Morphological and Molecular Phylogenetic Analysis of a Lemanea Specimen (Batrachospermales, Rhodophyta) from China

: The genus Lemanea is an evolutionally derived lineage of freshwater Rhodophyta which has a rare distribution. Morphological characterization and molecular phylogenetic analyses were conducted for a Lemanea specimen collected from Guilin, China, in this study. The results based on morphological observation and molecular evidence, including rbc L and SSU sequences, showed it was a new record of L. manipurensis Ganesan, West, Zuccarello et Rout in China. The specimen in this study was morphologically characterized by branches that were positioned in the lower part of the thallus, hair cells absent around the outer cortex, ellipsoidal and single carpospores at the top of the gonimoblast filaments and spermatangia formed in continuous wide rings on the thallus nodes. By comparing the morphology of specimens collected from Guilin , China, and the holotype speci ‐ men in India, it was found that the spermatangial shapes were variable among different popula ‐ tions. It was found that the presence or absence of hair cells around the outer cortex was not a reliable characteristic for the identification of the genus Lemanea based on a comparison between L. manipurensis collected in Guangxi and in India and another four Lemanea species previously rec ‐ orded in China. The results of this study provided molecular evidence and a theoretical basis for molecular phylogenetic research on the genus Lemanea and enriched the species diversity and geo ‐ graphical distribution of Lemanea in China.


Introduction
Lemanea Bory (1808) belongs to the Batrachospermales, Nemaliophycidae, Florideophyceae, Eurhodophytina.It is characterized by tufts of cartilaginous, tubular, pseudoparenchymatous gametophytic thalli, lacking cortical filaments, around a central, uniseriate axis.The outer cortex closely abuts T-or L-shaped ray cells.The thallus color is blue-green to olive when young, becoming rusty-brown to black at maturity [1].This genus is the most derived lineage in the Batrachospermales, which is exclusively distributed in freshwater [2].It is widely distributed in fast streams and rivers with typically high current velocities as an important constituent in aquatic plant ecosystems.There are currently 16 species currently accepted in Algaebase based on traditional morphological identification [3].Molecular evidence is accessible for only six species: Lemanea borealis Atkinson, L. fluviatilis (L.) C. Agardh, L. fucina Bory, L. manipurensis Ganesan, JA West, Zuccarello, J Rout, L. occidentalis ML Vis and KM Müller and L. condensata Israelson [4].Species identification for this genus is based on characteristics including thallus height, branch density and spermatangial morphology [5].The systematics of Lemanea are con-Citation: Nan, F.; Zhao, Y.; Feng, J.; Lv, J.; Liu, Q.; Liu, X.; Xie, S.

Morphological and Molecular
Phylogenetic Analysis of a Lemanea Specimen (Batrachospermales, Rhodophyta) from China.Diversity 2022, 14, 479.https://doi.org/10.3390/d14060479 Academic Editor: Renhui Li troversial due to its simple and plastic morphology, and the lack of comparative anatomical and reproductive characteristics [6][7][8].In recent years, it was found that species identified by morphology were not in the same clade identified using molecular evidence [9].The morphology of Lemanea varies with the season and water conditions including salinity, temperature and flow velocity [10,11].The systematics and diversity of Lemanea based only on traditional morphology were not reliable and thus needed revision using molecular methods.With the rapid development of molecular technologies, identification and phylogenetic analyses of Rhodophyta based on gene sequences became common [12][13][14], except for a few reports for Lemanea [15].Li analyzed the phylogeny of Batrachospermales based on rbcL sequences using Lemanea specimens from the USA and Canada, and concluded that the Lemaneaceae were a derived lineage in this order [16].Ganesan collected specimens from India and analyzed the morphology, reproductive characteristics and molecular phylogeny of L. australis Atkinson, L. catenata Kützing, L. fluviatilis, L. mamillosa Kützing and L. torulosa, and described a new species, L. manipurensis [12].
The genus Lemanea is distributed widely in North America and Europe [17][18][19][20][21][22][23], whereas it is rarely reported in Asia, except India and Israel [12,24,25].It has also been reported rarely in China.Jao reported two new species of Lemanea from China in 1941 [26].Xie reported four species in China including L. sinensia Jao from Yunnan, L. crassa Xie et Shi and L. simplex Jao from Chongqing, and L. ramose Xie et Shi from Fujian [27].Additionally, the distribution of Lemanea in Jialing River, Lancang River and Mengxing River was also reported in previous studies [28][29][30].To date, the reports of Lemanea in China have all been based on morphological observations and lack molecular evidence.Thus, they are all regarded as doubtful species [4].As an important lineage in Rhodophyta, the species diversity and distribution of Lemanea in China needs to be explored further, and their molecular sequences and phylogenetics need to be studied.
A Lemanea specimen was collected in Guilin, China.Morphological characterization and molecular phylogenetic analysis were conducted on this material as part of a survey of freshwater red algae in China, adding to the species and distribution diversity of this genus.

Materials and Methods
The Lemanea specimen was collected from Lingchuan, Guilin, China (25°42′ N, 110°32′ E) growing on stones in a clean stream (Figure 1).It was encoded as GLYZC.The sample was transferred to the laboratory as soon as possible and washed with sterilized water carefully to remove the epiphytes.The portion of the samples used for morphological observation was fixed in a centrifuge tube with a 4% formalin solution.The samples used for DNA extraction were preserved in a silica desiccant.For morphological observation, both fresh and formalin-preserved thalli were examined under a BX-51 Olympus microscope equipped with a charge-coupled device (DP72; Olympus, Tokyo, Japan) for photographing.
Total DNA was extracted from the desiccated thalli following the protocol described by Saunders with modifications as described by Vis and Sheath [31,32].Polymerase chain reaction (PCR) amplifications were performed using the primers F577 and FR753 for rbcL [33], and G01 and G07 for SSU [34].The PCR products were purified using a SanPrep column DNA gel purification kit (Sangon, Shanghai, China) and then were sent to BGI Tech Corporation (Beijing, China) for sequencing on an ABI 3730XL sequencer.Both amplification primers were used to determine the sequences.The sequences were manually inspected with Sequencher Version 4.14 (http://sequencher.software.informer.com/,accessed on 1 May 2022).The DNA sequence data generated from this study have been deposited in GenBank (accession numbers: MT297643 for rbcL and MT294296 for SSU).
The sequences obtained in this study and the sequence data obtained by nucleotide blasting downloaded from GenBank were assembled in Clustal-X 2.0 [35].Ambiguous bases on both ends were deleted to produce an identical length alignment.Two datasets were used for the phylogenetic analyses: the rbcL dataset included 42 taxa and 1 Tuomeya as an outgroup, and the SSU dataset included 13 taxa and 2 Balbiania taxa as outgroups.The outgroup taxa were selected according to previous studies [10,12].The pairwise genetic distances between each specimen were computed in MEGA 5.0 [36].The optimal substitution model for each marker was estimated using Modeltest [37].Maximum likelihood trees were built using PHYML software [38,39].The bootstrap analysis was conducted using 1000 replicates.Additionally, Bayesian inference was performed in Mr. Bayes version 3.1.2[40].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 20% trees of burn-in.The resulting phylogenetic trees were edited using Figtree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 1 May 2022).

Morphological Characterization
The specimen was green to purple-brown in color, sessile, unbranched in the base of the thallus, and sparsely and unregularly branched in the middle part.The branch was up to 10 cm in height and tapered toward apices (Figure 2a,b).The nodes were obviously inflated and 110-276 μm in diameter.The spermatangia were positioned at the outer cortex and formed a circular ring around the nodes (Figure 2c,d).The thallus was pseudoparenchymatous and the outer cortex consisted of two or three layers, and the cells of the outermost layer were irregular in shape and hairless (Figure 2e).The central axis was uniseriate and lacked cortical filaments (Figure 2f).Carposporophytes were growing from the outer cortex to the central cavity, with short and sparsely branched gonimoblast filaments (Figure 2g).The ellipsoidal carposporangium was at the top and single, 41-52 μm in length and 12-25 μm in diameter (Figure 2h).Morphological comparisons between specimens collected in this study and those reported previously in China and India are listed in Table 1.

Molecular Analysis
The length of the rbcL fragment used for phylogenetic analysis in this study was 996 bp, of which 274 bp (27.51%) were variable sites and 221 bp (22.19%) were informative for parsimony.The length of the SSU marker used for phylogenetic analysis in this study was 932 bp, of which 177 bp (18.99%) were variable sites and 98 bp (10.52%) were parsimony sites.The p-distance of the rbcL gene between GLYZC and the L. manipurensis specimen from India was 0.01 (vs.0.08-0.09between GLYZC and other species in the phylogenetic tree).The p-distances of the SSU gene between GLYZC and the other Lemaneaceae species were 0-0.01 (vs.0.01-0.04 between GLYZC and Batrachospermaceae species).
All three methods including maximum likelihood, neighbor joining and Bayesian inference produced similar topologies.Therefore, only the Bayesian trees for rbcL and SSU are shown in Figures 3 and 4, respectively, with all the supporting values included on the nodes.In rbcL phylogenetic tree (Figure 3), there were two main branches consisting of the genera Lemanea and Paralemanea , with high supporting values of 0.98/87.2/93and 1.00/100/100., respectively The GLYZC specimen collected in this study clustered with L. manipurensis from India, supported by 1.00/100/100.In the SSU phylogenetic tree (Figure 4), there was only one accessible sequence of the genus Lemanea in the GenBank database (AF026051 for L. fluviatilis).The GLYZC formed a sister relationship with AF026051 but with low values.Three Paralemanea sequences formed a monophyletic clade supported by 1.00/91.5/95.

Discussion
The typical characteristics of the genus Lemanea are a thallus growing in tufts with a naked central axis.The morphology of GLYZC collected in this study was consistent with this characteristic.The rbcL phylogeny showed a close relationship between GLYZC and L. manipurensis, a new species reported in 2015 [12].L. manipurensis was reported in Manipur in northeast India and characterized by a carposporophyte with very short gonimoblast filaments and a single, large, elongate carpospore at the terminal cell of each branch [12].The morphological characteristics observed in GLYZC collected in this study was consistent with these characters; therefore, GLYZC was identified as a new record of L. manipurensis in China.According to the morphological comparison, the L. manipurensis distributed in China was larger in thallus height than the type specimen (10-13 cm for GLYZC vs. 10 cm for L. manipurensis in India).The branches were at the lower part of L. manipurensis collected in this study and at the base of the thallus in the type specimen.The shapes of the spermatangium of L. manipurensis were slightly different between the specimen collected in this study and that distributed in India.The spermatangia were continuous wide rings on the nodes in both young and mature branches in L. mani- purensis collected in this study, whereas they occurred in isolated, low and indistinct patches in L. manipurensis from India.The carposporophytes of L. manipurensis collected from China were 41-52 μm in length and 12-25 μm in diameter, which were much smaller than those of L. manipurensis from India [12].Although there are a few morphological discrepancies between GLYZC and the type specimen of L. manipurensis, the material from China was identified as L. manipurensis, mainly on the basis of the molecular evidence.It can be inferred that the morphological characteristics including the thallus height, the position of the branches, the shape of spermatangia and the size of carposporophytes are variable among different populations within a species.Validation of the reliability of these characteristics for species identification needs observation on more specimens and molecular information in further studies.
The family Lemaneaceae consists of the genera Lemanea and Paralemanea.Based on traditional morphological systematics, the genus Lemanea is characterized by the occurrence of hair cells in the outer cortex.However, there were no hair cells in the outer cortex of L. manipurensis collected in this study, indicating that hair cells in the outer cortex cannot be considered as a reliable criterion for the genus Lemanea.The morphological characteristics for the identification of the genus Lemanea were expanded in our study.Up to now, there have been four species reported in China, including L. sinica, L. simplex, L. ramosa and L. crassa, all of which are endemic to China and were identified based on morphology with no molecular information.These four species have numerous characteristics in common with each other and L. manipurensis, and all five species are from Asia [4].According to the morphological comparison listed in Table 1, the newly recorded L. manipurensis was similar to L. sinica and L. crassa in thallus height, while the thallus was obviously larger than that of L. simplex and L. ramose [27].The position and density of the branches were different between L. manipurensis and the other species, with the branches on the lower part in L. manipurensis, no branch in L. crassa and L. simplex, and branches on the base position in L. sinica and the upper part in L. ramose.The carposporangial size of L. manipurensis was smaller than that of the other four species reported in China.Morphological characters including the shape of spermatangium, thallus height and branching are critical for identification of the genus Lemanea [5,27].The shapes of the carposporangium were similar in L. manipurensis and L. sinica and L. simplex.There were large similarities between L. manipurensis and L. sinica, except for the stalk and branching.Therefore, the molecular sequence of L. sinica was needed to validate if they were the same species.This also suggests the necessity to supply molecular sequences of the genus Lemanea currently found in China to elucidate the species distinctness.
The rbcL gene is a common DNA barcode for phylogenetic analyses in freshwater Rhodophyta at the systematic level including the order, family, genus and species [13][14][15]41,42].It is also the most commonly used in molecular phylogenetic analyses of the genus Lemanea [10,12,15,43].The phylogeny constructed from the rbcL gene is more reliable and can be used for species identification for the genus Lemanea.By comparison, the application of the SSU sequence in Lemanea has been infrequent.Accessible SSU sequences in the GenBank database are limited and resulted in the low supporting values of the phylogenetic tree.This suggests the urgent need for molecular phylogenetic analyses of Lemanea species worldwide.The first molecular information of Lemanea species from China was provided in our study, contributing data and a theoretical basis for phylogenetic analyses of this genus, and supplementing the species and distribution diversity of the genus Lemanea in China.

Conclusions
Morphological comparisons and molecular analyses both supported the new record of L. manipurensis in China, the fifth reported species of the genus Lemanea in China.The specimen in this study was morphologically characterized by branches that were positioned in the lower part of the thallus, hair cells absent around the outer cortex, carpospores that were ellipsoidal and single at the top of the gonimoblast filaments and spermatangia that formed continuous wide rings on the thallus nodes.Given the morphological discrepancies between GLYZC and type specimen of L. manipurensis from India, the material from China was identified mainly through molecular evidence.By comparing the morphology of specimens collected from Guilin, China, and the holotype specimen in India, it was found that spermatangial shapes are variable among different populations of L. manipurensis.The first molecular information of Lemanea species from China was provided in our study, providing molecular evidence and a theoretical basis for molecular phylogenetic research on the genus Lemanea, and enriching the species diversity and geographical distribution of Lemanea in China.

Figure 1 .
Figure 1.Map showing approximate location of the sample investigated in this study.

Figure 2 .
Figure 2. Morphological characteristics of Lemanea collected in Guilin, China.(a) Morphology of the entire thallus.(b) Axes narrow toward the apices.(c) Spermatangium growing continuously on the thallus nodes.(d) Cross section of the spermatangium.(e) Cells of the outer cortex.(f) Naked axial filaments with no surrounding cortical filaments.(g) Carposporophyte projecting into the center of the thallus.(h) Single carpospores terminal on the carposporophyte branches.Scale bar: (b,c,e,f) 100 μm; (d,g,h) 20 μm.

Figure 3 .
Figure 3.The phylogenetic tree constructed from rbcL sequences.The numbers on the branch nodes represent the Bayesian posterior probabilities, maximum likelihood bootstrap tree support values andneighbor joining bootstrap tree support values.Supporting values below 50% are noted with "-".The Lemanea specimen collected in this study is indicated by a black box.

Figure 4 .
Figure 4.The phylogenetic tree constructed from SSU sequences.The numbers on the branch nodes represent the Bayesian posterior probabilities, maximum likelihood bootstrap tree support values and neighbor joining bootstrap tree support values.Supporting values below 50% are noted with "-".The Lemanea specimen collected in this study is indicated by a black box.

Table 1 .
Morphological comparisons of Lemanea species distributed in China and India.