Phycologia Volume 53 (4), 329–341 Published 12 June 2014 Diversity of freshwater red algae (Rhodophyta) in Malaysia and Indonesia from morphological and molecular data EMILY T. JOHNSTON1, PHAIK-EEM LIM2, NURLIAH BUHARI3, EMILY J. KEIL1, M. IQBAL DJAWAD4 AND MORGAN L. VIS1* 1 2 Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701 USA Institute of Biological Sciences and Institute of Ocean and Earth Sciences (IOES), University of Malaya, 50603 Kuala Lumpur, Malaysia 3 Mataram University, West Nusa Tenggara, Indonesia 4 Hasanuddin University, Makassar, South Sulawesi, Indonesia ABSTRACT: Both Malaysia and Indonesia have high biodiversity for a variety of organisms, including freshwater red algae. The type localities of over a dozen freshwater red algal taxa are located in Malaysia and Indonesia. These species were described prior to the advent of molecular systematics, and no molecular data were available for specimens from these two countries. Therefore, the goal of this study was to visit type and other locales in Malaysia and Indonesia to recollect freshwater red algal taxa for both morphological and molecular studies. A total of 11 previously published species were identified, with eight taxa belonging to the Batrachospermales, and one each in the Compsopogonales (Compsopogon caeruleus), Ceramiales (Caloglossa beccarii) and Thoreales (Nemalionopsis shawii). The rbcL gene provided numerous insights including two new species, Batrachospermum phangii sp. nov. from Malaysia and Kumanoa celebes sp. nov. from Indonesia. The placement of Batrachospermum cylindrocellulare in section Aristata rather than section Batrachospermum was clarified. Specimens from Malaysia identified as Sirodotia delicatula were distantly related to specimens from South America, suggesting a cryptic species in South America. Likewise, Balliopsis prieurii from Malaysia was distantly related to Balliopsis prieurii from South America. A gametophyte specimen and numerous chantransia stage specimens were conspecific with Batrachospermum macrosporum from South America, and this is a new record of this taxon in Malaysia. Chantransia stage sporophyte specimens from Indonesia had a similar sequence to Sheathia arcuata from Hawaii. The sequence placement of N. shawii from Indonesia points to the need for further systematic research on this genus. Although Kumanoa gibberosa was not recollected at the type location in Malaysia, it was found in Indonesia; likewise, the type locality in Indonesia did not yield S. delicatula but this species was present in Malaysia. Given the previously described diversity, the two new taxa proposed in this study and the insights gained from the present molecular data, we suggest that future focus on freshwater red algae from Southeast Asia will yield considerable knowledge of the flora of the region and freshwater red algal diversity in general. KEY WORDS: Balliopsis, Batrachospermum, Caloglossa, Compsopogon, Kumanoa, LSU rDNA, Nemalionopsis, rbcL, Sheathia, Sirodotia INTRODUCTION Most rhodophyte taxa inhabit marine environments, with freshwater red algae accounting for only ~ 3–5% of the species diversity (Sheath 1984). Nevertheless, freshwater red algae are taxonomically diverse, with members in nine orders (Kumano 2002). Three orders, the Batrachospermales, Thoreales and Balbianiales, are exclusively freshwater and contain the bulk of the species diversity. However, there are many taxa scattered in primarily marine orders, such as Caloglossa in the Ceramiales. Many of these species, including Caloglossa beccarii (Zanardini) De Toni, have been reported from both brackish and freshwater (Kamiya et al. 2003). In freshwater, many of the species within the Batrachospermales occur only in flowing water, with a few reported from lentic waters (Sheath & Hambrook 1990). Other taxa, such as Compsopogon caeruleus (Balbis ex C. Agardh) Montagne, Compsopogonales, are often reported from a variety of habitats with little flow (Sheath 1984). * Corresponding author (vis-chia@ohio.edu). DOI: 10.2216/13-223.1 Ó 2014 International Phycological Society Freshwater red algae are reported from Africa, Asia, Australasia, Europe, North America and South America, as well as Pacific, Indian and Caribbean oceanic islands (Kumano 2002). Despite historical and modern collections, we are far from understanding their true diversity and biogeography. For example, Kumanoa has been the subject of intense morphological and molecular study recently, yielding numerous new species and confirming the majority of the previously described species (Necchi & Vis 2012; Vis et al. 2012). Many previously presumed cosmopolitan taxa have restricted distributions, e.g. Batrachospermum gelatinosum (Linnaeus) De Candolle (Vis & Entwisle 2000) and Kumanoa ambigua (Montagne) Entwisle, M.L. Vis, W.B. Chiasson, Necchi & A.R. Sherwood (Vis et al. 2012). Other taxa, such as Compsopogon caeruleus, appear to be truly cosmopolitan, with little genetic variation among specimens from Asia, Australia, Europe, North and South America and Pacific Islands (Carlile & Sherwood 2013; Necchi et al. 2013). There are regions that may have high degrees of endemism, e.g. the numerous Australasian endemic taxa (Vis & Entwisle 2000). Molecular data suggest many of these endemics are phylogenetically related. Conversely, Sirodotia suecica Kylin diverged from its closest known relatives following a long-distance dispersal event (Vis & Entwisle 329 330 Phycologia, Vol. 53 (4) 2000; Entwisle et al. 2009; Lam et al. 2012). Malaysia is one of the most studied countries for freshwater red algal diversity and has many endemic taxa (Table S1; Kumano 2002 and the references therein). Additionally, Indonesia, the Philippines and Thailand also have many type locations (Table S1 for Indonesian type locations; Skuja 1934, 1938; Kumano & Liao 1987; Traichaiyaporn et al. 2008). Therefore, Southeast Asia may be a hot spot for freshwater red algal diversity, just as the region is known for vascular plant biodiversity (Mutke & Barthlott 2005). The history of freshwater red algal studies in Indonesia and Malaysia is sparse, punctuated by brief periods of concentrated research. The first reported freshwater red alga was Caloglossa beccarii described from Sarawak, Malaysia, on the island of Borneo (Zanardini 1872). A number of reports followed years later from Indonesia: Thorea zollingeri Schmitz from Java (Schmitz 1892) and two species in the Batrachospermales, Batrachospermum lochmodes Skuja and Sirodotia delicatula Skuja (Skuja 1938). To date, no further investigations of the freshwater red algal flora of Indonesia have been published. However, 12 new species from the genera Thorea, Balliopsis, Batrachospermum and Kumanoa have been described from Malaysia (Table S1). In total, 27 taxa have been reported from these two countries, with many (13) potential endemics, known from only a few locations or the type localities (Table S1). There are only a few taxa that appear to have a wider distribution, such as S. delicatula, having been recorded numerous times from South America (Necchi et al. 2007; Lam et al. 2012) and C. beccarii reported from India, Northern Australia, the Panama Canal, Singapore, Thailand and Vietnam (Lin et al. 2001; Kumano 2002; Kamiya et al. 2003). For the past 20 yr there has not been a focus on Malaysian freshwater red algae, and the time period is much longer for Indonesia. The aim of this study was to visit as many type localities as possible in Malaysia and Indonesia, along with additional sites, to collect both previously described and potentially unknown taxa. Both morphological features and molecular data, primarily the rbcL gene, were used to characterize the taxa, determine their phylogenetic affinities and make biogeographic inferences. MATERIAL AND METHODS Sites in Sulawesi and Java, Indonesia, as well as peninsular Malaysia, were investigated (Fig. S1). Thirty-four freshwater red algal samples (macroscopically identified) were collected (Table S2). Water temperature, pH and conductivity were recorded at each site using handheld meters (Oakton Waterproof ECTestr low and Waterproof pH Testr, Oakton Instruments, Vernon Hills, Illinois, USA) (Table S2). Visible epiphytes were removed, and the sample was divided into four or six portions. One portion was dried in silica desiccant for DNA extraction, one was placed in 95% ethanol for morphological examination and the remaining portions were pressed as herbarium voucher specimens. For samples collected in Indonesia, one herbarium sheet was deposited at the Floyd Bartley Herbarium at Ohio University, USA (BHO), and the second was deposited in Research and Development of Marine, Coast and Small Islands, Hasanuddin University, Indonesia. For samples collected from Malaysia, two additional herbarium sheets were made when enough material was available, and these were deposited in the University of Malaya Seaweeds and Seagrasses Herbarium (KLU) and the National Herbarium of Malaysia at the Forest Research Institute Malaysia (KEP) (Table S2). Holotype specimens were prepared from the BHO material and deposited at the University of Michigan, USA (MICH). For morphological identification, preserved specimens were examined in a BX-40 compound microscope (Olympus America Inc., Lake Success, New York, USA). Diagnostic morphological features observed in the microscope were photographed and measured using an attached SC20 camera (Olympus America Inc.) and the software MicroSuiteTM Special Edition Five (Olympus America Inc.). All specimens were identified to the lowest possible taxonomic unit using numerous relevant literature sources but in particular Kumano (2002), Kamiya et al. (2003) and Necchi & Vis (2012). For DNA extraction, tissue dried in silica was either homogenized by grinding in liquid nitrogen with a mortar and pestle or by pulverizing with beads in the TissuelyserLT (Qiagen Inc., Valencia, California, USA). DNA was extracted using the NucleoSpin Plant Genomic DNA kit (Macherey-Nagel, Bethlehem, Pennsylvania, USA) following the manufacturer’s protocol, with the following modification: the cell lysis step was increased to 30 min. A portion (1282 bp or 1340 bp) of the chloroplast ribulose-1,5bisphosphate carboxylase/oxygenase large subunit gene (rbcL) was amplified using the primers listed in Table S3. The following three cocktails were used to obtain polymerase chain reaction (PCR) product: AmpliTaq Gold (Applied Biosystems, Carlsbad, California, USA), with 11 lL dH2O, 1.5 lL of each primer, 15 lL AmpliTaq Gold master mix and 1 lL template DNA; Ex Taqt DNA polymerase (Clontech Laboratories, Inc., Mountain View, California, USA), with 36.75 lL dH2O, 1.5 lL of each primer, 4 lL dNTP, 5 lL 103 buffer, 0.25 lL Ex Taq and 1 lL template DNA; or Illustrae PuReTaq Ready to Go PCR Beads (GE Healthcare, Piscataway, New Jersey, USA), with 19 lL dH2O, 2 lL of each primer and 2 lL template DNA. Amplification was conducted in a 2720 Thermocycler (Applied Biosystems) using the following cycle: 958C for 1 min, 35 cycles of 938C for 30 s, 508C for 30 s, 688C for 1 min, a final hold for 10 min at 728C. All products were visualized using gel electrophoresis. For Caloglossa specimens, multiple products were observed for a single reaction, and the band with the appropriate size was excised from the gel and cleaned using the GelElute Extraction Kit (5Prime, Gaithersburg, Maryland, USA) following the kit protocol, with one modification: the drying step was increased to 25–30 min. All other PCR products were purified using the UltraCleane PCR Clean-up DNA Purification Kit (Mo Bio Laboratories, Carlsbad, California, USA). The following modifications were made to the kit protocol: the second centrifuge cycle was increased to 60 s and, for reactions that produced a faint PCR band, 30 lL instead of 50 lL extraction buffer was added in the final step. To further confirm the identification of the Caloglossa specimens, a portion of the 26S rDNA gene (LSU) was PCR amplified Johnston et al.: Freshwater red algae in Indonesia and Malaysia using the primers T05 (5 0 -GCAACGGGCAAAGGGAATCC-3 0 ) and T15 (5 0 -TGATAGGAAGAGCCGACATCGA-3 0 ) from Harper & Saunders (2001) and AmpliTaq Gold cocktail and PCR and purification protocols previously described. The purified PCR products were submitted to the Ohio University Genomics Facility for sequencing using an ABI 3100 Genetic Analyzer (Applied Biosystems), using both the PCR primers and internal primers (Table S3) to obtain the sequence data for both the sense and antisense strands. Contigs were assembled and edited using either Geneious Pro version 5.5.6 (Biomatters, Ltd., New Zealand, Drummond et al. 2011) or Sequenchere 4.10.1 (Gene Codes Corp., Ann Arbor, Michigan, USA). All sequence data were compared with previously published sequences using a BLASTn search in GenBank (Benson et al. 2011). For the Batrachospermales and Thoreales specimens, rbcL sequence data were combined with previously published sequences of closely related taxa within those orders available through GenBank. The Balbianiales [Balbiania investiens (Lenormand ex Kützing) Sirodot KF944666 and Rhododraparnaldia oregonica Sheath, Whittick & K.M. Cole AF029156] was used as an outgroup for phylogenetic analyses. Models of sequence evolution were examined using jMODELTEST (Guindon & Gascuel 2003; Posada 2008). Phylogenetic inferences were made using Bayesian inference (BI) with MrBayes version 3.2 (Ronquist et al. 2012) and maximum likelihood (ML) analysis using the RAxML 7.2.6 (Stamatakis 2006; Stamatakis et al. 2008). Both BI and ML analyses were conducted using a general time reversible model with a gamma distribution, as suggested by the jMODELTEST results. The BI analysis was run for 5,000,000 generations, until the standard deviation of the split frequencies was less than 0.01 and the first 10% of sampled trees were discarded as burnin. The ML analysis was conducted using the rapid bootstrapping and best tree search algorithm (-f a) for 1000 repetitions. RESULTS Eight samples were collected from Indonesia and 26 samples were collected from Malaysia. Two Malaysian samples (MR-24 and MR-26) contained two morphologically distinct entities when viewed in the stereomicroscope, bringing the total for this country to 28 red macroalgae (Table S2). Eleven species were readily identified, with eight taxa belonging to the Batrachospermales and one each in the Compsopogonales, Ceramiales and Thoreales (Figs 1– 26). One specimen from Malaysia (MR-15) was identified as Batrachospermum section Turfosa but no existing species epithet could be assigned (Figs 27–31). Likewise, two specimens (IR-27 and IR-28) had features that placed them in Kumanoa but could not be ascribed to an existing species using the key in Necchi & Vis (2012) (Figs 32–38). From the Batrachospermales, there were three Batrachospermum species, B. beraense, B. cylindrocellulare, and B. macrosporum; two Kumanoa species, K. gibberosa and K. tortuosa; Sirodotia delicatula; Balliopsis prieurii 331 (Kützing) G.W. Saunders & Necchi; and two distinct chantransia stage sporophytes (analogous to the tetrasporophyte phase of marine red algae). Batrachospermum beraense Kumano was collected from the type locality, Tasek Bera (Lake Bera), in Malaysia and was a member of section Aristata having long carpogonial branches and short carpogonia compared with other species of the section (Figs 1–3). Batrachospermum cylindrocellulare Kumano was also collected from its type locality, Tasek Bera, in Malaysia. This taxon had spherical carposporophytes in the outer part of the whorl and carpogonia on undifferentiated branches but differed from other species by having cylindrical fascicle cells (Figs 4–6). Specimens of B. macrosporum were intermixed with B. beraense and had a similar whorl appearance but differed in the shape of the carpogonium and the much larger carposporangia (Figs 7– 9). Kumanoa gibberosa (Kumano) Necchi & M.L. Vis was distinct with highly reduced whorls, large carposporophytes and carpogonia on a compacted, coiled branch (Figs 10, 11). This species, originally described from Malaysia, was reported for the first time in Indonesia (Tables S1, S2). Diminutive thalli of K. tortuosa (Kumano) E.T. Johnston, P.-E. Lim & M.L. Vis comb. nov., were collected intermingled with the chantransia stage sporophytes of B. macrosporum growing on vascular plants in their type locality, Tasek Bera, Malaysia. These K. tortuosa specimens had smaller whorl size than the protologue but the carposporophyte and carpogonial characteristics were similar (Figs 12, 13). Sirodotia delicatula, originally described from Indonesia, was collected in Malaysia and showed the characteristic whorl morphology, gonimoblast filaments arising from the protuberant side of the carpogonium and creeping, indeterminate carposporophytes (Figs 14–16). The small crimson thalli of Balliopsis prieurii were collected at Ampang Park in the city of Kuala Lumpur at both the inflow and outflow of a reservoir. This taxon was distinguished from the only other species described in the genus, Balliopsis pinnulata (Kumano) G.W. Saunders & Necchi, by having branched branchlets and relatively large monosporangia (Figs 17, 18). Two distinct chantransia stage sporophytes, distinguished by the size of the vegetative cells and monosporangia, were collected. The chantransia stage of B. macrosporum, having larger cells and monosporangia, was collected from Malaysia (Fig. 19). The sporophyte specimens from Indonesia with smaller vegetative cells and monosporangia were not assigned to a taxonomic entity because sporophytes with this morphology have been linked to numerous species in the Batrachospermales and Thoreales using molecular data (Fig. 20). A single member of each order, Compsopogonales, Ceramiales and Thoreales, was identified. The monospecific Compsopogon caeruleus (Compsopogonales) was collected from both Malaysia and Indonesia (Table S2). This taxon had a typical smooth thallus appearance in most locations but in one location (MR-16) the thalli had spines that had been attributed to Compsopogon aeruginosus, now a synonym of C. caeruleus (Figs 21, 22). Caloglossa beccarii (Ceramiales) was found in four locations with low conductivity ( 50 lS/cm2) (Fig. 23; Table S2). Nemalionopsis 332 Phycologia, Vol. 53 (4) Figs 1–13. Morphological features of Batrachospermum beraense, B. cylindrocellulare, B. macrosporum, Kumanoa gibberosa and K. tortuosa. Figs 1–3. Batrachospermum beraense MR-24 BHO voucher. Fig. 1. Distinct barrel-shaped whorls with a peripheral small spherical carposporophyte (arrowhead). Scale bar ¼ 300 lm. Fig. 2. Carpogonium with a long stalked trichogyne (arrowhead) at the apex of a long carpogonial branch (double arrowhead). Scale bar ¼ 10 lm. Fig. 3. Compact carposporophyte with obovoid carposporangia (arrowheads) at the tips of the gonimoblast filaments. Scale bar ¼ 20 lm. Figs 4–6. Batrachospermum cylindrocellulare MR-25 BHO voucher. Johnston et al.: Freshwater red algae in Indonesia and Malaysia shawii Skuja (Thoreales) was recorded from one location in Indonesia (Table S2). Morphological characteristics observed included a thick medulla with compact assimilatory filaments, carpogonia with thin long trichogynes and putative spermatangia (Figs 24–26). Within the 28 samples from Malaysia, nine distinct taxa were identified (Table S2). Batrachospermum macrosporum was the most abundant, with the chantransia stage sporophyte collected in 12 locations and the gametophyte in one location for a total of 13 observations. Caloglossa beccarii was reported from four locations, Compsopogon caeruleus from three and Balliopsis prieurii from two. All other taxa were collected in one location: Sirodotia delicatula, Batrachospermum beraense, Batrachospermum cylindrocellulare, Batrachospermum phangii sp. nov. and Kumanoa tortuosa comb. nov. Only eight samples were collected in Indonesia but five distinct taxa were identified. Compsopogon caeruleus, Kumanoa celebes sp. nov. and chantransia stage sporophyte were each collected in two locations, while Kumanoa gibberosa and Nemalionopsis shawii were collected from single locales (Table S2). Kumanoa celebes sp. nov. and N. shawii were from the island of Sulawesi, and the other specimens were from Java. Sequence data of the rbcL gene were obtained for as many specimens as possible (Table S2). For Batrachospermum macrosporum chantransia stage sporophytes, the rbcL sequences for six specimens were identical. The sequence data for the two sporophytes from Indonesia were identical to each other and differed from those of B. macrosporum. Likewise, the two sequences from Caloglossa beccarii specimens were identical to each other. Among the three Compsopogon caeruleus specimens, there was only 1 bp difference between MR-16 and the other two. Single sequences were obtained for Batrachospermum cylindrocellulare, Batrachospermum phangii sp. nov., Kumanoa gibberosa, Kumanoa celebes sp. nov., Sirodotia delicatula, Balliopsis prieurii and Nemalionopsis shawii. Unfortunately, after multiple attempts, no molecular data could be acquired for Batrachospermum beraense and Kumanoa tortuosa. BLAST searches of GenBank showed some of the newly produced sequences to be identical or near identical to previously published data. The sequences from the Batra- 333 chospermum macrosporum chantransia stage sporophytes were identical to the B. macrosporum sequences for specimens from Bolivia and Brazil (AY423417 and AY423416, respectively) and 1 bp different from French Guiana specimens (AY423413 and AY423414). The sequence of the chantransia stage sporophytes from Indonesia was 2 bp different from Sheathia arcuata (Kylin) Salomaki & M.L. Vis (as Batrachospermum arcuatum) EF116873 from Hawaii. The Nemalionopsis shawii specimen differed by 3 bp from a previously published sequence DQ296122 identified as Nemalionopsis tortuosa Yoneda & Yagi. As well, the Compsopogon caeruleus sequence for two of the specimens was identical to C. caeruleus JX028157 and MR-16 was only 1 bp different. All other sequences were at least 2% different from other sequences in GenBank. There were no rbcL sequence data available for Caloglossa beccarii. Therefore, a 748 bp portion of the LSU gene was sequenced and compared with the closely related taxa C. beccarii (AF522207) and Caloglossa ogasawaraensis Okamura (AB669608). Our specimens (MR-07 and MR-09) differed by 1 bp and were 99.5% similar (4–5 bp) to C. beccarii and only 98.8% similar (9–10 bp) to C. ogasawaraensis. A phylogeny was produced from ML and BI analyses because many of the rbcL sequences of batrachospermalean taxa and Nemalionopsis (Thoreales) differed from previously published data. The tree topologies were similar, and only the ML tree was shown with bootstrap values and BI posterior probabilities (Fig. 39). The genus Batrachospermum was polyphyletic, with sections being variously related to other genera in the Batrachospermales. Batrachospermum cylindrocellulare was in a well-supported clade with Batrachospermum cayennense Montagne ex Kützing specimens from section Aristata (Fig. 39). Batrachospermum phangii sp. nov. was in a well-supported clade with specimens of Batrachospermum turfosum Bory from section Turfosa. The Sirodotia delicatula specimen was well supported as part of the Sirodotia clade but not monophyletic with a previously published sequence of S. delicatula from South America. Balliopsis prieurii was on a long branch, sister to B. prieurii from South America but with low support in either analysis. Kumanoa gibberosa was placed within the clade of species from this genus but was not closely related to other taxa. Likewise, Kumanoa celebes sp. nov. was within the Kumanoa Fig. 4. Distinct whorls with one or two peripheral small spherical carposporophytes (arrowheads). Scale bar ¼ 100 lm. Fig. 5. Separate whorls of primary fascicles with sparse branching composed of cylindrical cells. Scale bar ¼ 100 lm. Fig. 6. Carpogonium with a clavata trichogyne (arrowhead) and attached spermatium (double arrowhead) at the tip of a carpogonial branch obscured by involucral filaments (arrow). Scale bar ¼ 10 lm. Figs 7–9. Batrachospermum macrosporum MR-24 BHO voucher. Fig. 7. Large distinct barrel-shaped whorls with small spherical carposporophytes (arrowheads). Scale bar ¼ 300 lm. Fig. 8. Carpogonium with obovoidal trichogyne (arrow). Scale bar ¼ 20 lm. Fig. 9. Compact carposporophyte with large obovoid carposporangia (arrowheads) at the tips of gonimoblast filaments. Scale bar ¼ 20 lm. Figs 10, 11. Kumanoa gibberosa IR-30 BHO voucher. Fig. 10. Elongate, obconic and reduced whorls with large dense carposporophyte (arrowhead) that is higher than the whorl. Scale bar ¼ 100 lm. Fig. 11. Carpogonium with lanceolate bent trichogyne (arrowhead) at the tip of a highly compacted coiled carpogonial branch (double arrowhead). Scale bar ¼ 10 lm. Figs 12, 13. Kumanoa tortuosa comb. nov. MR-26 BHO voucher. Fig. 12. Indistinct whorls with large axial carposporophyte (arrowhead). Scale bar ¼ 50 lm. Fig. 13. Carpogonium with club-shaped trichogyne (arrowhead) on a coiled carpogonial branch forming a rosette (double arrowhead). Scale bar ¼ 10 lm. 334 Phycologia, Vol. 53 (4) Figs 14–26. Morphological features of Sirodotia delicatula, Balliopsis prieurii, Batrachospermum macrosporum chantransia stage sporophyte and chantransia stage sporophyte (identified as Sheathia arcuata by rbcL sequence data), Compsopogon caeruleus, Caloglossa beccarii and Nemalionopsis shawii. Figs. 14–16. Sirodotia delicatula MR-08 BHO voucher. Fig. 14. Well-developed obconic whorls that are confluent due to secondary filament development. Scale bar ¼ 150 lm. Fig. 15. Carpogonium with a stalked cylindrical trichogyne (arrowhead), attached spermatium (double arrowhead) and gonimoblast filaments initiating from the protuberant side of the carpogonial base (arrow). Scale bar ¼ 10 lm. Fig. 16. Carposporangia (arrowheads) terminal on lateral branches of a prostrate gonimoblast (double arrowhead). Scale bar ¼ 10 lm. Johnston et al.: Freshwater red algae in Indonesia and Malaysia clade and within a well-supported clade with numerous other species, some of which are endemic to Australasia and others that are more cosmopolitan (Fig. 39). The chantransia stage sporophytes from Malaysia that were identified morphologically as Batrachospermum macrosporum were confirmed as this species being within a clade of sequences from this taxon. The chantransia stage sporophyte from Indonesia that could not be placed with a taxon name based on morphology was within a clade of Sheathia arcuata sequences. Finally, Nemalionopsis shawii was sister to Nemalionopsis tortuosa from Taiwan and not closely related to previously published sequences of N. tortuosa (syn. N. shawii forma caroliniana) from North American and Hawaii (Fig. 39). Taxonomic proposals Batrachospermum phangii E.T. Johnston, P.-E. Lim & M.L. Vis sp. nov. Figs 27–31 DESCRIPTION: Whorls well developed, confluent, dense and compressed, 650–750 lm in diameter. Loose cortication around the main axis. Primary fascicles straight, 8–10 cell storeys, cells cylindrical, elliptical with proximal cells often teardrop shaped and 7–13 lm in diameter and 27–63 lm long, L/D ¼ 4.2; branching dichotomous near the tips but irregular on the lower parts of the thallus. Secondary fascicles abundant covering the whole internode and as long as primary fascicles, 8–10 cell storeys. Monoecious. Spermatangia spherical to subspherical, abundant, terminal or subterminal on primary and secondary fascicles, 5.5–6 lm in diameter. Carpogonial branches short, three to seven cells, arising from the pericentral cell; involucral filaments clustered near the base of the carpogonium; carpogonia 34–36 lm, trichogynes club shaped to somewhat cylindrical, sometimes with a basal knob, indistinctly stalked 5–7 lm in diameter. Carposporophytes one per whorl, loose, forming a V shape and can be as high as the whorl, 225–357 lm in diameter and 250–285 lm high; gonimoblast filaments 5–6 cell storeys, cells elliptical; carposporangia ovoid, 11–15 lm long, 9–10 lm in diameter. HOLOTYPE: Deposited at MICH (coll. 1 September 2011); a single specimen pressed and mounted on a herbarium sheet. ISOTYPES: BHO A-0979, KLU 12149, KEP 216686. 335 TYPE LOCALITY: 03839 0 18.48 00 N, 101819 0 11.04 00 E, drainage ditch outside peat swamp alongside road, North Selangor, Malaysia. ETYMOLOGY: This species is named in honour of Dr Siew-Moi Phang for her pioneering work and significant contributions to algal research in Malaysia. Kumanoa celebes E.T. Johnston, N. Buhari & M.L. Vis sp. nov. Figs 32–38 DESCRIPTION: Plants moderately mucilaginous, delicate; branching irregular, frequent near base but less frequent in upper part of thallus, 5.5–8.5 cm high, 425–575 lm in diameter. Whorls well developed, obconic and confluent. Internode 370–400 lm. Pericentral cells ovoid with three to four fascicles. Primary fascicles straight, 9 to 13 cell storeys, proximal cells cylindrical or elliptical, 6.8 lm in diameter, 29– 37.5 lm long, L/D 4–6; distal cells obovoid to pear shaped, 7–8.7 lm in diameter, 11.5–12.8 lm long, L/D 1.5–2; branching di- or trichotomous. Secondary fascicles abundant covering the whole internode and as long as primary fascicles. Monoecious. Spermatangia spherical to subspherical, abundant, terminal or subterminal on primary and secondary fascicles, 5.5–7 lm in diameter. Carpogonial branches curved, arising from the pericentral cell; involucral filaments forming a mass of cells near the base of the carpogonium; carpogonia 40–51 lm, trichogynes club shaped, indistinctly stalked 7.5–11.0 lm in diameter. Carposporophytes one per whorl as high as whorl radius, dense and hemispherical, 315–420 lm in diameter and 240–390 lm high; gonimoblast filaments 4–5 cell storeys, cells cylindrical to elliptical; carposporangia obovoid, 15–20 lm long, 11–12 lm in diameter. HOLOTYPE: Deposited at MICH (coll. 09 August 2011); a single specimen pressed and mounted on a herbarium sheet. ISOTYPES: BHO specimen number A-1000, a specimen deposited at the Research and Development of Marine, Coast and Small Islands Hasanuddin University, Indonesia. TYPE LOCALITY: Sungai Tomehipi near Kolori Village, Bomba Valley, Central Sulawesi, Indonesia, near 1828 0 29.72 00 S, 120811 0 20.55 00 E (estimated from Google Maps 2013). ETYMOLOGY: This species is named in honour of the island Sulawesi, formally known as Celebes, and in honour of the people of this region for their amazing generosity, kindness and assistance with our fieldwork. Figs 17, 18. Balliopsis prieurii. Fig. 17. Balliopsis prieurii MR-14 BHO voucher showing the thallus habit and prominent monosporangia (arrowheads). Scale bar ¼ 150 lm. Fig. 18. Balliopsis prieurii MR-06 BHO voucher showing a thallus apex with dichotomously branched laterals (arrowheads). Scale bar ¼ 50 lm. Fig. 19. Batrachospermum macrosporum chantransia stage sporophyte MR02 BHO voucher with terminal monosporangia (arrowheads). Scale bar ¼ 30 lm. Fig. 20. Chantransia stage sporophyte identified as Heterocorticum arcuatum from molecular data IR-31 BHO voucher with terminal monosporangia (arrowheads). Scale bar ¼ 20 lm. Figs 21, 22. Compsopogon caeruleus. Fig. 21. Compsopogon caeruleus IR-33 BHO voucher showing typical thallus habit. Scale bar ¼ 100 lm. Fig. 22. Compsopogon caeruleus MR-16 BHO voucher showing thallus with spine-like branches. Scale bar ¼ 100 lm. Fig. 23. Caloglossa beccarii MR03 BHO voucher showing habit of thallus. Scale bar ¼ 300 lm. Figs 24–26. Nemalionopsis shawii IR-29 BHO voucher. Fig. 24. Thallus showing thick medulla and compact assimilatory filaments. Scale bar ¼ 200 lm. Fig. 25. Carpogonium with somewhat swollen base (arrowhead) and long thin trichogyne (double arrowhead). Scale bar ¼ 20 lm. Fig. 26. Spermatangia (arrowheads) at the tips of assimilatory filaments. Scale bar ¼ 10 lm. 336 Phycologia, Vol. 53 (4) OTHER LOCATIONS: Sungai Tomehipi near Kolori Village, downstream from the population represented by IR-27, Bomba Valley, Central Sulawesi, Indonesia, near 18 28 0 29.72 0 0 S, 1208 11 0 20.55 0 0 E (estimated from Google Maps 2013). Kumanoa tortuosa (Kumano) E.T. Johnston, P.-E. Lim & M.L. Vis comb. nov. BASIONYM: Batrachospermum tortuosum S. Kumano. Botanical Magazine of Tokyo, 91, p. 101 (1978). This taxon was not transferred previously to the genus Kumanoa because of lack of characters observed (Necchi & Vis 2012). After careful morphological examination, we concluded it was a morphologically distinct taxon, and it had a twisted carpogonial branch characteristic of Kumanoa. DISCUSSION Much of the freshwater red algal diversity was in the order Batrachospermales. The phylogeny based on the rbcL data showed the genus Batrachospermum to be paraphyletic, and this result is congruent with all previous molecular systematic studies of this order (Vis et al. 1998; Entwisle et al. 2009). Researchers have recognized this paraphyly, and to date, the genera Kumanoa and Sheathia have been described. Work to describe new genera from detailed studies of Batrachospermum sections is ongoing and outside the scope of this study (Entwisle et al. 2009; Salomaki et al. 2014). In the present study, the new sequences for Batrachospermum species have been placed in sections of Batrachospermum so that they can be included in future revisions. To that end, B. cylindrocellulare when first described was placed in section Batrachospermum but the author noted that the early stages of development of the carpogonial branch allied it to B. cayennense in section Aristata, and he considered it to be an intermediate between these sections (Kumano 1993). The molecular data generated in this study of B. cylindrocellulare from the type locality clearly refute its placement in section Batrachospermum and confirm its membership in section Aristata closely allied to B. cayennense. Therefore, when a new genus is described for that clade, B. cylindrocellulare should be transferred. Batrachospermum phangii, described here from peninsular Malaysia, represents previously undescribed diversity in Batrachospermum section Turfosa, from which there were previously only five species recognized: B. keratophytum Bory emend. Sheath, Vis & Cole, B. orthostichum Skuja, B. periplocum (Skuja) Necchi, B. tapirense Kumano & Phang and B. turfosum Bory emend. Sheath, Vis & Cole (Kumano 2002). Batrachospermum tapirense has only been reported from the type location in Malaysia. The distinguishing character for this species, the carpogonial branch descending from the periaxial cell, was not observed in B. phangii; as well, B. tapirense has reduced whorls with fascicle cells 4–5 cell storeys, and B. phangii has well-developed whorls with primary fascicles composed of 8–10 cell storeys. Similarly, B. orthostichum and B. keratophytum have reduced whorls [(2–) 3–6(7) cell storeys and (3–)4–7(8) cell storeys, respectively] Figs 27–31. Batrachospermum phangii sp. nov. MR-15 BHO voucher. Fig. 27. Indistinct confluent whorls with axial triangular carposporophytes (arrowheads). Scale bar ¼ 300 lm. Fig. 28. Three carpogonia (arrowheads) arising from the same whorl. Scale bar ¼ 10 lm. Fig. 29. Carpogonium with a club-shaped trichogyne (arrowhead) on a carpogonial branch with many involucral filaments forming a rosette at the base of the carpogonium. Scale bar ¼ 10 lm. Fig. 30. Spermatangia (arrowheads) in clusters at the tips of fascicles. Scale bar ¼ 10 lm. Fig. 31. Loose carposporophyte with carposporangia (arrowheads) at the tips of many-celled gonimoblast filaments. Scale bar ¼ 20 lm. unlike the presently described species. Batrachospermum periplocum has well-developed whorls like B. phangii but has monosporangia, which was not seen in the new species. Batrachospermum turfosum has well-developed whorls but differs from B. phangii in that B. turfosum has abortive carposporophytes and B. phangii has fully developed carposporophytes that produce carposporangia. In addition, the proximal fascicle cells in B. turfosum are reported to be ellipsoidal, and in B. phangii they are more often teardrop shaped. Therefore, B. phangii is morphologically distinct from all other species of section Turfosa, and its rbcL Johnston et al.: Freshwater red algae in Indonesia and Malaysia 337 Figs 32–38. Kumanoa celebes sp. nov. IR-27 BHO voucher. Fig. 32. Confluent barrel- to obconic-shaped whorls with loose carposporophytes (arrowheads) that are as high as the whorl. Scale bar ¼ 400 lm. Fig. 33. Immature carpogonium (arrowhead) on a twisted carpogonial branch (double arrowheads). Scale bar ¼ 10 lm. Fig. 34. Carpogonium with cylindrical trichogyne (arrowhead). Scale bar ¼ 20 lm. Fig. 35. Carpogonium with asymmetrical club-shaped trichogyne (arrowhead), attached spermatia (double arrowheads) and putative gonimoblast filament (arrow) arising from the carpogonium base. Scale bar ¼ 20 lm. Fig. 36. Carpogonium with symmetrical club-shaped trichogyne (arrowhead) and attached spermatia (double arrowhead). Scale bar ¼ 20 lm. Fig. 37. Spermatangia (arrowheads) clustered at the tips of fascicle branches. Scale bar ¼ 20 lm. Fig. 38. Carposporangia (arrowheads) at the tips of gonimoblast filaments. Scale bar ¼ 20 lm. sequence is distinct from specimens of B. turfosum. The type localities of both B. phangii and B. tapirense are in Malaysia, and B. turfosum (as B. vagum) was reported from this country, such that half of the known species diversity (three of six species) for the section has been reported for Malaysia (Kumano 1978). The new species, Kumanoa celebes, was placed in a clade with K. globospora (Israelson) Entwisle, M.L. Vis, W.B. Chiasson, Necchi & A.R. Sherwood; K. novaecaledonensis M.L. Vis, Necchi, W.B. Chiasson & Entwisle; K. virgatodecaisneana (Sirodot) Entwisle, M.L. Vis, W.B. Chiasson, Necchi & A.R. Sherwood; K. mahlacensis (Kumano & W.A. Bowden-Kerby) M.L. Vis, Necchi, W.B. Chiasson & Entwisle and K. australica (Kützing ex Entwisle & Foard) Entwisle, M.L. Vis, W.B. Chiasson, Necchi & A.R. Sherwood based on the molecular data. Kumanoa celebes differed from each of these taxa in at least one key morphological feature as follows: K. globospora in carpogonium length [40– 51 lm vs 20–34(40) lm, respectively], K. novaecaledonensis in carposporophyte arrangement (dense vs loose, respectively) and carpogonium length (40–51 lm vs 20–25 lm, respectively), K. virgatodecaisneana in carpogonium length (40–51 lm vs 19–35 lm, respectively), K. mahlacensis in whorl morphology [well-developed, primary fascicles 9–13 cell storeys vs reduced, 4–7(9) cell storeys, respectively], K. australica in fascicle cell shape (distal fascicle cells obovoid to pear shaped L/D 1.5–2 vs distal fascicle cells elliptical to fusiform L/D 4–7, respectively) (Necchi & Vis 2012). In 338 Phycologia, Vol. 53 (4) Johnston et al.: Freshwater red algae in Indonesia and Malaysia morphology this taxon was closest to K. abilii (Reis) Necchi & M.L. Vis from the key in Necchi & Vis (2012) but molecular data showed these two taxa to be distantly related within the genus. The morphological similarities are probably due to convergent evolution. In terms of biogeography, the specimens in the clade with K. celebes were collected from three distant geographic regions, North America (K. mahlacensis, K. virgatodecaisneana), Australasia (K. australica, K. novaecaledonensis) and South America (K. globospora), such that no affinity with the flora of a particular region can be discerned. Interestingly, the only other Kumanoa specimen, K. gibberosa, sequenced from Southeast Asia was far removed from this clade. Kumanoa tortuosa comb. nov. (as Batrachospermum tortuosum) was listed as a doubtful species in Necchi & Vis (2012) because key features were missing in the description and type material as well as a potential synonymy with K. montagnei Entwisle, M.L. Vis, W.B. Chiasson, Necchi & A.R. Sherwood. We collected specimens morphologically identical to the protologue (Kumano 1978) from the type locality (Fort Iskander, Tasek Bera, Pahang, Malaysia). From our new observations, K. tortuosa differs from K. montagnei by having dense, compact carposporophytes, smaller carposporangia (8–6 lm diameter, 8.5–10.5 lm length) and smaller cylindrical carpogonia (5–6 lm diameter, typically ~ 33 lm length but can be up to 40 lm length). Unfortunately, we could not sequence the rbcL gene from the K. tortuosa material but based upon new morphological observations, we have transferred it to the genus Kumanoa. It is still known only from the type locality. Several names have been previously ascribed to Sirodotia species from Southeast Asia. Two species of Sirodotia have been reported from Malaysia and Indonesia: S. ateleia and S. delicatula, respectively (Skuja 1938). More recent studies have questioned the validity of S. ateleia: Umezaki (1960) and Necchi (1991) placed it in synonymy with S. delicatula. However, Necchi et al. (1993), after exhaustive examination of the type specimens of Sirodotia, overturned the synonymy of S. ateleia with S. delicatula and synonymized it with the older name S. huillensis based on whorl morphology. The type specimens of S. huillensis and S. ateleia were found to have truncate-pyramidal whorls; whereas, the type specimen of S. delicatula had barrel-shaped or obconic whorls. We distinguished our specimen (MR-08) as S. delicatula and not S. huillensis based on whorl morphology. Therefore, based on the literature, it appears there are currently two distinct species, S. delicatula and S. huillensis (as S. ateleia), in Southeast Asia. We believe that there is still a question concerning the number of species (1, 2 or 3) because S. ateleia has been variously synonymized based on morphology but there may be cryptic species, and there is only sequence data for a single specimen resembling S. delicatula from this region. Future research with more sequence data 339 from specimens in the region is needed to provide the answer as to the number of species. Although our molecular data for Sirodotia did not clarify the number of species for the genus in Southeast Asia, the results did show that the specimen from Malaysia (MR-08) and specimens from South America (DQ646475) attributable to S. delicatula based on morphology were not monophyletic. The type location at Bogor Botanical Gardens, Bogor, Indonesia, was visited but S. delicatula was not found. The Malaysian specimen is geographically closer to the Indonesian type locality than the South American specimen, and therefore we suggest that the Malaysian specimen represents S. delicatula, and the South American specimen represents a cryptic species. Balliopsis prieurii was previously reported from Malaysia (Kumano & Phang 1990), and the specimens from the current study were similar in morphology. Both accounts are similar to reports from South America (Saunders & Necchi 2002); however, the rbcL sequences from our study diverge considerably from the South American specimens (Vis et al. 2007). It is possible that Balliopsis is the chantransia stage of a batrachospermalean taxon of which the gametophyte has not yet been sequenced, analogous to the findings for freshwater Ptilothamnion (Vis et al. 2006). These researchers showed with sequence data that the Ptilothamnion morphology was a chantransia stage of Batrachospermum antipodites Entwisle & Foard and an unknown Kumanoa species. Therefore, no new species epithet is proposed, even though there was sequence divergence, since this may serve to only perpetuate superfluous names in the literature. Nemalionopsis shawii (Thoreales) was originally described from the Philippines (Skuja 1934), and we report it here for the first time from Indonesia. Molecular analyses showed that the Indonesian specimen was sister to a specimen identified as N. tortuosa from Taiwan (DQ296122) and only distantly related to a North American and a Hawaiian specimen each identified as N. tortuosa (syn. N. shawii forma caroliniana, AB159658 and EF116879, respectively). Bourrelly (1970) and Howard & Parker (1979) discussed the possibility that N. tortuosa and N. shawii were synonymous taxa. However, very few sequence data are available, no holotype was designated for N. tortuosa and the taxonomy of this genus is in need of much work. For now, we choose to leave this specimen with the name N. shawii since its morphology is similar to the protologue and overlaps with all characters provided by previous, morphological examination of isotype material of N. shawii (Sheath et al. 1993). The sequenced data for two chantransia stage sporophyte specimens from Java, Indonesia, confirm them to be the sporophyte of Sheathia arcuata; these specimens were only 2 bp different from the sequence for specimens from the Hawaiian Islands (Chiasson et al. 2007; Salomaki et al. 2014). Interestingly, this taxon was collected on Kauai, Oahu, Molokai, and Hawaii but only sporophytes, no Fig. 39. Maximum likelihood (ML) tree (ln likelihood ¼ 20880.803788) based in rbcL sequence data showing the relationships of Batrachospermales and Thoreales species. Classification in the Batrachospermales follows Entwisle et al. (2009) and Salomaki et al. (2014); B. ¼ Batrachospermum. The outgroup taxa (Balbiania investiens and Rhododraparnaldia oregonica) were pruned for presentation. An asterisk denotes . 80% ML bootstrap support and . 0.90 posterior probabilities from BI analysis. Specimens from this study in bold (see Table S2). 340 Phycologia, Vol. 53 (4) gametophytes, were observed despite substantial sampling effort (Carlile & Sherwood 2013). Similar sequences are also known from Taiwan, where gametophytes were collected and sequenced (Chou & Wang 2006; Vis et al. 2010). It should be noted that the chantransia stage sporophyte represents the sporophyte of a number of batrachospermalean taxa such that sequence data are needed to confirm the species identity (Chiasson et al. 2005). Compsopogon caeruleus (Compsopogonales) was previously reported from Malaysia and Indonesia (Skuja 1938; Ratnasabapathy & Kumano 1982), and we collected it from both countries. Sequence data from three of the five collections support previous findings that this taxon is genetically similar across its broad geographic range (Carlile & Sherwood 2013; Necchi et al. 2013). In addition, our samples supported the conclusion that this taxon is morphologically plastic; both the typical and the spiny morphology were observed, and the rbcL sequences were similar. Although many taxa appear to be endemic to the region, the cosmopolitan species Compsopogon caeruleus was collected, and Batrachospermum macrosporum was shown to have a wider geographic distribution than previously thought. To our knowledge, neither the gametophyte nor the sporophyte stage of B. macrosporum has been previously reported from Malaysia. Yet, in this survey, 12 collections of the sporophyte and one collection of gametophyte were made. Six of the sporophyte specimens were sequenced and were unequivocally identified as B. macrosporum because the rbcL sequences were identical to specimens from Brazil and Bolivia, and only 1 bp different to specimens from French Guiana, the type locality for this species. Batrachospermum hypogynum was first described from Malaysia and is morphologically similar to B. macrosporum; the two species overlap with regard to carpogonia size and shape, and both have large carposporangia but differ in the maximum number of cells in the carpogonial branch (Kumano 2002). Sheath et al. (1994) recorded similar carpogonial branch cell numbers but distinguished these two taxa based on abundant or sparse secondary fascicles; however, whorl morphology can be quite variable, even on the same thallus, in Batrachospermum species. These two species may or may not be synonymous based on morphology, and molecular data would be useful in determining their relationship (no molecular data for B. hypogynum in GenBank). Unfortunately, specimens for DNA from the type locality for B. hypogynum could not be obtained, since we were informed the site is now private land that could not be accessed during the study. Sampling the B. hypogynum type specimens was not attempted since they were placed in fixative prior to pressing (S. Kumano, personal communication). Therefore, conspecificity of B. hypogynum with B. macrosporum (name with precedence) remains an open question. Nevertheless, insights were gained regarding B. macrosporum, and the close sequence identity between the Malaysian and South American collections point to this taxon being widespread in tropical and subtropical regions but to date it has rarely been reported outside of North and South America (Kumano 2002). Although 13 distinct taxa were collected, there were still some species that we were unable to recollect. The type localities for several species, Batrachospermum bakarense, B. crispatum, B. hirosei, B. hypogynum, B. tapirense, B. tiomanense and Thorea prowsei, could not be visited, and large areas of both countries could not be sampled. We visited the type location (Gombak River in Selangor, Malaysia) and surrounding areas for Balliopsis pinnulata, Batrachospermum gombakense, and Thorea clavata but did not find them; their occurrence may be seasonal and outside of our collecting period. Likewise, Batrachospermum lochmodes was described in the 1930s from springs on the Dieng Plateau in central Java, Indonesia. This location was visited, and the taxon was not found, possibly because the land has been heavily modified from a natural state. The type localities for Kumanoa gibberosa and Sirodotia delicatula were visited but they were not found at these localities. However, they were collected in Indonesia and Malaysia, respectively, which extended their ranges beyond the type localities. ACKNOWLEDGEMENTS We thank Dr Gene Ammarell for facilitating our collaboration, Dr Christine Fletcher for assistance and advice in the search for Kumanoa gibberosa, Dr Siripen Traichaiyaporn for her advice and insights concerning the Thoreales, Fisher Saint Amour for field and photographic assistance, Dr Yong Hoi Sen for his invaluable guidance in locating sites in Malaysia, Dr Kathy Yule for her generous hospitality and advice, Chou Lee Yiung and Ong Sue Peng for their help in the field, and Dr Dwia Aries Tina for sample shipping recommendations. This research was funded by an Ohio University Student Enhancement Award to ETJ and Research Incentive funds from Ohio University to MLV. SUPPLEMENTARY DATA Supplementary data associated with this article can be found online at http://dx.doi.org/10.2216/13-223.1.s1. 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