Phytotaxa 423 (2): 068–074
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
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,
* Corresponding author (e-mail: firstname.lastname@example.org)
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
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).
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,
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 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
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.
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.
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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