PhytoKeys 193: 107-123 (2022) as A peer-reviewed open-access journal doi: 10.3897/phytokeys. |93.79667 RESEARCH ARTICLE eel h y toKe y S https:/ / Pp hyto keys -pen soft.net Launched to accelerate biodiversity research Thorea baiyunensis sp. nov. (Thoreales, Rhodophyta) and T. okadae, a new record from China Jinfen Han', Fangru Nan’, Jia Feng', Junping Lv', Qi Liu', Xudong Liu', Shulian Xie' I School of Life Science, Shanxi Key Laboratory for Research and Development of Regional Plants,Shanxi University, Taiyuan 030006, China Corresponding author: Shulian Xie (xiesl@sxu.edu.cn) Academic editor: Juliet Brodie | Received 22 December 2021 | Accepted 13 March 2022 | Published 1 April 2022 Citation: Han J, Nan E Feng J, Lv J, Liu Q, Liu X, Xie S (2022) Thorea baiyunensis sp. nov. (Thoreales, Rhodophyta) and 7. okadae, a new record from China. PhytoKeys 193: 107-123. https://doi.org/10.3897/phytokeys.193.79667 Abstract The freshwater red algal order Thoreales has a triphasic life history, of which the “Chantransia’” phase is a small filamentous sporophyte. The “Chantransia” stage is difficult to distinguish from species in the genus Audouinella by its morphological characteristics. In this study, five “Chantransia” isolates (GX41, GX81, GD224, GD225, GD228) were collected from Guangxi Zhuang Autonomous Region and Guangdong Province in China. Based on morphological data, all five isolates were similar to A. pygmaea, whereas sequence data from the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) gene and the 5’ region of the mitochondrial cytochrome oxidase I gene (COI-5P) determined that these specimens represented the “Chantransia’ stage of two species in the genus 7/orea rather than Audouinella. Phylogenetic analyses of the concatenated genes supported the proposal of a new species, 7’ baiyunensis, and a new geographic record of 7’ okadae, a species previously described only in Japan. Therefore, combined with previous records, four species of this genus are now recognized in China, including 7! hispida, T’ violacea, T. baiyunensis and T. okadae. Keywords COL5P, freshwater Rhodophyta, new taxon, phylogeny, rbcL, Thorea Copyright Jinfen Han et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 108 Jinfen Han et al. / PhytoKeys 193: 107-123 (2022) Introduction Currently, more than 7,000 red algal species are reported worldwide, of which freshwater species only account for 3%. In the Rhodophyta, only four orders (Balbianiales, Batrachospermales, Compsopogonales, and Thoreales) have all members that are strictly freshwater species. Among the freshwater red algae, the genus Thorea Bory was established by Bory de Saint-Vincent (1808), since when its taxonomic status has undergone numerous changes. Based on the pit plug structure, Pueschel and Cole (1982) removed the Thoreaceae and Batrachospermaceae from the Nemaliales and established the order Batrachospermales. However, molecular research showed that species in the Thoreaceae were distantly related to the other Batrachospermales taxa (Vis et al. 1998). Subsequently, based on DNA sequence data, secondary structure of the SSU gene and characteristics of the outer layer of the pit plug, Miiller et al. (2002) established the Thoreales. Species of this order are characterized by having multiaxial gametophytes, a uniaxial “Chantransia” stage, and pit plugs with two cap layers, the outer one of which is usually plate-like. As currently recognized, the Thoreales contain a single family Thoreaceae with two genera, Jhorea and Nemalionopsis. The main difference between the genera Thorea and Nemalionopsis is that Thorea has assimilatory filaments that are not contained in a common gelatinous matrix with reproductive structures (carpogonia, spermatangia, carposporangia and monosporangia) at their base and a lower ratio of sporangial branch to assimilatory filament length and loose aggregation (Skuja 1934; Sheath and Cole 1993). In a recent review of Thorea, sixteen species were recognized worldwide (Johnston et al. 2018). However, only two species (7! hispida (Thore) Desvaux, T. violacea Bory) of this genus have been reported in China (Shi 2006). The type species of the genus, 7’ hispida, was widely distributed in China, while 7’ violacea was only reported in Guizhou Province (Xie and Shi 2003; Shi 2006). Asexual reproduction by monosporangia is commonly reported in Thorea, while sexual reproduction is observed in a few species, including 7’ hispida, T: conturba Entwisle & Foard, T’ okadae Yamada, TL. bachmannii C.Pujals ex R.G.Sheath, M.L.Vis & K.M.Cole, 7’ kokosinga-pueschelii E.T Johnston & M.L.Vis, and 7 mauitukitukii E.T.Johnston, K.R.Dixon, J.A.West & M.L.Vis (Kumano 2002; Johnston et al. 2018). Among them, 7’ okadae is widely distributed in Japan (Yamada 1949; Johnston et al. 2018), but so far, this species has not been reported in other countries and regions. In addition, according to Kumano (2002), 7’ okadae is the largest species in the genus Thorea, and the length of its gametophyte often exceeds 1 m, and may reach 3 m. Like other sexually reproductive species of freshwater red algae, 7horea species have a triphasic life history, including gametophyte, carposporophyte, and a diminutive diploid sporophyte termed “Chantransia” stage. Recently, several studies have focused on the phylogenetic affinities of the “Chantransia” stages of Thorea and the relation- ship between the isolates in this stage and a phylogenetically distant genus Audouinella Bory (Necchi and Oliveira 2011; Han et al. 2021). Based on these studies, new species were proposed, new distributions were found, and higher Thorea species diversity was Thorea baiyunshanensis sp. nov. and T! okadae a new record from China 109 revealed (Johnston et al. 2018; Han et al. 2021). In terms of morphological charac- ters, the “Chantransia’” stages of the Thoreales are very similar to those of Audouinella taxa, although some studies have indicated that thallus color can be used as a reliable character (Zucchi and Necchi 2003) to distinguish true Audouinella (reddish) from “Chantransia’ (bluish). However, “Chantransia’ stages of the Thoreales can be brown- ish (Chiasson et al. 2007), and some species of Sheathia can be brownish to reddish in addition to bluish (Han et al. 2020). Thus, morphological characteristics that can un- equivocally distinguish them have not been found (Pueschel et al. 2000; Chiasson et al. 2005). It was not until the emergence of molecular data that the “Chantransia” stage could be used for species identification. Recently, researchers have shown that some “Chantransia’ isolates of 7horea were misclassified as Audouinella (Han 2012; Chen et al. 2014; Nan et al. 2016). Based on different gene markers, Han (2012), Chen et al. (2014) and Nan et al. (2016) proposed that A. sinensis C.-C.Jao and A. heterospora S.L.Xie & Y.J.Ling were “Chantransia” of 7. hispida rather than belong to Audouinella. Besides, numerous surveys (Pueschel et al. 2000; Zucchi and Necchi 2003; Chiasson et al. 2005, 2007; Necchi and Oliveira 2011; Han et al. 2020) demonstrated that eleven species, including members of the Thoreales (7! hispida and N. tortuosa Yoneda & Yagi), were associated with “A. pygmaea”. Furthermore, the molecular phylogeny based on the rbcL, COI-5P and the plastid 23S rRNA (UPA) genes unequivocally demonstrated that a sample morphologically similar to A. macrospora (Wood) Sheath & Burkholder represented the “Chantransia” phase of 7! hispida (Han et al. 2021). It is therefore evident that the morphological difference between “Chantransia” stages and Audouinella is not clear. Relying solely on traditional morphological meth- ods can often cause confusion in the classification and identification of Audouinella- like freshwater red algae. All samples in this study are “Chantransia” stages, meaning that key diagnostic morphological characters were unavailable. Therefore, the present work has attempted to use two molecular markers (rbcL and COI-5P) to infer the phylogenetic position of all isolates in this study. In addition, a secondary aim of this investigation is to provide reference for local resource survey of freshwater red algae biodiversity in China. Methods Five “Chantransia’” specimens (GX41, GX81, GD224, GD225 and GD228) were collected from Guangxi Zhuang Autonomous Region and Guangdong Province in China (Fig. 1, Table 1). Handheld meters (YSI Professional Plus Multiparameter Water Quality Instrument 19E102487, YSI Incorporated, Brannum Lane Yellow Springs, Ohio, USA) were used to measure water quality parameters, including water temperature and pH. Materials were picked up carefully using a knife and tweezers and transferred to the laboratory as soon as possible. After transfer to the laboratory, the samples were rinsed with sterile water to remove impurities. Other algae attached to the samples were carefully removed using tweezers and other tools. Then microscopic 110 Jinfen Han et al. / PhytoKeys 193: 107-123 (2022) examination was undertaken to ensure that all epiphytic algae were removed. Morphological and genetic analysis was performed for each sample. For observations and measurements, we used a BX-51 Olympus microscope equipped with a charge- coupled device (DP72; Olympus, Tokyo, Japan). Panay Map review No. GS(2021)5451 ~ Map review No. GS(2021)5442 1:41 000 000 i] i} i f < HUNAN .\4_--qiancxi J i GUIZHOU i 5 ; C J va / ‘4 FUJIAN Ns 4 aN Eta ~~ S mq ae § z. Ay il “N d os \ va - t ie, 4 \ ¢ i 4 GUANGXI ~~ GUANGDONG - @ cx D ZHUANGZU ( ZIZHIQU @ 2224, cp225, co28 S ; GUANGXI ZHUANGZU ZIZHIQU 5 4 @ xsi VIET NAM HAINAN Map review No. GS(2019)3266- 744 Map review No. GS(2019)3266 1: 4300 000 Figure |. Map showing approximate locations of samples investigated in this study. More detailed location information is provided in Table 1. The red circles and the green circles indicate the locations of Thorea okadae in China and Japan, respectively. Table |. Collection information and sequence accession numbers for taxa analyzed in this study. Isolate Locality with longitude and latitude Collection date Collector Voucher number GX41 _ Baimo Cave, Bama County, Guangxi Zhuang Autonomous Region, 22 December 2019 Kunpeng Fang GX19041 China (24°18.03'N, 107°05.96'E) GX81 —__Tongling Great Falls, Jinxi County, Guangxi Zhuang Autonomous 23 December 2019 Kunpeng Fang GX19081 Region, China (23°43.10'N, 106°39.77'E) GD224 Baiyun Mountain, Guangzhou, Guangdong province, China 22 November 2020 Jinfen Hanand GD20224 (23°36.00'N, 113°49.53'E) Kunpeng Fang GD225 Baiyun Mountain, Guangzhou, Guangdong province, China 22 November 2020 Jinfen Hanand GD20225 (23°36.00'N, 113°49.53'E) Kunpeng Fang GD228 Baiyun Mountain, Guangzhou, Guangdong province, China 22 November 2020 Jinfen Hanand GD20228 (23°36.00'N, 113°49.53'E) Kunpeng Fang Thorea baiyunshanensis sp. nov. and T’ okadae a new record from China 111 Table 2. GenBank accession numbers of rocL and COI-5P sequences generated in this study. Taxon Isolate rbcL accession No. COI-5P accession No. Thorea okadae GX41 MZ648088 MZ676778 T. okadae GX81 MZ648089 MZ676779 T. baiyunensis GD224 MZ648090 MZ676780 T. baiyunensis GD225 MZ648091 MZ676781 T. baiyunensis GD228 MZ648092 MZ676782 Total DNA was extracted following the protocol originally described by Saunders (1993) and revised by Vis and Sheath (1997). Two molecular markers, rocL and COI-5P, were amplified using the primers and protocols described by Vis et al. (1998) and Saunders (2005). The PCR products with their amplification primers were sent to BGI Tech Corporation (Beijing, China) for sequencing on an ABI 3730XL sequencer. Sequences generated in this study were submitted to the GenBank databases (Table 2). Additional related sequence data of the Thoreales order and outgroup taxa Batrachospermum were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/) (Suppl. material 3: Table S1). The 58 rbcL, and 47 COI-5P sequences were aligned by CLUSTAL-X 2.0 (Thompson et al. 1997) and MEGA 5.0 (Tamura et al. 2011). Pairwise distance and the number of nucleotide variances for the taxa’s molecular markers were calculated in MEGA 5.0. For phylogenetic analyses, the appropriate models for the sequence evolution were determined by MODELTEST 3.7 (Posada and Buckley 2004). The parameters for the concatenated sequences (7bcL and COI-5P) maximum likelihood (ML) analyses were as follows: GTR+I+G model; gamma distribution=0.5411; proportion of invariable sites= 1.1344; base frequencies A=0.3352, C=0.1247, G=0.1641, and T=0.3760; and rate matrix A—C=3.7852, A-G=9.4223, A-T=1.1874, C—G=0.6077, and C—-T=20.8378. PHYML software (Felsenstein 1981; Guindon and Gascuel 2003) was utilized to construct the ML trees with 1,000 replicates of bootstrap analysis. Bayesian Inferences (B.I.) were performed in MRBAYES VERSION 3.1.2 (Ronquist and Huelsenbeck 2003) and runs 5,000,000 generations sampling every 1,000 generations until the standard error was lower than 0.01. The resulting phylogenetic trees were edited using FIGTREE 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/). Results Molecular analysis The rbcL data matrix included 56 specimens of Thoreales and 2 outgroup taxa, consisting of 1203 characters, of which 410 (34.08%) were variable and 380 (31.59%) were parsimony informative. The rbcL p-distance among the five “Chantransia” isolates and other specimens of order Thoreales (Suppl. material 4: Table $2) showed that divergences between the isolates (GX41 and GX81) collected from Guangxi Zhuang Autonomous Region and five T. okadae specimens previous reported from Japan were 0%—1%, corresponding to 4—6-bp 112 Jinfen Han et al. / PhytoKeys 193: 107-123 (2022) differences, the average distance between these two samples (GX41 and GX81) and the other species of genus Thorea was 8.5%. These results supported the close relationship between the two isolates (GX41 and GX81) and 7 okadae and further substantiated that GX41 and GX81 were the “Chantransia’ of 7’ okadae. However, three other isolates (GD224, GD225, and GD228) collected from Guangdong Province had unique sequences. The distances between them and the other species of 7horea were higher than the intraspecific distances of this genus (10.88% VS 8.28%). The COI-5P sequence determined for the five “Chantransia” isolates from this study were 676 bp, of which 297 (43.93%) were variable and 273 (40.38%) were parsimony informative. The pairwise distance among the “Chantransia’ isolates collected in this study and other Thoreales taxa (Suppl. material 5: Table $3) showed that isolates GX41 and GX81 were closely related to 7’ okadae from Japan. ‘The mean p-distance between samples from China and Japan was 3%. The intraspecific p-distances of species in this genus were 0—6%, whereas the interspecies p-distances of COI-5P sequences among the species of Zhorea were 7.3-18.0%. For the remaining three isolates GD224, GD225, and GD228, their pairwise distances with other Thoreales specimens also supported the unique taxonomic status of GD224, GD225 and GD228: the p-distance between them and the other species of Thorea was 14.17%, which was larger than the intraspecific distance of this genus (0—6%). Phylogenetic analyses based on single gene and concatenated genes produced trees with similar tree topologies, such that only the concatenated B.I. tree with supporting values calculated from two methods was displayed (Fig. 2). The single gene phylogenetic trees based on récL and COI-5P are shown in the supporting materials (Suppl. material 1, 2: Figs S1, $2). The phylogenetic analyses strongly supported the monophyly of the Thoreales, Nemalionopsis and Thorea (Fig. 2). The Thorea clade contained two major subclades, one comprising four species: 7’ guisqueyana E.T.Johnston & M.L.Vis, T- riekei Bischoff, LT. gaudichaudii C.Agardh, and T’ mauitukitukii (1.00/99.9); and the second subclade including six species (7’ baiyunensis, T; bachmannii, T: kokosinga-pueschelii, T’ okadae, I. indica Necchi, E.K.Ganesan & J.A.West, and T° hispida) and an undetermined species (1.00/90.3). Isolates from this study were in the second subclade and formed two distant clusters. The isolates GX41 and GX81 were within a clade with five 77 okadae specimens collected from Japan. This relationship was well supported by Bayesian posterior probabilities (100%) and ML bootstrap (100%). The remaining three isolates, GD224, GD225, and GD228 were an independent clade distantly related from any of the previously described species with high support values (1.00/90.3). Morphological observations The morphometric data showed that all samples used in this study fit the morphological description of Audouinella pygmaea (Roth) Duby, although the tuft length of GX81 is significantly smaller than others (Fig. 3A—T, Table 3). Their characteristics are as follows: tuft-shape, 1.2—25.9 mm length, bluish or brownish in color; basal portion consisting Thorea baiyunshanensis sp. nov. and T’ okadae a new record from China 113 of an irregular prostrate system of densely aggregated filaments; erect filaments dense, irregular branched, apical cells obtuse, without hair; vegetative cells of main branch- es cylindrical, 8.2-68.2 um in length and 7.2—22.7 um in diameter; monosporangial branches short, mostly grow at the tip of vegetative branch, with few small branches; monosporangia are obovoidal, 11.8—22.7 um in length and 7.2—17.3 um in diameter. T. quisqueyana (Dominican Republic)|T. quisqueyana 1.00/100 . T. riekei (Mexico) . T. riekei (USA) 1.00/99.9]f. T. riekei (USA) T. riekei T. riekei(USA) |"? . T. riekei (USA) . T. riekei (USA) 1.00/99.9 ig §.85/71.1] Tt... 1.00/97.5 1.00/100} - T. mauitukitukii (Vanuatu) 1.00/100!~ 1.00/100}.. 1,00/100 1.00/90.3 0.52/.] !}00/100 1.00/99.8,- 1.00/100| © 0.51/- 1.00/89.8 (China) (China) (China) - T. okadae (Japan) T. gaudichaudii (Federated States of Micronesia) T. gaudichaudii (Vanuatu) T. gaudichaudii (Vanuatu) T. gaudichaudii (Guam) T. gaudichaudii (Guam) T. gaudichaudii (Japan) T. gaudichaudii (Japan) T. gaudichaudii (Japan) T. gaudichaudii (Japan) T. gaudichaudii (Philippines) UPNDYDIPNdS I |z- mauitukitukii T. mauitukitukii (Vanuatu) T. baiyunensis sp. nov. T. bachmannii (Brazil) T. bachmannii (Brazil) T. sp. (USA) IT. sp. T. kokosinga- pueschelii (USA) T. kokosinga- pueschelii (USA) 7: bachmannii |r kokosinga-pueschelii GX8 I T. okadae (Japan) T. okadae T. okadae (Japan) T. okadae (Japan) . T. okadae (Japan) 1.00/99.9 T. indica (India) |7. indica -- T. hispida (Australia) 1.00/100 -. T. hispida (China) - T. hispida (China) 0.50/- . T. hispida . T. hispida (Germany) . T. hispida (USA) 1.00/100 - T. hispida (Spain) - T. hispida (Japan) - T. hispida (Great Britain) |T. hispida - T. hispida (Great Britain) - T. hispida (Great Britain) - T. hispida (Italy) . T. hispida (Japan) - T. hispida (Japan) -- T. hispida (China) -- T. hispida (USA) 0.98/93.2 1.00/98.7F- 1.00/100 1.00/99.8 1.00/100 Outgroup 0.1 N. shawii (Indonesia) N. shawii (Japan) . N. shawii (Japan) -- N. shawii (Japan) -- N. shawii (Japan) -- N. shawii (Nepal) - N. parkeri (USA) . N. parkeri (USA) . N. parkeri (USA) .- N. parkeri (USA) -- N. parkeri (USA) -- N. parkeri (USA) N. shawii N. parkeri N. parkeri (USA) Figure 2. Bayesian inference tree for Zhorea and Nemalionopsis based on concatenated sequences from rbcL and COI-5P genes. Support values for all analyses are shown as follows: Bayesian posterior prob- abilities/ML bootstrap. ‘-’ denotes <50% support for those analyses at that node. All new sequences generated in this study are indicated in red boxes. Jinfen Han et al. / PhytoKeys 193: 107-123 (2022) 114 Thorea baiyunshanensis sp. nov. and T’ okadae a new record from China 115 Taxonomic proposals The genetic distance and phylogenetic analysis based on rbcL, COI-5P and the concatenated genes all supported the identification of new species described below. Thorea baiyunensis Han, Nan & Xie, sp. nov. Fig. 3I-T Description. Known only from the “Chantransia” sporophyte generation. Plant macroscopic, up to 25.9 mm, bluish; basal portion consisting of an irregular prostrate system of densely aggregated filaments; lateral branches developing at angle < 25°. Vegetative cells of main branches cylindrical, (12.7) 13.6-63.6 (68.2) um long and (8.2) 10.9-20.9 (22.7) uum in diameter. Monosporangia numerous, mostly grow at the tip of 1—5-celled, short branchlets, singly or in clusters, obovoidal, (11.8) 12.7—21.8 (23.6) um long and (10.9) 11.8-16.4 (17.3) um in diameter. Chloroplasts laminate or irregularly lobed, 2-4 per cell. Diagnosis. Diagnostic DNA sequence: rocL and COI-5P (accession number: MZ648090, MZ.648091, and MZ648092 for rbcL and MZ676780, MZ676781, and MZ676782 for COI-5P). Type. Cuina, Guangdong Province, Baiyun Mountain, 540 m alt., 23°36.00'N, 113°49.53'E: epilithic on the rocks in spring water, November 2020, J.R. Han & K.P Fang (Holotype: SXU-SAS18040; Paratype: SXU-SAS18041). Deposited in Herbari- um of Shanxi University (SXU), Shanxi University, Taiyuan, Shanxi Province, China. Habitat and distribution. Baiyun Mountain (23°36.00'N, 113°49.53'E), Guang- dong Province, China: on the rocks in spring water; water temperature 17.3—20.6 °C, pH 6.5-7.6 (Figs 1, 4A—C). Etymology. The species epithet refers to the type locality (Baiyun Mountain, China). Authentic strain. SXU-GD20224. Figure 3. Morphological characters of samples investigated in this study A—D sample GX41 A morpho- logical observation of the tufts of erect filament, bluish in color B monosporangial branch with ovoid mono- sporangium (black arrow) C filament showing branch angle < 25° (black arrow) D erect filaments arise from basal system consisting of a prostrate mass with sparse rhizoids (black arrowhead) E—H sample GX81 E mor- phological observation of the tufts of erect filament, brownish in color F monosporangial branch with ovoid monosporangium (black arrow) G filament showing branch angle < 25° (black arrow) H cells have parietal laminate or irregularly lobed Chloroplast (white arrow) I-L sample GD224 I morphological observation of the tufts of erect filament, bluish in color J monosporangial branch with obovoidal monosporangium (black arrow) K filament showing branch angle < 25° (black arrow) L cells have parietal laminate or irregularly lobed Chloroplast (white arrow) M—O sample GD225 M morphological observation of the tufts of erect filament, bluish in color N monosporangial branch with obovoidal monosporangium (black arrow) O filament show- ing branch angle < 25° (black arrow) P cells have parietal laminate or irregularly lobed Chloroplast (white arrow) Q=T sample GD228 Q morphological observation of the tufts of erect filament, bluish in color R monosporangial branch with obovoidal monosporangium (black arrow) $ filament showing branch angle < 25° (black arrow) T cells have parietal laminate or irregularly lobed Chloroplast (white arrow). 116 Jinfen Han et al. / PhytoKeys 193: 107-123 (2022) Table 3. Morphological characteristics of specimens in this study. Thalli GXx41 GX81 GD224 GD225 GD228 characteristics Color Bluish Brownish Bluish Bluish Bluish Height (mm) (4.8) 5—9.4 (10.0) (1.2) 1.5-3.5 (4.4) (3.4) 4.1-9.3 (10.8) (4.1) 5.4-22.9 (25.9) (8.5) 8.8-12.9 (13.6) Branch angle* < 25° < 25° < 25° < 25° < 25° Vegetative cells (8.2) 10-28.2 (31.8) (20.9) 22.7-54.5 (12.7) 13.6-63.6 (21.8) 22.7— (22.7) 23.6-43.6 Length (ym) (59.1) (68.2) 59.1(60.9) (45.5) Vegetative cells (7.2) 8.2-10.9 (11.8) (8.2) 9.1-10.9 (11.8) (8.2) 10.9-15.5 (10.9) 11.8-15.5 (11.8) 12.7-20.9 Diameter (11m) (16.4) (16.4) (22.7) Monosporangia Obovoidal Obovoidal Obovoidal Obovoidal Obovoidal Shape Monosporangia (12.7) 13.6-14.5 (12.7) 13.6-18.2 (11.8) (12.7) 13.6-17.3 (13.6) 14.5-21.8 Length (ym) (1525) (19.1) 12.7-21.8 (22.7) (19.1) (23.6) Monosporangia (7.2) 8.2-14.5 (15.5) (8.2) 9.1-12.7 (13.6) (10.9) 11.8-15.5 (10.9) (10.9) 11.8-16.4 Diameter (jum) (16.4) 11.8-13.6 (14.5) (17.3) Chloroplast 24 2-4 2-4 2-4 2-4 number Chloroplast laminate or laminate or laminate or laminate or laminate or shape irregularly lobed irregularly lobed irregularly lobed irregularly lobed irregularly lobed * Definition follows Necchi and Zucchi (1995). Discussion Several studies have confirmed that it is difficult to distinguish “Chantransia’” stages from true Audouinella based only on morphological characteristics, which often hinders species identification and brings confusion to the classification system of freshwater red algae (Chiasson et al. 2005; Necchi and Oliveira 2011; Han et al. 2020). More surprising is that even individuals with completely different gametophyte morphology, belonging to different species, genera or orders, may also produce “Chantransia” stages with extremely similar morphology. In this study, morphological data showed that all samples collected from South China were similar and within the circumscription of A. pygmaea. However, molecular analysis confirmed that they were not Audouinella taxa but the “Chantransia” stages of two different Thorea species, T’ okadae and TL. baiyunensis. Based on sequence data and culture studies (Pueschel et al. 2000; Zucchi and Necchi 2003; Chiasson et al. 2005, 2007; Necchi and Oliveira 2011; Han et al. 2020), eleven species of orders Batrachospermales and Thoreales have been confirmed to form “Chantransia’ stages similar to A. pygmaea in morphology. ‘Thus, at least thirteen species of the orders Batrachospermales and Thoreales are associated with “A. pygmaea’ so far. It is clear that the morphological characteristics of the “Chantransia” stages cannot be used to determine the identity individual species (Necchi and Oliveira 2011; Han et al. 2021). However, how genetic information and environmental factors regulate the morphological expression of “Chantransia” stages remain unclear. With the widespread application of molecular data in the taxonomy of freshwater red algae, the view that morphologically simple organisms have considerable genetic diversity and species richness has been widely recognized (Johnston et al. 2018; Han et al. 2020, 2021). In the past few years, several new species of the Batrachospermales and Thorea baiyunshanensis sp. nov. and T’ okadae a new record from China 117 Thoreales were proposed based on the “Chantransia” sporophyte generation, such as 7’ quisqueyana, Sheathia shimenxiaensis J.-F. Han, E-R.Nan et S.-L.Xie, S. jiugongshanensis J.-EHan, E-R.Nan et S.L.Xie, and S. ginyuanensis J.-F.Han, F-R.Nan et S.L.Xie (Johnston et al. 2018; Han et al. 2020, 2021). In this article, based on the rbcL and COI-5P sequences, all five samples utilized in this study were unquestionably proved to be the “Chantransia” stages of two species of genus Thorea, whose gametophytes have never been reported in China before. Therefore, taking into account the two stages, the geographical distribution of genus 7horea would be larger, and the number of species would be more than that identified only by morphological data. Combined with previous reports in China, four species of genus T7horea have been recognized, including T° hispida, T’ violacea, T’ baiyunensis, and T! okadae. At the same time, the number of the Yhorea distribution sites is proposed to have expanded to eight, including Shanxi, Jiangsu, Yunnan, Guizhou, Hunan, Henan, Guangdong provinces and Guangxi Zhuang Autonomous Region. ‘This study reinforces the importance of collecting and sequencing specimens of “Chantransia” stages as a tool to reveal the hidden diversity of freshwater red algae of the orders Batrachospermales and Thoreales in different regions of the world. In 1949, Yamada reported a new species, 7’ okadae, when studying the specimens of genus 7horea in Kagoshima prefecture, Japan (Yamada 1949). According to Ku- mano (2002) and Kozono (2020), 7’ okadae is the largest species known in the genus Thorea and is widely distributed in different areas of Honshu Island and Kyushu Island in Japan. This species has long been considered endemic to Japan. However, in this study we reported two “Chantransia” stages (GX41 and GX81) of 77 okadae from Guangxi Zhuang Autonomous Region, China. Sequence analysis based on rbcL and COI-5P showed that there were only a few base differences between 7’ okadae from China and Japan. As shown in Fig. 1, the distribution of 7’ okadae in China (red cir- cle) is far away from that in Japan (green circle). However, these regions all belong to the subtropical monsoon climate, which means that these places have similar climatic conditions, including light, temperature and precipitation. According to Higa et al. (2007), both gametophytes and “Chantransia” stage specimens of 7’ okadae were ob- served in Kikuchi River, Japan where water pH is 6.6—7.5, temperature is 26.3 °C in summer and 9.5 °C in winter. In addition, the mean day length of this place ranged from 9.9 h in December to 14.3 h in June. In this study, the “Chantransia” isolates of 7! okadae were collected in Baimo Cave and Tongling Great Falls (Fig. 4D, E). The habitat characteristics of these two places are as follows: mean water temperature ranged from 14 °C to 21 °C and pH values fluctuated between 7.2 and 7.8, while the day length is about 10.7 h in December and 13.5 in June. The environmental factors of Baimo Cave and Tongling Great Falls were similar to those of Kikuchi River, thus, it is not surprising that 7’ okadae was found in Guangxi Zhuang Autonomous Region, China. It is therefore reasonable to infer that 77 okadae also occurs in other areas of China with the same regional climate. In a study on the seasonality of gametophyte occurrence, maturation and fertilization of the freshwater red alga 7’ okadae, Higa et al. (2007) pointed out 118 Jinfen Han et al. / PhytoKeys 193: 107-123 (2022) - ~ — eae a —_ = Be A Figure 4. Habitat of Zhorea baiyunensis and T: okadae A-C habitat of Thorea baiyunensis D, E habitat of T. okadae. that gametophytes generally form from December and can be observed from early autumn to late spring, while “Chantransia” stages (sporophytes) last throughout the year. The “Chantransia” stages of 7’ okadae in this study were collected in December 2019, but we did not find its gametophytes. We speculate that this may be due to Thorea baiyunshanensis sp. nov. and T’ okadae a new record from China 119 the following reasons: firstly, since the samples were only collected in December, the gametophytes of 7’ okadae may have just begun to form. 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Yamada Y (1949) On the species of Thorea from the far eastern Asia. Shokubutsu Kenkyu Zasshi 24: 155-159. https://doi.org/10.15281/jplantres1887.62.18 Zucchi MR, Necchi Jr O (2003) Blue-greenish acrochaetioid algae in freshwater habitats are “Chantransia stages of Batrachospermales sensu lato (Rhodophyta). Cryptogamie, Algologie 24(2): 117-131. Supplementary material | Figure S1 Authors: Jinfen Han, Fangru Nan, Jia Feng, Junping Lv, Qi Liu, Xudong Liu, Shulian Xie Data type: COL Explanation note: Bayesian inference tree based on the rbcL gene sequences. Support values for all analyses are shown as follows: Bayesian posterior probabilities/ML bootstrap. ‘-’ denotes <50% support for that analysesat that node. All new sequences generated in this study are indicated in red boxes. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/phytokeys.193.79667.suppl1 122 Jinfen Han et al. / PhytoKeys 193: 107-123 (2022) Supplementary material 2 Figure S2 Authors: Jinfen Han, Fangru Nan, Jia Feng, Junping Lv, Qi Liu, Xudong Liu, Shulian Xie Data type: COL Explanation note: Bayesian inference tree based on the COI-5P gene sequences. Support values for all analyses are shown as follows: Bayesian posterior probabilities/ ML bootstrap. ‘-’ denotes <50% support for that analyses at that node. All new sequences generated in this study are indicated in red boxes. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/phytokeys.193.79667.suppl2 Supplementary material 3 Table S1 Authors: Jinfen Han, Fangru Nan, Jia Feng, Junping Lv, Qi Liu, Xudong Liu, Shulian Xie Data type: docx Explanation note: Specimen information of sequences downloaded from the GenBank database. “—” denotes no related information for the specimen. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/phytokeys.193.79667.suppl3 Thorea baiyunshanensis sp. nov. and T’ okadae a new record from China 123 Supplementary material 4 Table S2 Authors: Jinfen Han, Fangru Nan, Jia Feng, Junping Lv, Qi Liu, Xudong Liu, Shulian Xie Data type: xls Explanation note: Pairwise distance (lower-left matrix) and number of nucleotide variance (upper-right matrix) of rbcL sequence among the taxa in this study. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/phytokeys.193.79667.suppl4 Supplementary material 5 Table S3 Authors: Jinfen Han, Fangru Nan, Jia Feng, Junping Lv, Qi Liu, Xudong Liu, Shulian Xie Data type: xls Explanation note: Pairwise distance (lower-left matrix) and number of nucleotide variance (upper-right matrix) of COI-5P sequence among the taxa in this study. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/phytokeys.193.79667.suppl5