Diversity of freshwater red algae (Rhodophyta) in Malaysia and

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 ([email protected]).
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
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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
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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.
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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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Received 31 August 2013; accepted 5 January 2014