Phylogenetic Analysis of Bangiadulcis Atropurpurea (A

Arch. Biol. Sci., Belgrade, 67(2), 445-454, 2015 DOI:10.2298/ABS140902009C

PHYLOGENETIC ANALYSIS OF BANGIADULCIS ATROPURPUREA (A. ROTH) W. A. NELSON AND BANGIA FUSCOPURPUREA (DILLWYN) LYNGBYE (BANGIALES, RHODOPHYTA) IN TAIWAN

Jui-Yu Chou1, Shao-Lun Liu2, Yu-Der Wen1 and Wei-Lung Wang1,*

1 Department of Biology, National Changhua University of Education, Changhua 500, Taiwan 2 Department of Life Science, Tunghai University, Taichung 407, Taiwan

*Corresponding author: [email protected]

Abstract: Samples of the freshwater red algae, Bangiadulcis atropurpurea, collected from the mountain waterfalls and its close species, Bangia fuscopurpurea, collected from coasts, were phylogenetically analyzed. The sequences of the rbcL gene and RuBisCO spacer region of the freshwater Bangiadulcis atropurpurea species were identical or similar to those of B. atropurpurea from Japan, North America and Europe. This result indicated that the freshwater Bangiadulcis species from Taiwan shared a common ancestor with the three above-mentioned populations and formed a distinct clade from the marine Bangia species in the phylogenetic trees. It is suggested that all the previous records on marine Bangia species should be revised and the name B. fuscopurpurea be used for the marine species in Taiwan. In this study, the freshwater alga B. atropurpurea presents a new record in the algal flora of Taiwan. This finding is important for the protection of the biodiversity of red algal flora, and provides useful information on the ecological conservation of the species in Taiwan. Key words: Bangiadulcis atropurpurea; Bangia fuscopurpurea; rbcL gene; Rhodophyta; RuBisCO spacer Received September 2, 2014; Revised October 13, 2014; Accepted October 15, 2014

INTRODUCTION purea, which was later included in the genus Bangia by C. Agardh (1824). Geesink (1973) The red-algae genus Bangia Lyngbye is wide- concluded that the freshwater B. atropurpurea spread along the coast of Taiwan. In the marine and the marine B. fuscopurpurea (Dillwyn) Lyn- environment, Bangia generally occupies a habitat gbye are conspecific. On the basis of karyology in the high intertidal zone (Sheath, 1984). Ban- and DNA sequence analysis, freshwater Bangia giadulcis atropurpurea (A. Roth) W. A. Nelson is sp. are placed on a separate and well-supported reported from freshwater, and it was originally branch, and the name B. atropurpurea has been described by Roth (1806) as Conferva atropur- proposed to represent this lineage (Müller et al.,

445 446 Chou et al.

1998, 2003). Later, Nelson (2007) indicated that were collected from marine environments, and we the freshwater species could be distinguished believe that the name used in these studies was from other members of the Bangiales based on incorrect. Molecular tools have recently proved cytological, morphological, ecological, and phy- useful in various studies of algae, facilitating the logenetic characters. Thus, Nelson used Ban- elucidation of relationships among taxa, especially giadulcis to describe the freshwater species. among red algae (Coleman and Goff, 1991; Mül- ler et al., 1998, 2003; Wang et al., 2005). However, In Taiwan, B. atropurpurea has been recorded there has not been any molecular method-based in many studies (Okamura, 1936; Shen and Fan, taxonomic or phylogenetic study of Bangia spe- 1950; Tanaka, 1950, 1952; Fan, 1953; Tokida, cies from Taiwan. The present study was under- 1954; Rho, 1958; Chiang, 1962, 1973; Taniguti, taken to re-examine the Bangia species found in 1976; Sheath and Cole, 1984). All the specimens Taiwan. We collected marine Bangia species from

Fig. 1. Bangiadulcis atropurpurea (A. Roth) W. A. Nelson. A. Unbranched filaments. Young filaments initially comprising a single row of cylindrical cells (arrowheads). Mature filaments become polysiphonous by longitudinal cell divisions (arrows). B. Close-up of young filaments, showing a single row of cylindrical cells. C. Close-up of mature filaments, showing longitudinal cell divisions. D. Rhizoidal part of a plant. E. Asexual reproduction by monospore(s) (arrow). F. Unknown bud-like structure on a filament. Bangiadulcis atropurpurea and Bangia fuscopurpurea in Taiwan 447 the coasts of northern Taiwan and Kinmen County MATERIALS AND METHODS (the county lies in the sea roughly 190 km west of the Taiwan) and two samples of freshwater Bangia Morphological observations species from the mountain waterfalls of northern and central Taiwan. In addition to confirming their Samples of marine Bangia species were collected phylogenetic relationship based on the rbcL gene from three sites in northern Taiwan and Kinmen sequence, the RuBisCO spacer region and mor- County. Two samples of freshwater Bangiadulcis phological characters are also described in detail. sp. were collected, one from the Youling Waterfall,

Table 1. Location (abbreviation), collection information, and GenBank accession number of Bangia species used in this study.

Location GenBank accession number Collection information (abbreviation) RuBisCO spacer rbcL GLD Disko Island, Greenland. Coll. C. Wiencke. 19941 AF007849 AF043366 NWT Rankin Inlet, NWT, Canada. Coll. R. Sheath and K. Müller. Dec. 12, 19961 AF007849 AF043366 NF Holyrood, NF, Canada. Coll. R. Hooper. Oct. 1, 19931 AF007852 - NH Hampton Beach, NH, USA. Coll. R. Sheath. May 19, 19921 AF007849 AF043366 MA Woods Hole, MA, USA. Coll. M. A. Allen. Summer, 1954. University of Texas AF007848 AF043369 Culture Collection (UTEX) LB 7411 RI Narragansett, RI, USA. Coll. R. Sheath. Apr. 28, 19921 AF007849 AF043378 NC South Masonboro Inlet Jetty, NC, USA. Coll. W. Freshwater. June 14, 19961 AF007848 AF043368 TX Port Aransas, TX, USA. Coll. R. Sheath and M. Vis. Dec. 4, 19931 AF007854 AF043377 VIS7 Tortola, British Virgin Islands. Coll. R. Sheath. Dec. 19, 19951 AF007850 - AK Chaichei Islands, AK, USA. Coll. S. Lindstrom. June 12, 19951 AF007849 AF043366 BC1 Triple Island, BC, Canada. Coll. L. Golden. Oct. 1, 19931 AF007850 AF043372 BC2 Ogden Point, Victoria, BC, Canada. Coll. B. Oates. May 31, 19961 AF007853 AF043376 OR Lincoln City, OR, USA. Coll. R. Sheath. Apr. 2, 19921 AF007851 AF043367 CA Shell Beach, CA, USA. Coll. S. Sparling. Mar. 7, 19941 AF007853 AF043374 NJ Data from National Center for Biotechnology Information - AF169328 NS Seaview Memorial Park, Halifax, NS, Canada. Coll. A. Sherwood. Dec. 26, 19972 - AF169331 KM1 Tianbu, Kinmen County. Coll. J. Y. Chou and W. L. Wang3 DQ408159 DQ408158 KL Pinglang Bridge, Keelung City, Taiwan. Coll. J. Y. Chou3 DQ408161 - KM2 Fongshang, Kinmen County. Coll. J. Y. Chou and W. L. Wang3 DQ408160 DQ408163 NL* Ysselmeer, Enschede, The Netherlands. Coll. K. Müller and A. Sherwood. May - AF169330 24, 19982 AT17* Traunsee, Gmunden, Austria. Coll. R. Sheath. June 24, 19982 - AF169327 AT22* Bodensee, Bregenz, Austria. Coll. R. Sheath. July 5, 19982 - AF169333 BI12* River Thames, England. Coll. R. Sheath. May 24, 19961 AF007855 AF043373 IT* Near Lake Garda, Italy. SAG culture 1351-11 AF007855 AF043370 IR* River Shannon, Ireland. Coll. R. Sheath. July 11, 19971 AF043545 AF043371 JP1* Oi-gawa, Ikawa, Shizuoka, Japan4 - AB114641 JP2* Amahata-gawa, Hayakawa, Yamanashi, Japan4 - AB114640 TY* Youling Waterfall, Taoyuan County, northern Taiwan. Coll. J. Y. Chou and Y. DQ408157 DQ408155 H. Huang. Feb. 5, 20053 NT* Caihong Waterfall, Nantou County, central Taiwan. Coll. J. Y. Chou and Y. H. DQ408156 DQ408162 Huang. Jan. 1, 20063

1Müller et al. 1998; 2Müller et al. 2003; 3This study; 4Hanyuda et al. 2004; *freshwater population 448 Chou et al.

Taoyuan County, northern Taiwan, and the other ed according to the Kimura two-parameter model from the Caihong Waterfall, Nantou County in (Kimura, 1980). The robustness of tree topolo- central Taiwan. Specimens used in the morpho- gy was estimated by 100 replicates of bootstrap logical study were preserved in 5-10% formalin/ resampling, using the maximum parsimony (MP) freshwater or dried as herbarium specimens, and method. The A+T content (%) was calculated us- those used in the molecular study were preserved ing the GeneDoc software (Nicholas et al., 1997). in 95% ethanol. The vegetative and reproductive structures were examined using a light microscope (Carl Zeiss, Axioskop 2, Germany) under RESULTS 200× or 400× magnification and a dissecting microscope (Carl Zeiss, Stemi SV11, Germany), respectively. Photographs were taken using a cooled Morphological observations CCD digital camera system (Pixera, Penguin 600CL with Auto-Montage software; CA, USA) Bangiadulcis atropurpurea attached to the microscope. Species identifica- (Roth) W. A. Nelson 2007 tion using morphological methods was based on Basionym − Conferva atropurpurea the description in Marhold and Hindak (1998), Roth 1806: 208, pl. 6 Kumano (2002), Kusber et al. (2005) and Nelson (2007). The specimens used in this study were de- Description − Plants are filamentous, with un- posited at the Department of Biology, National branched dark-purple filaments. Young filaments Changhua University of Education, Taiwan. initially comprise a single row of cylindrical cells (Figs. 1A, B). Mature filaments become polysi- Molecular analyses phonous by longitudinal cell divisions (Figs. 1A, C). The plants are anchored to the substratum Genomic DNA was extracted using the DNeasy by a rhizoidal part, which grows downward from Plant Mini Kit (Qiagen, Valencia, CA, USA) ac- the lower cells through the common outer wall cording to the instructions of the manufacturer. (Fig. 1D). Asexual reproduction occurs by mono- Total DNA served as the template for double- spores (Fig. 1E). An unknown bud-like structure stranded polymerase chain reaction (PCR). The was observed on a filament (Fig. 1F). primers and protocols for gene amplification and automated sequencing of the rbcL gene and Specimens − (1) Collected from Youling Wa- RuBisCO spacer region were those outlined by terfall, Taoyuan County, northern Taiwan, by Wang et al. (2005) and Kamiya et al. (1998), re- Chou J. Y. and Huang Y. H., 5 Feb. 2005, NCUE- spectively. Most parsimonious trees were gener- JYC-940205. (2) Collected from Caihong Wa- ated by performing a heuristic search under the terfall, Nantou County, central Taiwan, by constraints of 100 replicates of random sequence Chou J. Y. and Huang Y. H., 1 Jan. 2006, NCUE- addition, steepest descent and tree bisection- JYC-950101. reconnection (TBR) branch swapping in the Phylogenetic Analysis Using Parsimony (PAUP) Type locality − Bremen, Germany. program (Swofford, 2003). Each character in the DNA sequence analysis was analyzed with equal Holotype − Roth’s collection was destroyed in weight. The distance-matrix values were estimat- World War II (Koster, 1957). Bangiadulcis atropurpurea and Bangia fuscopurpurea in Taiwan 449

Neotype − Designated by Müller et al. (2003). Chialing River near Pehpei, Szechwan, China; Collected from Ysselmeer, Enschede, the Nether- Amahata-gawa, Yamanashi, Japan; Great Lakes lands, by Müller K. M. and Sherwood A. R. May except Lake Superior, Canada; North America; 24, 1998. The Natural History Museum, (earlier, and Taiwan. British Museum [BM]), London; BM accession number: BM000637980. Habitat − Plants are epilithic, growing on stones submerged in a well-aerated, moderately World distribution − Belgium, Serbia and the agitated, and clear pool under a waterfall, at a Netherlands in Europe (as Bangia atropurpurea); mean depth of 2-5 cm. In Taiwan, they are found

Fig. 2. Bangia fuscopurpurea (Dillwyn) Lyngbye. A. Unbranched filaments (arrows). B. Mature filaments become polysiphonous by longitudinal cell divisions. 450 Chou et al. in Youling Waterfall, Taoyuan County, northern Remarks − Bangia fuscopurpurea is dioecious. Taiwan, and Caihong Waterfall, Nantou County, Spermatangia develop in packets by division of central Taiwan. an undifferentiated cell, and carpogonia produce a small protrusion for the attachment of sperma- Remarks − B. atropurpurea presents a new re- tia. Asexual reproduction occurs by vegetatively cord in the algal flora of Taiwan. Asexual repro- produced monospores. This species occurs only duction occurs by vegetatively produced mono- in marine habitats. spores, and natural populations of this species have been reported to reproduce only asexually. The rbcL gene This species occurs only in freshwater habitats. A set of 23 rbcL sequences of selected species Bangia fuscopurpurea (Dillwyn) Lyngbye 1819 (Table 1), including 1 020 aligned sites with 289 potentially parsimony-informative characters, Basionym − Conferva fuscopurpurea was analyzed. Five most parsimonious trees were Dillwyn 1807: 1802-1809, pl. 92 obtained in the MP analysis based on a heuristic search performed using the stepwise addition Descriptions − Plants are filamentous, with un- of 100 random replications. One of these trees branched dark-purple filaments. Young filaments is shown in Fig. 1, wherein we have shown the initially comprise a single row of cylindrical branches supported by ≥50% bootstrap values in cells (Fig. 2A). Mature filaments become polysi- the MP analysis. The tree length was 524 steps, phonous by longitudinal cell divisions (Fig. 2B). with a consistency index (CI) of 0.7023 and re- Asexual reproduction is not observed. tention index (RI) of 0.8497. We encountered difficulty in amplifying the rbcL gene for the Specimens − (1) Collected from Tianpu, Kin- men County, by Chou J. Y. and Wang W. L., 5 Feb. collection from Pinglang Bridge, Keelung City, 2005, NCUE-JYC-940205. (2) Collected from Taiwan, and therefore the related information Fengshang, Kinmen County, by Chou J. Y. and was not included in the analysis. The freshwater Wang W. L., 5 Feb. 2005, NCUE-JYC-940205. (3) Bangiadulcis species clade was well supported by Collected from Pinglang Bridge, Keelung City, by bootstrap analysis (100%), and as shown in Fig. Chou J. Y., 5 Feb. 2005, NCUE-JYC-940205. 3, it represents a distinct clade from the marine Bangia species. Type locality − Dunraven Castle, Glamorgan, Wales, UK. RuBisCO spacer region

World distribution − Ireland, Europe; western The RuBisCO spacer region in the specimens of Africa; eastern North America; eastern South Amer- both the marine and freshwater Bangiadulcis spe- ica; Caribbean Basin; Mediterranean Basin; eastern cies was 77-bp long and A/T rich, with a nucleo- Asia; western North and central America; Southeast tide composition ranging from 70.13% to 77.92% Asia; western South America; and Atlantic Islands. A+T. Both the freshwater specimens from Taiwan (TY* and NT*) were found to be identical. The Habitat − Plants are epilithic, growing on freshwater specimens from Ireland (IR*) differed rocks in the high intertidal zone. from those collected from Taiwan by 1 bp; this Bangiadulcis atropurpurea and Bangia fuscopurpurea in Taiwan 451 difference was due to an A↔C transversion. Fur- DISCUSSION thermore, the freshwater specimens from Amer- ica and Europe differed from those collected The genus Bangia has a long evolutionary history from Taiwan by 1 bp; this difference was due to (1.2 × 109 y; Butterfield et al., 1990; Butterfield, an A↔G transition. Both the marine specimens 2000) and a divergence time of 174 × 106 to 265 from Kinmen County (KM1 and KM2) were × 106 y between freshwater and marine species identical to that from Canada (NF) and differed (Müller et al., 1998). Based on the rbcL gene se- by 3 bases from the marine specimen from Tai- quence analysis in this study, we found consid- wan (KL; 8.29% sequence divergence), which was erable sequence divergence between the marine identical to the specimens from North Carolina and freshwater species. (NC) and Massachusetts (MA) (Table 2). In this study, the values of A-T richness and We have not shown the trees inferred from length of the RuBisCO spacer region of the ex- the parsimony analysis of the RuBisCO spacer amined species were within the range reported region because of the lack of parsimony-infor- for Rhodophyta (Destombe and Douglas, 1991; mative sites. Parsimony analysis of the RuBisCO Kamiya et al., 1998). Due to homoplasy and the spacer region resulted in 12 most parsimonious small number of phylogenetically informative trees (data not shown). These trees were based on characters, we could not clarify the relationship 7 potentially parsimony-informative characters among the various Bangia species. However, we within the 77-bp region of the spacer; the tree observed some basic trends that are consistent length was 27 steps, with CI = 0.8889 and RI = with those indicated by the rbcL gene analysis. 0.8000. There was considerable homoplasy in 6 For instance, the 2 freshwater specimens col- characters, because of which we could not clarify lected from northern and central Taiwan (TY* the relationship among these specimens. and NT*, respectively; Fig. 3) were identical with

Fig. 3. A set of 23 rbcL sequences of selected species, including 1020 aligned sites with 289 potentially parsimony-informative characters, was analyzed. This figure shows 1 of the 5 most parsimonious trees generated by performing a heuristic search with the stepwise addition of 100 random replications. Bootstrap values are shown above the branches that were supported by ≥50% bootstrap values in the maximum parsimony (MP) analysis. The tree length was 524 steps, with the consistency index (CI) of 0.7023 and the retention index (RI) of 0.8497. 452 Chou et al. regard to the spacer region, as were the marine until the complex relationship among these spe- specimens collected from Alaska, Northwest Ter- cies can be resolved (Sheath and Müller, per- ritories, Greenland, New Hampshire and Rhode sonal communication). Besides confirming the Island (AK, NWT, GLD, NH and RI, respective- taxonomic relationship among the first-recorded ly). Furthermore, all the freshwater specimens of freshwater Bangiadulcis species, the present study Bangia species were nearly identical, as revealed indicates that the marine Bangia species are dis- by the analyses of two genes in a previous study tinct from the freshwater Bangiadulcis species (Müller et al., 2003). found in Taiwan. It is suggested that all the previous records of marine Bangia species should be The finding of monophyly of the freshwa- revised and the name B. fuscopurpurea be used ter Bangiadulcis species based on the rbcL gene for the marine species in Taiwan. analysis in this study was consistent with that based on independent analyses of the rbcL and The role of taxonomy should never be un- 18S RNA genes and a combined analysis of the derestimated. We need more taxonomic data and two genes in a previous study (Müller et al., 2003, convenient methods to tackle algal identification. Nelson, 2007). From this study and those of Mül- Here we use the rbcL gene and RuBisCO spacer ler et al. (1998, 2003), it is clear that all the fresh- region to distinguish Bangiadulcis atropurpurea water species formed a separate lineage. With from its close species. regard to the marine Bangia species, several distinct entities were revealed by the 18S RNA gene Bangiadulcis atropurpurea (as B. atropur- analysis performed in a previous study (Müller et purea) has been indicated as an endangered al., 2003), but not by the rbcL gene analysis con- species in Serbia (Simić et al., 2007), Slovakia ducted in this study. However, all marine Bangia (Marhold and Hindak, 1998) and Germany species should be recognized as B. fuscopurpurea (Kusber et al., 2005). It has even been declared

Table 2. Pairwise comparison of distance matrices and variation in the aligned sequences of the RuBisCO spacer region (77 bp) among Bangiadulcis and Bangia populations. The lower matrix shows the number of base-pair changes between entities. The upper matrix shows sequence divergence between entity pairs, calculated using the Kimura two-parameter model.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 TY*1 - 0.000 0.0132 0.0131 0.0682 0.0265 0.0682 0.1450 0.0682 0.0265 0.0967 0.0539 0.1595 0.0539 2 NT*1 0 - 0.0132 0.0131 0.0682 0.0265 0.0682 0.1450 0.0682 0.0265 0.0967 0.0539 0.1595 0.0539 3 BI12* and IT*2 1 1 - 0.0265 0.0829 0.0402 0.0829 0.1620 0.0829 0.0402 0.1121 0.0682 0.1431 0.0682 4 IR*2 1 1 2 - 0.0542 0.0132 0.0542 0.1298 0.0542 0.0132 0.0824 0.0402 0.1439 0.0402 5 KM11 5 5 6 4 - 0.0402 0.0000 0.1620 0.0000 0.0402 0.1439 0.0682 0.1127 0.0132 6 KL1 2 2 3 1 3 - 0.0402 0.1137 0.0402 0.0000 0.0974 0.0265 0.1278 0.0265 7 KM21 5 5 6 4 0 3 - 0.1620 0.0000 0.0402 0.1439 0.0682 0.1127 0.0132 8 AK, NWT, GLD, 10 10 11 9 11 8 11 - 0.1620 0.1137 0.1765 0.1286 0.2483 0.1450 NH, and RI2 9 NF2 5 5 6 4 0 3 0 11 - 0.0402 0.1439 0.0682 0.1127 0.0132 10 NC and MA2 2 2 3 1 3 0 3 8 3 - 0.0974 0.0265 0.1278 0.0265 11 TX2 7 7 8 6 10 7 10 12 10 7 - 0.0829 0.2276 0.1278 12 BC1 and VIS72 4 4 5 3 5 2 5 9 5 2 6 - 0.1595 0.0539 13 CA and BC22 11 11 10 10 8 9 8 16 8 9 15 11 - 0.0974 14 OR2 4 4 5 3 1 2 1 10 1 2 9 4 7 - 1This study; 2Müller et al. 1998; *freshwater population Bangiadulcis atropurpurea and Bangia fuscopurpurea in Taiwan 453 that B. atropurpurea is already extinct in Poland Eloranta, P. and J. 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