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Morphological and molecular investigation of freshwater Hildenbrandia (Hildenbrandiales, Rhodophyta) with a new species reported from Japan

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The morphological characterization and phylogenetic analysis based on molecular methods were executed on the freshwater Hildenbrandia specimens collected in this study. The cell size and filament height of the specimen collected from Pipa Lake, Japan were larger than the widely recognized species Hildenbrandia angolensis and Hildenbrandia rivularis, whereas overlapped with Hildenbrandia jigongshanensis. Based on molecular evidences of rbcL phylogenetic analysis and comparison of ITS1 secondary structure, specimen collected from Shiga Prefecture, Japan was proposed as a new species Hildenbrandia japananense. It brings the freshwater Hildenbrandia species in Japan to two. The specimens collected from Niangziguan, Shanxi province, China represent two new records of H. jigongshanensis. It enriches the species diversity of genus Hildenbrandia and increases the distribution diversity of this freshwater red algal taxon.
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Phytotaxa 423 (2): 068–074
https://www.mapress.com/j/pt/
Copyright © 2019 Magnolia Press Article PHYTOTAXA
ISSN 1179-3155 (print edition)
ISSN 1179-3163 (online edition)
68 Accepted by Thomas Pröschold: 20 Oct. 2019; published: 4 Nov. 2019
https://doi.org/10.11646/phytotaxa.423.2.2
Morphological and molecular investigation of freshwater Hildenbrandia
(Hildenbrandiales, Rhodophyta) with a new species reported from Japan
FANG-RU NAN1, JIN-FEN HAN1, JIA FENG1, JUN-PING LV1, QI LIU1, XU-DONG LIU1 & SHU-LIAN XIE1*
1 School of Life Science, Shanxi Key Laboratory for Research and Development of Regional Plants, Shanxi University, Taiyuan 030006,
China
* Corresponding author (e-mail: xiesl@sxu.edu.cn)
Abstract
The morphological characterization and phylogenetic analysis based on molecular methods were executed on the freshwater
Hildenbrandia specimens collected in this study. The cell size and filament height of the specimen collected from Pipa
Lake, Japan were larger than the widely recognized species Hildenbrandia angolensis and Hildenbrandia rivularis,
whereas overlapped with Hildenbrandia jigongshanensis. Based on molecular evidences of rbcL phylogenetic analysis and
comparison of ITS1 secondary structure, specimen collected from Shiga Prefecture, Japan was proposed as a new species
Hildenbrandia japananense. It brings the freshwater Hildenbrandia species in Japan to two. The specimens collected from
Niangziguan, Shanxi province, China represent two new records of H. jigongshanensis. It enriches the species diversity of
genus Hildenbrandia and increases the distribution diversity of this freshwater red algal taxon.
Keywords: Red algae, rbcL, ITS1, Morphology
Introduction
Genus Hildenbrandia Nardo belongs to Rhodophyta, Florideophyceae, Hildenbrandiales, Hildenbrandiaceae. It is
morphologically characterized by simple crustose thallus construction with a single basal layer and a derived vertical
cell files (Bird and Mclachlan 1992). It inhabited in both marine and freshwater environments. The freshwater
Hildenbrandia is specific in habitat selection and only found at streams with high water temperatures and high specific
conductance values (Sheath et al. 1993). Therefore population of freshwater Hildenbrandia is increasingly rare with
the serious pollution of water environment. The freshwater species reproduce by asexual means including stolon,
fragmentation and gemmae (Nichols 1965).
Currently, five freshwater species have been described including H. rivularis (Liebmann) Agardh (1851: 379),
H. angolensis Welwitsch ex West & West (1897: 3), Hildenbrandia arracana Zeller (1873: 192), Hildenbrandia
ramanaginaii Khan (1974: 238) and H. jigongshanensis Nan & Xie (2017: 245) (Agardh 1851, Zeller 1873, West and
West 1897, Kahn 1974, Nan et al. 2017). The most extensively reported species were H. rivularis and H. angolensis.
H. rivularis is most commonly reported in Europe, also in North America, South America, Asia and Australia (West
and West 1897, Bourrelly 1985, Necchi 1987, Sheath et al. 1993, Sherwood and Sheath 1999, 2000). H. angolensis is
a common freshwater species in North America (Sheath et al. 1993, Sherwood and Sheath 1999) and was first reported
from Europe in 1997 (Ros et al. 1997). H. arracana was only reported in Burma and H. ramanaginaii was described
from India, whereas their taxonomic identity is unclear due to the unavailability of the type materials (Zeller 1873,
Kahn 1974). The fifth freshwater species H. jigongshanensis was described from China (Nan et al. 2017).
The widely reported freshwater Hildenbrandia species H. angolensis and H. rivularis are distinguished by
morphometric measurements, with H. angolensis characterized by smaller mean cell and filament dimensions (cells
4.0 × 4.4 µm, filaments 46.5 µm) and H. rivularis by larger parameters (cells 5.8 × 6.6 µm, filaments 55.3 µm)
(Sherwood and Sheath 2003). H. jigongshanensis is separated from other freshwater members by significantly larger
cell dimensions (9.8–19.6) × (9.9–10.4) µm and filament height (364–409 µm) (Nan et al. 2017). However, thallus
thickness has been known to vary with the age of thallus and cell dimensions are variable in different parts of the
INVESTIGATION OF FRESHWATER HILDENBRANDIA Phytotaxa 423 (2) © 2019 Magnolia Press 69
thallus (Starmach 1969). Therefore, species identification of genus Hildenbrandia based on traditional morphometric
analysis is difficult.
Molecular data have been used widely in genus Hildenbrandia for species identification and phylogenetic analysis
(Sherwood and Sheath 1999, 2000, Sherwood et al. 2002). The chloroplast rbcL gene sequences have been used
for investigation on systematics of Hildenbrandiales in North America and Europe, and proved powerful in species
relationship inference (Sherwood and Sheath 1999, 2000). Sherwood and Sheath investigated 57 Hildenbrandia
samples collected from North America, South America, Europe and Africa based on morphometric and molecular
analysis in 2003 and concluded that H. rivularis was monophyletic while H. angolensis was genetically heterogeneous
(Sherwood and Sheath 2003). Nan et al. identified a freshwater specimen from Henan province, China using gene
sequences of rbcL and 18S rRNA and proposed a new species H. jigongshanensis (Nan et al. 2017).
Species diversity and geographical distribution of freshwater genus Hildenbrandia are under estimated and need
to be explored further. We execute morphological and molecular investigation on freshwater Hildenbrandia specimens
collected in this study to determine their taxonomic attribution. Based on morphological characterization and molecular
evidences, a new species collected from Japan and two new records of genus Hildenbrandia in China were reported.
Materials and methods
Algal specimens collected in a flowing stream in Samegai trout farm, Shiga Prefecture, Japan (35°17’57.7”N,
136°20’18.4”E) on Sep. 3rd, 2018 were coded as YZJP, and specimens collected at different sites of Niangziguan
Lake, Shanxi province, China (37°58’24.6”N, 113°53’27.9”E) on Jul. 7th, 2016 were coded as YZNZG01, YZNZG02.
Morphological characters of fresh Hildenbrandia algae were observed under a BX-51 Olympus microscope equipped
with a charge-coupled camera and cellSensStandard software for photographing (DP72; Olympus, Tokyo, Japan).
The morphological examination were executed under the magnification of 10x and 40x without immersion oil. The
thalli morphology were analyzed by measuring cell size and filament height. Total DNA was extracted from the fresh
thalli following the protocol described by Saunders (1993) with modifications following Vis and Sheath (1997). For
amplification of rbcL and ITS1 gene sequences, primers HILF1 and HILR1, ITSH.1 and ITS10 were used respectively
(Sherwood et al. 2002, Nan et al. 2017). PCR amplifications were conducted in 20 μL volumes containing 12.5 μL
ddH2O, 2.0 μL 10×buffer, 2.0 μL 2.5 mM dNTPs, 0.2 μL Taq DNA polymerase (all from Sangon Biotech Co., Ltd.,
China), 2.0 μL of each primer (10 mM), and 1.0 μL of genomic DNA. PCR products were visualized on 2% agarose
gels, and successful amplification products were purified using a SanPrep column DNA gel purification kit (Sangon,
China). The sequencing was performed on an ABI 3730XL sequencer using both amplifying primers. Sequences
obtained in this study and sequence data for genera Hildenbrandia and Porphyridium (used as outgroup) downloaded
from GenBank (listed in Supplemental Table S1) were assembled in Clustal-X 2.0 (Thompson et al. 1997). The data
matrix was used to construct phylogenetic trees after alignment. Modeltest was used to calculate the optimal substitution
model for rbcL gene sequence (Posada and Buckley 2004). The Neighbor-joining method was performed in the MEGA
5.0 (Tamura et al. 2011) with 1,000 bootstrap repetitions. PHYML software was used to construct maximum likelihood
trees (Felsenstein 1981, Guindon and Gascuel 2003). Bayesian inferences were developed in MrBayes version 3.1.2
(Ronquist and Huelsenbeck 2003). A Markov chain Monte Carlo (MCMC) was initiated in the Bayesian inference
and run for 5,000,000 generations; the trees were sampled every 1000 generations. A consensus tree was summarized
after 1,000 trees of burn-in. The resulting phylogenetic trees were edited using Figtree1.4.2 (http://tree.bio.ed.ac.uk/
software/figtree/). The secondary structures of ITS1 sequence were folded using the RNAfold Web Server by choosing
the minimum free energy algorithm and the option to avoid isolated base pairs (Gruber et al. 2008). The predicted
secondary structures were visualized using the program RNAviz (De Rijk et al. 2003).
Results
Taxonomy
Hildenbrandia japananense F. NAN, J. HAN, J. FENG, J. LV, Q. LIU, X. LIU & S. XIE, sp. nov. (Fig. 1)
Etymology:—The species epithet refers to the Holotype locality (Shiga Prefecture, Japan).
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70 Phytotaxa 423 (2) © 2019 Magnolia Press
Diagnosis:Freshwater alga, thallus palmelloid, bright red, forming crustose thalli on the surface of rocks in
flowing water (Fig. 1a). This species is characterized by medium cell size (10.5 × 8.4 μm) and filament height (308–
491 μm) (Fig. 1b-j). Molecular-assisted identification is necessary for identification of this species.
Type:Japan, Shiga Prefecture, a flowing stream in Samegai trout farm (136.3384E, 35.2994N): growing on the
stones in flowing water, September 3rd, 2018, Ren-Hui Li (Holotype: SXU-JAP18001; Paratype: SXU-JAP18002). The
spescimens were deposited in Herbarium of Shanxi University (SXU), Shanxi University, Taiyuan, Shanxi Province,
China.
Authentic strain:—SXU-JAP18001, Herbarium of Shanxi University (SXU), Shanxi University.
FIGURE 1. Morphological structures of H. japananense. Scale bar 20 μm. a. Specimens collected in flowing water in Shiga Prefecture,
Japan; Crustose thalli growing epilithic on the surface of rocks; b–c. Branching of closely associated filaments; d–f. Monostromatic and
polystromatic areas of the thallus; g–i: Cell shapes internal to the marginal area of the thallus; j: Transection of a colony near the margin
of the thallus.
INVESTIGATION OF FRESHWATER HILDENBRANDIA Phytotaxa 423 (2) © 2019 Magnolia Press 71
Phylogenetic analysis
Phylogenetic tree based on rbcL sequence was illustrated in Fig. 2. The freshwater species H. rivularis formed an
independent branch with strong support values (0.96/912/98), whereas specimens of H. angolensis were polyphyletic
and distributed dispersedly. Specimens collected in Niangziguan Lake, Shanxi province YZNZG01, YZNZG02 formed
sister cluster with H. jigongshanense, the new species reported from China. Specimen collected in Japan (YZJP) formed
an independent branch and distributed at the basal position. The secondary structures of ITS1 sequence for specimens
collected in this study were depicted in Fig. 3. Specimens from Niangziguan Lake YZNZG01, YZNZG02 are identical
in ITS1 sequence and share the same secondary structure (Fig. 3a). The secondary structure of H. jigongshanense was
illustrated in Fig. 3b. Secondary structure of H. japananense was shown in Fig. 3c. The helix numbers of specimens
in Niangziguan and H. jigongshanense were both 10, and nucleotide composition of helix 1, 2, 3, 5, 7 were the same.
Helix number of H. japananense (YZJP) were 12 and nucleotide composition of all helix were different from that of
specimens in Niangziguan and H. jigongshanensis.
FIGURE 2. Bayesian inference tree based on rbcL sequences from species of Hildenbrandia. Support values for all analyses are shown
as follows: Bayesian posterior probabilities / ML bootstrap/ NJ distance bootstrap. ‘-’ denotes <50% support for that analyses at that node;
the new species proposed in this study was highlighted in black.
FIGURE 3. Predicted ITS1 secondary structures of the freshwater specimens. a. Predicted ITS1 secondary structure of specimens collected
in Niangziguan, China; b. Predicted ITS1 secondary structure of H. jigongshanensis; c. Predicted ITS1 secondary structure of specimens
collected in Shiga Prefecture, Japan.
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72 Phytotaxa 423 (2) © 2019 Magnolia Press
Discussion
Morphologically, the cell size (8.4 × 10.5 μm) and filament height (308–491 μm) of the new species proposed in this
study is larger than H. angolensis (cells 4.0 × 4.4 µm, filaments 46.5 µm) and H. rivularis (cells 5.8 × 6.6 µm, filaments
55.3 µm) (Sherwood and Sheath 2003, Nan et al. 2017). Whereas compared with H. jigongshanensis (cells (9.8–19.6)
× (9.9–10.4) μm, filaments (364–409 μm)), a new species reported from China, the morphological measurements were
overlapping (Nan et al. 2017). Another two freshwater Hildenbrandia species were originally reported based on only
morphology description and with no molecular data. H. arracana was only reported in 1873 from Burma characterized
by filament height of 36.3–42.3 μm, which was smaller than the new species in this study (Zeller 1873). H. ramanaginaii
was firstly described from India and characterized by olive-green color, which is evidently different from the new
species described in this study (Kahn 1974). According to previous literatures, freshwater species of Hildenbrandia
are delineated primarily by cell size, filament dimensions and thallus thickness (Nichols 1965). However, it was also
reported that cell dimensions are variable in different parts of the thallus (Starmach 1969). Consequently, criteria of
cell dimensions and filament height used for species discrimination among genus Hildenbrandia are problematic. The
new species proposed in this study was in many cases difficult to distinguish with unique morphological characters.
Molecular methods assisted systematics of genus Hildenbrandia have showed the monophyly of H. rivularis
(Sherwood and Sheath 1999, 2000, Sherwood et al. 2002). The new species H. jigongshanensis reported from China
was also proposed by the evidence of rbcL sequence (Nan et al. 2017). Phylogenetic analysis based on rbcL gene
supported the new species H. japananense in this study. It has been reported that identification of new species in
freshwater red algae can only base on molecular markers. Molecular evidences are necessary for identification of
H. japananense, as the same with the proposal of another two freshwater red algal new species Sheathia americana
and Batrachospermum shanxiense (Salomaki et al. 2014, Chapuis et al. 2017). The secondary structure of ITS1
sequence has also been used for phylogenetic analysis in the freshwater algal genus Ulvella and proved powerful in
new species identification (Su et al. 2018). The ITS1 secondary structure of specimens collected in Japan exhibited
obvious difference in helix number and nucleotide composition with that of H. jigongshanensis, thus supporting it a
new species H. japananense.
Previous reports on distribution of genus Hildenbrandia in Japan were scarce, with only four marine populations
and one freshwater collection (Okamura 1936, Segawa 1981, Yoshida 1990, Yoshida 1998, Yoshida et al. 2015). The
freshwater collection corresponded to H. rivularis and was reported in 1977 (Hirose et al. 1977), since when there was
no reports on freshwater Hildenbrandia again. The new species H. japananense proposed in this study bring the total
number of freshwater Hildenbrandia species in Japan to two.
The distributions of freshwater genus Hildenbrandia in China were only reported in Shanxi, Fujian, Hubei, Xizang
and Taiwan corresponding to H. rivularis, in combination with Jigongshan, Henan province corresponding to H.
jigongshanensis (Shi et al. 2006, Anon 2012; Nan et al. 2017). The population of freshwater Hildenbrandia collected
in this study were reported in Niangziguan, Shanxi province for the first time and represented two new records of
H. jigongshanensis in China. Further detailed investigation should be executed to clarify the species diversity of
freshwater Hildenbrandia in China.
Conclusion
Morphological measurements and molecular-based phylogenetic analysis were implemented on freshwater
Hildenbrandia specimens collected in this study. Based on molecular sequences of rbcL and comparison of ITS1
secondary structure, a new species H. japananense was proposed collected from Shiga Prefecture, Japan. It brings
the freshwater Hildenbrandia species in Japan to two. The other two specimens collected from Niangziguan, Shanxi
Province, China were identified as H. jigongshanensis and represented two new records in China.
Acknowledgements
We appreciate professor Renhui Li from Institute of Hydrobiology, Chinese Academy of Sciences for collecting and
providing the samples. This study was funded by the National Natural Science Foundation of China (grant number
31670208, 41871037 to Shulian Xie and 31800172 to Fangru Nan) and the Fund for Shanxi “1331 Project”.
INVESTIGATION OF FRESHWATER HILDENBRANDIA Phytotaxa 423 (2) © 2019 Magnolia Press 73
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... Our current taxonomic knowledge of the genus Hildenbrandia accounts for 18 taxonomically accepted species including six freshwater species: H. angolensis Welwitsch ex West Agardh (widespread in Europe) (Guiry & Guiry 2020). Hildenbrandia japananensis and H. jigongshanensis were only recently described based on morphological characterization and molecular data of rbcL DNA sequence, as well as secondary structure of rDNA ITS1 for H. japananensis and 18S rDNA for H. jigongshanensis (Nan et al. 2019), and are the only species for which type material was sequenced. Sequence data are publicly available for four of the six currently described freshwater species: H. angolensis, H. rivularis, H. japananensis and H. jigongshanensis. ...
... To date, four species of Hildenbrandia were reported from Japan including two marine species, H. occidentalis Setchell (as H. yessoensis Yendo in Yendo (1920)) and H. rubra (Sommerfeld) Meneghini (Segawa 1981), and two freshwater species, H. rivularis (Hirose et al. 1977;Kumano 2002;Kitayama 2014) and H. japananensis (Nan et al. 2019). The only molecular work done on Japanese Hildenbrandia was performed on H. japananensis (Nan et al. 2019) based on the specimens from a trout aquaculture farm (Samegai Trout Farm, Kaminyu, Maibara, Shiga Prefecture, Japan, upper stream of river flowing to Lake Biwa; incorrectly referred to as Lake Biwa (misspelled Lake Pipa) in Nan et al. (2019) abstract). ...
... To date, four species of Hildenbrandia were reported from Japan including two marine species, H. occidentalis Setchell (as H. yessoensis Yendo in Yendo (1920)) and H. rubra (Sommerfeld) Meneghini (Segawa 1981), and two freshwater species, H. rivularis (Hirose et al. 1977;Kumano 2002;Kitayama 2014) and H. japananensis (Nan et al. 2019). The only molecular work done on Japanese Hildenbrandia was performed on H. japananensis (Nan et al. 2019) based on the specimens from a trout aquaculture farm (Samegai Trout Farm, Kaminyu, Maibara, Shiga Prefecture, Japan, upper stream of river flowing to Lake Biwa; incorrectly referred to as Lake Biwa (misspelled Lake Pipa) in Nan et al. (2019) abstract). While the authors ruled out the conspecificity of the specimens collected from Japan and China with H. angolensis and H. rivularis based on molecular evidence, absence of molecular data from the other Asian species, H. ramanaginae, does not exclude the possibility that these species may be conspecific. ...
Article
The genetic diversity of freshwater Hildenbrandia species (Florideophyceae, Hildenbrandiales) from Japan was reassessed based on specimens collected from Kagoshima (Kyushu), Hyogo and Kanagawa Prefectures (Honshu), using rbcL and 18S rDNA sequences. Phylogenetic results suggested the occurrence of only one species of freshwater Hildenbrandia in Japan, H. japananensis. The clade H. jigongshanensis–H. japananensis is sister to H. rivularis. Hildenbrandia angolensis was paraphyletic and positioned basally among the freshwater Hildenbrandia clades. Further- more, the genetic similarity between H. jigongshanensis and H. japananensis determined by rbcL and 18S sequences as well as the intraspecific morphological plasticity led us to the conclusion that the species from China and Japan were conspecific. Accordingly, we propose the taxonomic lumping of H. jigongshanensis and H. japananensis. In addition, we recommend additional molecular and morphological studies on Asian freshwater Hildenbrandia to determine whether H. jigongshanensis is conspecific with H. ramanaginae previously described from India.
... Also, some genera can be found in freshwater, brackish or marine environments, but include stenohaline species that differ in their salinity affinities (e.g. Caloglossa J. Agardh, Hildenbrandia Nardo, Pneophyllum Kützing; Krayesky et al. 2012;Zuljevic et al. 2016;Nan et al. 2019;Vieira et al. 2021). More rarely, some red algal species tolerate a wide range of salinities and have been found in both marine and freshwater environments. ...
Article
Species discovery is facilitated by the application of molecular tools and the exploration of poorly studied habitats. Recent surveys in Bafa Lake, Turkey, which experienced a transition from oligo- to mesohaline conditions during the last 40 years, led to the finding of two species of the genera Lophosiphonia and Polysiphonia. Our molecular and morphological study showed that one of the species corresponded to L. obscura, while the other differs from previously described Polysiphonia species and is proposed as P. sukatarii sp. nov. The new species differs from related congeners by a rbcL sequence divergence ≥5.8% and is morphologically distinguished by having four pericentral cells and trichoblasts arranged several segments apart. Lophosiphonia obscura, and its closely related species L. hemisphaerica comb. nov. and L. boldii comb. nov., have been previously found in similar habitats in Europe and Atlantic North America. Along with its record from Bafa Lake, P. sukatarii sp. nov. has been discovered in open shore algal turfs from the Canary Islands, suggesting that it is a euryhaline species with probably a wide distribution. Our work highlights that poorly studied habitats still harbour undiscovered species and additional surveys are necessary to acquire a more comprehensive understanding of the macroalgal diversity.
... Phylogenetic data, however, indicate that populations labeled under either of these two epithets, H. angolensis and H. rivularis, constitute polyphyletic lineages, which are far less widespread than presently presumed based on these two names. Recent DNA-based studies described as new species some of these lineages previously labeled under the epithet H. rivularis based on morphological data (Caisová and Kopecký 2008;Nan et al 2017;Nan et al 2019;Vieira et al 2021). Hildenbrandia has a rather simple morphology with few diagnostic features and expresses large morphological variation (Sherwood and Sheath 2003;Vieira et al 2021). ...
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Chapter
Subphylum Eurhodophytina is classified into two classes: Bangiophyceae and Florideophyceae. Class Bangiophyceae has a single order Bangiales, which is divided into two families: Bangiaceae with the type genus, Bangia, and a single species Bangia atropurpurea as the only occurring in freshwaters; and Granufilaceae with the monospecific type genus, Granufilum rivularis exclusively of freshwaters. Class Florideophyceae has several subclasses and those treated in this chapter are: Corallinophycidae, Hildenbrandiophycidae, and Rhodymeniophycidae. Corallinophycidae has only one species in freshwaters (Pneophyllum cetinaensis). Hildenbrandiophycidae has marine and freshwater members, and three species have been recognized in freshwater (Hildenbrandia angolensis, H. jigongshanensis, and H. rivularis). Rhodymeniophycidae has freshwater representatives in two orders: Ceramiales with three genera: Bostrychia (seven species), Caloglossa (six species), and Polysiphonia (one species); Gigartinales with the single genus Sterrocladia (two species).
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Fifteen populations of the widespread freshwater 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 midwestern hemlock-hardwood populations were in one grouping, whereas the deciduous forest, coastal plain, and eastern hemlock-hardwood populations were in a second. However, 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 nucleotide variation in ITS sequences, we conclude that B. gelatinosum is a morphologically variable and geographically widespread species that is a valid taxonomic entity.
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Hildenbrandia rivularis was collected in Denmark and the United States. The life history of this encrusting red alga is described on the basis of unialgal cultures maintained for more than 2 years. Significant changes in cellular and colonial morphology are reported. Events of the life history (i.e., reproduction by stolons, fragmentation, or gemmae; and manner of branching) which were previously in question or unknown are clarified. Mechanism(s) of certain morphogenetic phenomena remain obscure (cell wall and plastid ontogeny). These and related problems are now under investigation. Hildenbrandia should be added to the freshwater algal flora of the United States.
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Morphometric analysis and phylogenetic analysis of sequences of the rbcL chloroplast gene (which codes for the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme) and the nuclear 18S ribosomal RNA (rRNA) gene were carried out on 26 specimens of marine and freshwater Hildenbrandia from North America. Nineteen marine specimens were collected from Alaska to Costa Rica on the Pacific coast and from Newfoundland to Connecticut on the Atlantic coast. Seven freshwater samples were collected from Texas, Costa Rica, St Lucia and Puerto Rico. Three groups of samples were distinguished by morphometric analysis: one containing all freshwater samples (H. angolensis Welwitsch ex West et West), one consisting of a marine sample with parallel tetrasporangial divisions (H. occidentalis Setchell ex Gardner) and one group with non-parallel tetrasporangial divisions (H. rubra (Sommerfelt) Meneghini. These groupings were partially incongruent with those obtained by analyses of the molecular data. Parsimony and distance analyses of the rbcL gene resulted in trees in which Atlantic and Pacific clades were largely resolved. However, an Alaskan sample was included in the Atlantic group, which may indicate a trans-Arctic invasion event. The freshwater samples were paraphyletic for the rbcL gene, among the marine collections, which supports the concept of multiple invasions establishing the freshwater populations in North America. The 18S rRNA gene sequence data indicate that the freshwater samples are monophyletic with the exception of the unresolved position of the H. occidentalis sample. The freshwater samples form a monophyletic clade when multiple outgroups are used. The rbcL data appear to be mutationally saturated above approximately 17% divergence, which makes interpretation of phylogenetic signal among distant groups difficult. This may be a result of the asexual reproduction of the alga.
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Article
A new freshwater red alga, Hildenbrandia jigongshanensis, is described from materials collected on the surface of rocks in flowing water from Jigongshan Mountain, China. This new species is distinguished from the other freshwater members by significantly larger cell dimensions (9.8–19.6) × (9.9–10.4) μm and filament height (364–409 μm). Phylogenetic analysis based on sequences of the rbcL gene and the construction of 18S ribosomal RNA (rRNA) secondary structure demonstrated the separation between H. jigongshanensis and other freshwater Hildenbrandia species. Specimens of H. jigongshanensis formed a clade divergent from H. rivularis group with robust support values based on rbcL gene sequences, and was in the basal position relative to the monophyletic H. rivularis, whereas the species H. angolensis was paraphyletic and distributed among both the freshwater and marine clades. Primary structure of 18S rRNA produced phylogenetic tree with low resolutions and unresolved relationship among the freshwater Hildenbrandia species due to high sequence conservation. Secondary structure based on 18S rRNA sequences distinguished the new species by absence of 3 helices compared with common structure of H. rivularis. This species description brings the total number of recognized freshwater Hildenbrandia species in China to two.
Article
Twenty-one marine and freshwater specimens of Hildenbrandia from Europe were compared by analyses of morphometric data and sequences of the rbcL chloroplast gene (which codes for the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme) and the nuclear 18S ribosomal RNA (rRNA) gene. Eleven freshwater specimens, all corresponding to H. ribularis, were collected from the British Isles, Germany, France, Austria Spain and Italy. Six marine specimens, representing H. rubra, were obtained from Northern Ireland, Wales, France, Norway, Sweden and the Netherlands, while four marine specimens, corresponding to H. crouanii, were collected from Scotland and Germany. Morphometric analyses of vegetative and reproductive characters distinguished three groups of specimens, which correspond to the three species. Parsimony and distance analyses of the rbcL and 18S rRNA genes were performed on these samples and sequences of North American representatives were included to determine their inter-relationships. The rbcL gene sequence analyses separated the marine and freshwater specimens, with the H. rivularis and H. angolensis representatives forming a well-supported monophyletic clade. The rbcL sequence analyses did not resolve the groupings of marine specimens determined in the morphometric analysis. Analyses of the 18S rRNA gene sequences also supported separation of the marine samples from freshwater samples, although the bootstrap and decay support was not as strong as for the rbcL gene. H. rubra formed a monophyletic clade that was weakly supported. In terms of biogeographic trends, the fairly low intraspeci8e divergence of H. rivularis (0-1.9% for the rbcL gene and 0-3.6% for the 18S rRNA gene) and the positioning of these samples in the gene trees supports the concept that they are derived from marine populations.