Individuals appeared to be congregated into three distinct areas, with no plants between areas. Using three primers, 100 polymorphic bands were scored. Although it was hypothesized that genetic variation would reflect physical distance, the results showed six distinct genetic groupings, some of which consisted of physically distant individuals. Overall, genetic diversity was greater among individuals than between geographic and genetic groups. To investigate phylogeography, 11 stream reaches (3 from Ohio and one each from Connecticut, Massachusetts, Rhode Island, North Carolina, Tennessee, Indiana, Michigan and Louisiana) were sampled throughout the alga's known distribution in North America. Approximately 15 individuals were collected per stream for ISSR analysis. Five primers, yielding 243 polymorphic bands, were used to examine variation among all individuals. Neighbor-joining trees generated from the Dice, Jaccard and Excoffier coefficients showed that individuals from the same stream formed a clade. Among streams there was no distinct correlation between geographic distance and genetic similarity. Genetic groupings varied slightly among distance coefficients employed. Michigan and Tennessee, Connecticut and Rhode Island, and Indiana and Massachusetts streams consistently showed the same relationship, but the relationships among the three Ohio streams and Louisiana stream were variably related to each other.     

BATRACHOSPERMUM HETEROCORTICUM SP. NOV. AND POLYSIPHONIA SUBTILISSIMA (RHODOPHYTA) FROM FLORIDA SPRING-FED STREAMS1

Polysiphonia subtilissima Mont. Is reported for the first time from a freshwater environment. The presence of four pericentral cells, subdichotomous branching, apical trichoblasts and rhizoids arising from pericentral cells combined with a lack of cortication and reproductive cells is consistent with marine populations of this species. The range of filament length is 1.4–4.7 cm. Branch diameters are 38–76 μm and pericentral cell lengths are 58–125 μm. Batrachospermum heterocorticum sp. nov. is distinguished primarily by a developmental change in cortical filaments from typical cylindrical cells (5.0–7.9 μm diam in initial stages to enlarged, elliptical cells (12.9–24.1 μm diam) in mature axes. Another unique feature of this species is carpogonia with cylindrical, pedicellate trichogynes on stringht carpogonial branches in mid to outer portions of lateral whorls. Other characteristics of B. heterocorticum include the following: olive-green color, 2–6 cm length, dichotomous to trichotomous fascicles in 4–7 tiers, 385–647 μm whorl diameters, 109–198 μm carpospore diameters and relatively small “chantransia” filaments.