Porphyroglossum
is the last one of nine genera within the family Gelidiaceae that has yet to be analyzed by molecular markers. We analyzed
rbc
L and
cox
1 genes from
P. zollingeri
specimens collected near the type locality in Indonesia and compared them with other gelidioid algae. Thalli are cartilaginous, complanate, and up to 15 cm high. Abundant rhizoidal filaments are concentrated in the medullary layer. Tetrasporangial sori are on small, determinate ramuli. In all gene analyses,
P. zollingeri
consistently nested within
Gelidium.
The sister relationship of
P. zollingeri
to
G. floridanum
was well resolved. Because
Gelidium
has priority over
Porphyroglossum,
a new combination is proposed, viz.
Gelidium zollingeri
. Network analysis of the four
cox
1 haplotypes revealed many missing haplotypes, indicating high genetic diversity in the species.
INTRODUCTION
Most species of the family Gelidiaceae are familiar to molecular biologists because they produce commercially important agar for laboratory gels and bacterial growth media; they are also a food source. The Gelidiaceae is the largest family in the order and includes nine genera,viz.
Acanthopeltis
Okamura in Yatabe,
Beckerella
Kylin,
Capreolia
Guiry & Womersley,
Gelidium
Lamouroux,
Onikusa
Akatsuka,
Porphyroglossum
Kützing,
Ptilophora
Kützing,
Suhria
J. Agardh ex Endlicher, and
Yatabella
Okamura. The family is characterized by brush rhizoids and by endofibers in cortical and medullary layers (Perrone et al.2006).
Gelidium
is the largest genus within the family, comprising about 110 species widely distributed throughout tropical and temperate regions. Most members of the genus have large erect thalli with slightly protruding apical cells, tetrasporangia that are usually scattered on terminal branchlets, bilocular cystocarps with single, terminal carposporangia, and isomorphic life histories. Three large genera,
Gelidium, Pterocladiella
, and
Ptilophora,
are clearly established (e.g., Freshwater et al. 1995, Kim et al. in press), whereas smaller genera with one or few species are under taxonomic reconsideration. For example,Shimada et al. (1999) compared nuclear ribosomal cistron small subunit (SSU) and internal transcribed spacer (ITS) genes and plastid rbcL genes between
Acanthopeltis
and
Yatabella
and concluded that these entities are congeneric. Tronchin et al. (2002) reduced
Onikusa
and
Suhria
to synonymy under
Gelidium
because
rbc
L tree analysis nested both within
Gelidium; Onikusa
and
Suhria
have female reproductive and cystocarp systems very similar to those of
Gelidium.
Beckerella
has also been treated as a congener of
Ptilophora
based on analyses of the large subunit (LSU) of the nuclear cistron and
rbc
L (Tronchin et al. 2003). Kim et al. (in press) demonstrated through three-gene sequence analysis that
Acanthopeltis
is conspecific with
Gelidium.
In contrast,
Capreolia
is well supported by molecular data (Nelson et al. 2006).
Porphyroglossum
is the last genus within the family that has yet to be analyzed by molecular markers.
Porphyroglossum zollingeri
Kützing is a candidate species for mass culture in Indonesia.
Porphyroglossum zollingeri,
the only species in the genus, was described from specimens collected in Java, Indonesia (Kützing 1847). Later, Schmitz (1894) added a second species,
P. japonicum (= Suhria japonica
Harvey). However, Akatsuka (1986) moved this second species to his new genus
Onikusa,
leaving a single species in
Porphyroglossum.
Porphyroglossum
is distinguished by broad axes densely trimmed with slender lanceolate pinnules along the midline; it is attached to substrata by peg-like haptera (Kützing 1847, Kylin 1956, Hatta and Prud’homme van Reine 1991). The genus is currently recognized as an independent entity in the list of benthic marine algae in the Indian Ocean (Silva et al. 1996) and in AlgaeBase (Guiry and Guiry 2011). However, with their detailed observations of morphology, Hatta and Prud’homme van Reine (1991) cast doubt on the taxonomic validity of
Porphyroglossum.
Molecular markers have helped to correctly assign taxonomically confused taxa within the order Gelidiales. Earlier studies on gelidoid algae used only nuclear ribosomal cistron (small and large subunits and their spacer) and plastid
rbc
L genes for identification and phylogenet-
Locations of the Gelidium zollingeri sampled in Indonesia.
ic analysis (Freshwater and Rueness 1994, Freshwater et al. 1995, Millar and Freshwater 2005, Nelson et al. 2006). Recently, Kim et al. (in press) found that mitochondrial
cox
1 genes are also suitable for classifying these commercially important algae. In this study, we analyzed
rbc
L and
cox
1 from 16 specimens of
P. zollingeri
from Indonesia and reconstructed phylogenetic trees of sequence data for the two genes to determine whether
Porphyroglossum
is supported by molecular data.
MATERIALS AND METHODS
- Taxon sampling and morphology
Field collections of
Porphyroglossum
were made at three locations in Indonesia (
Table 1
,
Fig. 1
). Tissues were sectioned with a freezing microtome (FX-802A; Coper Electronics Co., Ltd., Kanagawa, Japan) and sections were stained with 1% aqueous aniline blue. Photographs were taken with an FX-35DX camera (Nikon, Tokyo, Japan) attached to a Vanox AHBT3 microscope (Olympus, Tokyo, Japan). Voucher specimens are housed at the herbarium of Chungnam National University, Daejeon, Korea (CNUK).
- DNA extraction and sequencing
Sixteen specimens were available for extraction of DNA (
Table 1
). DNA extraction, PCR amplification, and sequencing are described in Geraldino et al. (2010). Primer pairs for amplification and sequencing of each gene were as follows: for
rbc
L, F7-R753 and F645-RrbcS start (Freshwater and Rueness 1994, Lin et al. 2001, Gavio and Fredericq 2002); and for
cox
1, cox143F-cox11549R (Geraldino et al. 2006) and C622F-C880R (Yang et al. 2008).
Sixty-eight
rbc
L sequences (12 new and 56 published) from
P. zollingeri, Acanthopeltis
, and
Gelidium
including outgroups were collated using the Se-Al version 2.0 a11 software (Rambaut 1996) and aligned visually. Outgroup taxa included
Pterocladia, Pterocladiella
, and
Ptilophora.
Phylogenies were rooted with the distantly related genus
Gelidiella
Feldmann and G. Hamel (Freshwater et al. 1995, Kim et al. in press).
Materials of Gelidium zollingeri used in the study
Materials of Gelidium zollingeri used in the study
- Phylogenetic analyses
Maximum likelihood (ML) phylogenetic analysis of
rbc
L was performed using only the GTR + Γ + I model implemented in RAxML software (Stamatakis 2006). We used 100 independent tree inferences with the “number of run” option, with default optimized SPR rearrangement and 25 distinct rate categories to identify the best tree. Statistical support for each branch was obtained from 1,000 bootstrap replications using the same substitution model and RAxML program settings.
Maximum parsimony (MP) tree was constructed for each data set with PAUP* version 4.0b.10 software (Swofford 2002) using a heuristic search algorithm with the
Maximum likelihood tree of 68 rbcL sequences calculated using the GTR + Γ + I evolution model (-lnL = 10893.760045; substitution rate matrix RAC = 1.184322 RAG = 8.436870 RAT = 1.973412 RCG = 1.752931 RCT = 14.046278 RGT = 1; base frequencies πA = 0.306815 πC = 0.163813 πG = 0. 216361 πT = 0.313011; shape parameter [α] = 0.199229). Maximum likelihood and maximum parsimony bootstrap values are shown for each clade. Figures in parenthesis refer to the number of specimens with identical sequences. AU Australia; BR Brazil; CL Chile; CR Costa Rica; ES Spain; FR France; IE Ireland; IT Italy; JP Japan; KR Korea; MA Morocco; MX Mexico; NA Namibia; NZ New Zealand; NO Norway; OM Oman; PH Philippines; TH Thailand; TW Taiwan; UK United Kingdom; US United States; VE Venezuela; ZA South Africa.
Gelidium zollingeri (Kutzing) comb. nov.: network of four cox1 haplotyes (A-D). Small black circles correspond to missing haplotypes and the size of each circle is proportional to the number of individuals analyzed.
Porphyfollowing settings: 1,000 random sequence additions, tree bisection-reconnection (TBR) branch swapping, MulTrees, all characters unordered and unweighted, and branches with a maximum length of zero collapsed to yield polytomies. Bootstrap values for the resulting nodes were assessed using 1,000 bootstrapping replicates with ten random sequence additions, TBR, and MulTrees.
A statistical parsimony network of
cox
1 haplotypes was created using TCS version 1.21 software (Clement et al. 2000). Haplotype and nucleotide diversity measurements were performed using DNAsp software (Rozas and Rozas 1999).
RESULTS
- Molecular analyses
Sixty-eight sequences from 59 taxa of
Porphyroglossum, Gelidium
, and other genera within the family Gelidiaceae were aligned using a 1,266-nucleotide portion of
rbc
L. Variable sites occurred at 484 positions (38.2%), and 413 positions (32.6%) were parsimony-informative.
P. zollingeri
collections from Java Island in Indonesia formed a single monophyletic group with maximum support.
P. zollingeri
was sister to
G. floridanum
(51% for ML and 61% for MP) (
Fig. 2
). The genera
Gelidium
species and
Porphyroglossum
formed a single well supported clade (100% for ML and 95% for MP).
A 1,260-nucleotide portion of the
cox
1 gene was compared across 37 taxa of
Porphyroglossum, Gelidium,
Acanthopeltis, Gelidiella
, and
Pterocladiella.
Variable sites occurred at 454 positions (38.4%), and 417 positions (31.1%) were parsimony-informative. Sixteen samples of
P. zollingeri
were used for haplotype analyses of
cox
1. The nucleotide and haplotype diversities were 0.004 and 0.642, respectively. The statistical parsimony network revealed four haplotypes (
Fig. 3
). Haplotype A was found in eight specimens and haplotype B occurred in six specimens.
- Morphological observation
Thalli are cartilaginous, somewhat complanate when erect axes and branches are arranged in one plane, up to 15 cm high, arising from branched, recurved, stolon-like axes (
Fig. 4
A), and attached to the substratum by numerous peg-like haptera up to 0.5 mm in length. In a surface view, the cortical cells are rounded to ovate, mostly arranged in tetrads, 4-7 ㎛ in diameter. In cross section, the outer cortical cells are rounded, 4-8 ㎛ in diameter, gradually grading into larger cortical cells and then into medullary cells, 5-12 ㎛ in diameter. Rhizoidal filaments are abundant and interwoven in the longitudinal view (
Fig. 4
B). They are elongate and colorless, and concentrate around medullary cells (
Fig. 4
C). Erect axes are cylindrical at the base, progressively flattened above, ribbonlike in shape, up to 6 mm in width, ending in lanceolate, blunt, or truncate apices. The truncate ends often have ribbonlike proliferations similar in shape to the main axis but narrower. They arise in groups of two to four. The surfaces of main axes and proliferations are invested with longitudinally arranged rows of determinate, spatulate ramuli up to 1.5 mm in length (
Fig. 4
D). Tetrasporangial sori arise on small, determinate ramuli (
Fig. 4
E). Tetrasporangia are irregularly arranged in sori, rounded in surface view, and are 18-25 ㎛ in diameter (
Fig. 4
F). Mature tetrasporangia are cruciately divided.
DISCUSSION
This is the first report of gene sequences from
Porphyroglossum,
although many previous molecular analyses of other gelidioid algae have been performed (e.g., Freshwater and Rueness 1994, Freshwater et al. 1995, Shimada et al. 1999, Kim et al. in press). Through two-gene analyses and detailed morphological observations, we answered a question posed by Hatta and Prud’homme van Reine (1991) on the close relationships between
Porphyroglossum
and
Gelidium.
Phylogenetic analyses of plastid
rbc
L and mitochondrial
cox
1 sequences indicate that
Porphy-
Gelidium zollingeri (Kutzing) comb. nov.: morphology of (A) thallus with erect and creeping axes. (B) Longitudinal section cut in the center portion of the axis showing cortical cells and intermixed cylindrical rhizoidal filaments. (C) Transverse section of the central part of an axis showing cortex and medulla. (D) Ribbonlike proliferations on the surface. (E) Tetrasporangial sori on small determinate ramuli. (F) Cruciately divided mature tetrasporangia. Scale bars represent: A 2 cm; B 100 μm; C & F 40 μm; D 400 μm; E 200 μm.
roglossum
and
Gelidium
are not separate monophyletic groups. In all analyses of two individual genes and combined data,
Porphyroglossum
consistently nested within
Gelidium,
implying that
P. zollingeri
likely diverged from a common ancestral species of
Gelidium.
Our descriptions of morphology and reproduction in P. zollingeri are in agreement with those of previous studies (Weber-van Bosse 1921, Fan 1961, Akatsuka 1983, Hatta and Prud’homme van Reine 1991). One of the distinguishing features of
P. zollingeri
is the presence of abundant proliferations on broad axes (
Fig. 4
A & D). However, surface proliferations also occur in
Ptilophora
(Tronchin et al. 2003) and they cannot be considered autapomorphic for
Porphyroglossum.
Notably, rhizoidal filaments are so abundant and interwoven in the medullary layer that it makes discerning medullary cells difficult, a rare phenomenon in most species of
Gelidium.
We found no cystocarpic thalli in our collections of
P. zollingeri.
However, according to previous studies (Weber-van Bosse 1921, Fan 1961, Akatsuka 1986), cystocarps are bilocular with ostioles on both surfaces in flat or raised positions. The cystocarps are not different from those of
Gelidium
(Hatta and Prud’homme van Reine 1991). Male thalli have not been found in this species. Tetrasporangial characters of
P. zollingeri
in the present study are within the range of those in
Gelidium.
On the basis of
rbc
L and cox1 sequences data and inconsistencies in key morphological characters distinguishing
Porphyroglossum
from
Gelidium,
we conclude that their maintenance as separate genera is untenable. Accordingly, we propose that
Porphyroglossum
Kützing (1847) be merged with
Gelidium
Lamouroux (1813).
Gelidium zollingeri
is related to
G. floridanum
with moderate support (51% for ML and 61% for MP) in the
rbc
L tree, and to
G. serrulatum
without support. Determining which characters are synapomorphic for these species is difficult. However, they are well resolved in the largest clade within
Gelidium
(100% for both ML and MP) comprising
G. elegans, G. pacificum, G. linoides, G. tenuifolium, G. allanii, G. koshikianum, G. purpurascens, G. robustum, G. americanum, G. amansii, G. abbottiorum, G. pteridifolium,
and
G. proundum
. This clade (with the exception of
G. zollingeri
) has been identified in previously published molecular studies (Freshwater et al. 1995, Millar and Freshwater 2005, Nelson et al. 2006, Kim et al. in press).
Network analysis of four cox1 haplotypes of
G. zollingeri
revealed distantly related clusters, with connections of one (between haplotypes C and D) to 11 (between haplotypes A and C or D) missing haplotypes (
Fig. 3
). We believe that the high number of missing haplotypes may be an artifact of sampling and that an increased sampling effort from areas with isolated haplotypes will significantly reduce the number of steps linking the clusters. However, four haplotypes and a high number of missing haplotypes in a very limited area of sampling in Indonesia suggest that high haplotype diversity is likely. Haplotype diversity of the
cox
1 gene is also high in
Gelidiella fanii
S.-M. Lin (Wiriyadamrikul et al. 2010).
In summary, of nine genera within the family Gelidiaceae, only three (
Capreolia, Gelidium
[including
Acanth opeltis, Onikusa, Suhria, Porphyroglossum, Yatabella
], and
Ptilophora
[including
Beckerella])
remain as phylogenetically independent entities based on molecular marker(s). Phylogenetic studies using molecular markers have resulted in a broader concept of the genus
Gelidium.
The evolution of diverse morphologies, from cylindrical and compressed thalli (most species in
Gelidium
) via broad blades (
G. japonicum, G. zollingeri
) to sympodially growing axes with spirally arranged leaflike structures (species in the genus
Acanthopeltis),
may be the next focus of research on the genus
Gelidium.
- Taxonomic treatment
The formal synonym of
Porphyroglossum
follows:
Gelidium zollingeri (Kützing) K. M. Kim, G. S. Gerung and S. M. Boo comb. nov.
Basionym:
Porphyroglossum zollingeri
Kützing 1847: 775. Type: SE Java near Malang, Zollinger 2109 (L 941, 182-77).
Acknowledgements
We thank Il Ki Hwang and Jeong Kwang Park for help with the anatomical observations. This work was supported by Pegasus Int. Inc. to GSG and by the research fund of Chungnam National University in 2010 and by MarineBio21 program grants from the Ministry of Land, Transportation and Maritime Affairs, Korea to SMB.
View Fulltext
Akatsuka I
1983
The morphological relationships betweenGelidium japonicum(Harvey) Okamura andGelidium pristoides(Turner) Kützing
Nova Hedwigia
38
197 -
207
Akatsuka I
1986
Surface cell morphology and its relationship to other generic characters in non-parasitic Gelidiaceae (Rhodophyta)
Bot. Mar
29
59 -
68
DOI : 10.1515/botm.1986.29.1.59
Fan K. C
1961
Morphological studies of the Gelidiales
Univ. Calif. Publ. Bot
32
315 -
368
Freshwater D. W
,
Fredericq S
,
Hommersand M. H
1995
A molecular phylogeny of the Gelidiales (Rhodophyta) based on analysis of plastidrbcL nucleotide sequences
J. Phycol
31
616 -
632
DOI : 10.1111/j.1529-8817.1995.tb02558.x
Freshwater D. W
,
Rueness J
1994
Phylogenetic relationships of some EuropeanGelidium(Gelidiales Rhodophyta) species based onrbcL nucleotide sequence analysis
Phycologia
33
187 -
194
DOI : 10.2216/i0031-8884-33-3-187.1
Gavio B
,
Fredericq S
2002
Grateloupia turuturu(Halymeniaceae Rhodophyta) is the correct name of the non-native species in the Atlantic known asGrateloupia doryphora
Eur. J. Phycol
37
349 -
359
DOI : 10.1017/S0967026202003839
Geraldino P. J. L
,
Riosmena-Rodriguez R
,
Liao L. M
,
Boo S. M
2010
Phylogenetic relationships within the genusHypnea(Gigartinales Rhodophyta) with a description ofH. caespitosasp. nov
J. Phycol
46
336 -
345
DOI : 10.1111/j.1529-8817.2009.00804.x
Geraldino P. J. L
,
Yang E. C
,
Boo S. M
2006
Morphology and molecular phylogeny ofHypnea flexicaulis(Gigartinales Rhodophyta) from Korea
Algae
21
417 -
423
DOI : 10.4490/ALGAE.2006.21.4.417
Guiry M. D
,
Guiry G. M
2011
AlgaeBase
World-wide electronic publication National University of Ireland Galway
http://www.algaebase.org.
Hatta A. M
,
Prud'homme van Reine W. F
1991
A taxonomic revision of Indonesian Gelidiales (Rhodophyta)
Blumea
35
347 -
380
Kim K. M
,
Hwang I. K
,
Park J. K
,
Boo S. M
A new agarophyte speciesGelidium eucorneumsp. nov. (Gelidiales Rhodophyta) based on molecular and morphological data
J. Phycol
Kützing F. T
1847
Diagnosen einiger neuen ausländischen Algenspecies welche sich in der Sammlung des Herrn Kammerdirectors Klenze in Laubach befinden
Flora
30
773 -
776
Kylin H
1956
Die Gattungen der Rhodophyceen
CWK Gleerups Förlag
Lund
669 -
Lamouroux J. V. F
1813
Essai sur les genres de la famille des Thalassiophytes non articulées.
Ann. Mus. Hist. Nat. Paris
115-139 267-293
20
21 -
47
Lin S. -M
,
Fredericq S
,
Hommersand M. H
2001
Systematics of the Delesseriaceae (Ceramiales Rhodophyta) based on large subunit rDNA andrbcL sequences including the Phycodryoideae subfam.nov
J. Phycol
37
881 -
899
DOI : 10.1046/j.1529-8817.2001.01012.x
Millar A. J. K
,
Freshwater D. W
2005
Morphology and molecular phylogeny of the marine algal order Gelidiales (Rhodophyta) from New South Wales including Lord Howe and Norfolk islands
Aust. Syst. Bot
18
215 -
263
DOI : 10.1071/SB04041
Nelson W. A
,
Farr T. J
,
Broom J. E. S
2006
Phylogenetic diversity of New Zealand Gelidiales as revealed byrbcL sequence data
J. Appl. Phycol
18
653 -
661
DOI : 10.1007/s10811-006-9068-0
Perrone C
,
Felicini G. P
,
Bottalico A
2006
The prostrate system of the Gelidiales: diagnostic and taxonomic importance
Bot. Mar
49
23 -
33
DOI : 10.1515/BOT.2006.003
Rambaut A. E
1996.
Se-Al: sequence alignment editor
http://tree.bio.ed.ac.uk/software/seal/
Rozas J
,
Rozas R
1999
DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis
Bioinformatics
15
174 -
175
DOI : 10.1093/bioinformatics/15.2.174
Schmitz F
1894
Neue japanische Florideen von K. Okamura
Hedwigia
33
190 -
201
Shimada S
,
Horiguchi T
,
Masuda M
1999
Phylogenetic affinities of generaAcanthopeltisandYatabella(Gelidiales Rhodophyta) inferred from molecular analyses
Phycologia
38
528 -
540
DOI : 10.2216/i0031-8884-38-6-528.1
Silva P. C
,
Basson P. W
,
Moe R. L
1996
Catalogue of the benthic marine algae of the Indian Ocean
University of California Press
Berkeley
1259 -
Stamatakis A
2006
RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models
Bioinformatics
22
2688 -
2690
DOI : 10.1093/bioinformatics/btl446
Swofford D. L
2002
PAUP*:phylogenetic analysis using parsimony (*and other methods). v.4.0b10
Sinauer Associates
Sunderland MA
Tronchin E. M
,
Freshwater D. W
,
Bolton J. J
2003
A re-evaluation of the generaBeckerellaandPtilophora(Gelidiales Rhodophyta) based on molecular and morphological data
Phycologia
42
80 -
89
DOI : 10.2216/i0031-8884-42-1-80.1
Tronchin E. M
,
Freshwater D. W
,
Bolton J. J
,
Anderson R. J
2002
A reassessment and reclassification of species in the generaOnikusaAkatsuka andSuhriaJ. Agardh ex Endlicher (Gelidiales Rhodophyta) based on molecular and morphological data
Bot. Mar
45
548 -
558
DOI : 10.1515/BOT.2002.058
Weber-van Bosse A
1921
Liste des algues du Siboga 2. Rhodophyceae. Premiére partie. Protoflorideae Nemalionales Cryptonemiales
Siboga-Expeditie Monographie
59b
187 -
310
Wiriyadamrikul J
,
Park J. K
,
Lewmanomont K
,
Boo S. M
2010
Additional records ofGelidiella fanii(Gelidiales Rhodophyta) from the southeast Pacific based on morphologyrbcL andcox1 analyses
Bot. Mar
53
343 -
350
DOI : 10.1515/BOT.2010.037
Yang E. C
,
Kim M. S
,
Geraldino P. J. L
,
Sahoo D
,
Shin J.-A
,
Boo S. M
2008
Mitochondrialcox1 and plastidrbcL genes ofGracilaria vermiculophylla(Gracilariaceae Rhodophyta)
J. Appl. Phycol
20
161 -
168
DOI : 10.1007/s10811-007-9201-8