There are three carpet-like
Codium
species with a broadly expanded thallus reported from Korea and Japan:
C. latum
Suringar,
C. tapetum
Y. Lee, and
C. tenuifolium
Shimada, Tadano and J. Tanaka. During surveys of green algal diversity,we encountered a
Codium
species with carpet-like thalli from several sites on Jeju Island. To confirm the taxonomic identity of these specimens, we studied the morphological features and obtained
rbc
L gene sequences. The morphological and sequence data indicated that the carpet-like
Codium
specimens from Jeju are
C. tenuifolium
from Japan, but distant from Japanese
C. latum
. We propose that the carpet-like specimens from Jeju should be renamed
C. tenuifolium
,instead of
C. tapetum
or
C. latum. C. tenuifolium
is characterized by a tenuous, carpet-like and erect thallus with a very short and slightly compressed stipe arising from a discal holdfast, sub-pyriform and clavate utricles with a tumid and swollen apical head, and globular or sub-globular gametangia issued at the basal portion of the utricles.
INTRODUCTION
The green algal genus
Codium
occurs in various marine habitats, from estuarine to fully marine and from soft to hard substrata.
Codium
is cosmopolitan, except for the Arctic and Antarctic Oceans. Species range from crustose forms to erect highly branched or flattened forms. Nevertheless,most species have a dark green color, spongy texture, and multi-axial thalli up to approximately 1 m in length (Graham et al. 2009). Currently,
Codium
consists of 130 species, 14 subspecies, three varieties, and three forms (Guiry and Guiry 2011). The major morphological characters used for identification are shape of thalli,utricles, and gametangia as well as branching patterns(Yoshida 1998).
Codium
thalli are composed of numerous utricles with an interior medulla, consisting of an entangled mesh of siphon structures. The most apparent and variable attributes are external morphology in which thalli spread out over the surface as mats, finger-like, or flattened branches (Verbruggen et al. 2007). Some species are broadly expanded with a velvety and smooth texture.
In Korea, a carpet-like
Codium
has been reported as
Codium latum
Suringar (Kang 1968, Oh et al. 1987, Sohn 2000). Kang (1968) described its features based on specimens from Busan and Jeju, Korea (
Fig. 1
A) as follows:broad and flat, or long-spread thallus, with a silky texture,and cylindrical utricles with swollen and rounded apex. Sohn (2000) described the morphology and analyzed
trn
L gene sequences from
C. latum
collected from Jeju Island, Korea and Tateyama, Japan. Lee and Kang(2001) recorded
C. latum
in “A Catalogue of the Seaweeds in Korea.” Sixteen species were recently reported by Lee(2008), including a new carpet-like species,
Codium ta-
Map showing the distributions of carpet-like Codium species. (A) Sites reported by Kang (1968) in Korea (square marks) as C. latum. (B) Type locality (lozenge mark) and collecting sites by Shimada et al. (2007) in Japan (circle marks) as C. tenuifolium. (C) Sites reported by Lee (2008) (square marks) and collected for this study (arrows) on Jeju Island as C. tapetum.
petum
Y. Lee, collected from Jeju Island. Lee (2008) noted that
C. tapetum
was previously identified as
C. latum
, and characterized
C. tapetum
as having thalli with a broadly expanded frond with a short stipe, and sub-pyriform utricles with a slightly swollen apex.
In Japan, Shimada et al. (2004) conducted phylogenetic studies on 18
Codium
species, including carpet-like species, and concluded that
rbc
L gene regions are valuable for distinguishing between
Codium
species. Shimada et al. (2007) published a new carpet-like species as
C. tenuifolium
from Japan (
Fig. 1
B), rejecting the name
C. pseudolatum
, although they found
C. pseudolatum
named by Yamada in a herbarium specimen known as a nomen nudum. Because the name
Codium
pseudolatum was previously published by Nizamuddin (2001) from the Arabian Sea.
C. tenuifolium
is characterized by thin blades, clavate utricles with a tumid head, and variable reproductive structures at the utricle base (Shimada et al. 2007). Currently, there are three carpet-like
Codium
species,
C. latum, C. tapetum,
and
C. tenuifolium
, reported from Korea and Japan (Suringar 1867, 1870, Okamura 1915, Schmidt 1923, Kang 1968, Oh et al. 1987, Sohn 2000, Shimada et al. 2004, Shimada et al. 2007, Lee 2008).
During a survey of green algal diversity, we encountered
Codium
species with carpet-like thalli from several localities on Jeju Island, Korea. In this study, we confirmed the taxonomic position of the carpet-like species, currently named
C. tapetum
Y. Lee from Korea (2008) and
C. tenuifolium
Shimada, Tadano and J. Tanaka from Japan (2007). We examined morphological characteristics and sequenced
rbc
L gene using our recent specimen collections.
MATERIALS AND METHODS
The distributions of carpet-like
Codium
species from Korea and Japan can be seen on the map (
Fig. 1
A-C). Six samples of carpet-like
Codium
were collected from Jeju Island (
Fig. 1
C): 10TE001, 10TE002, April 6, 2010, Shinyang, J. C. Kang; 10TE003, June 6, 2010, Chaguido, Y. D. Koh; 10TE004 (gametangia), 10TE005 (gametangia), June 16, 2010, Sangmo-ri, E. G. Han; 10TE006, August 13, 2010, Sangmo-ri, E. G. Han. The samples were stored in silica gel for molecular studies, and DNA vouchers were prepared from two fresh samples. The remaining samples were preserved in formalin-seawater for morphological observations. Habit photographs were taken with a D-80 camera (Nikon, Tokyo, Japan), and internal morphology was photographed using QIMAGING 1394 camera (QImaging, Surrey, BC, Canada) with a BX50 microscope (Olympus, Tokyo, Japan). DNA vouchers and specimens were stored at the herbarium of Jeju National University (JNUB), Korea.
DNA was extracted from dry samples using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) following the manufacture’s protocol. Extracted DNA was stored at -20°C and was used to amplify
rbc
L gene. Polymerase chain reaction (PCR) amplification was performed using rbcL primers designed by Shimada et al. (2004). The forward primer was EadF (5′?GACTAACTTATTATACACCGG-3′; 21mer), and the reverse primer was EadR (5′?CACCAGTTTCAGATTGCGATT-3′; 21mer). PCR amplification was conducted in a total volume of 20 μL with AccuPower PCR PreMix (Bioneer, Daejeon, Korea). PCR was conducted with an initial denaturation at 95°C for 5 min, followed by 35 cycles of amplification (denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 2 min), with a final extension at 72°C for 7 min. The PCR products were purified using the AccuPrep PCR Purification Kit (Bioneer).
To confirm the taxonomic position of the carpet-like
Codium
from Jeju, maximum likelihood (ML) analysis was performed, using PAUP 4.0 (Swofford 2002). Six
rbc
L sequences of
Codium
were aligned with other Japanese
Codium tenuifolium Shimada Tadano and J. Tanaka. (A & C) Specimens from Shinyang Jeju on April 6 2010. (B & D) Specimens from Sangmo-ri on June 16 2010. Scale bars represent: 5 cm.
Codium
species. Two
Bryopsis
species were used as the outgroup. We performed a likelihood ratio test using MODELTEST (version 3.7) (Posada and Crandall 1998) to determine the best available model for our sequence data. The best model was a general time reversible (GTR) model with a gamma correction for among-site variation (Γ) and the proportion of invariable sites (I). ML trees were estimated using a heuristic search with 100 random addition sequence replicates, and tree bisection and reconnection (TBR) branch swapping. ML bootstrap analyses were performed with 1,000 replicates to test node stability.
RESULTS
- Morphological observations
Thalli arise from a discoid and concave holdfast attached to a shell or rock surface. Thalli have a stipe less than 1 cm long and 3-5 mm in diameter. The stipe is either terete or slightly compressed. The thallus is broadly expanded from a stipe and has a foliaceous and bi- or trifurcate shape, rather than branched, and is dark green in color (
Fig. 2
A-D). Thalli are 20-50 cm long, 10-25 cm wide, and 0.3-2.1 mm thick. The thallus has thin, fragile,
Codium tenuifolium Shimada Tadano and J. Tanaka. (A-C) Utricles having sub-pyriform and clavate shape but rarely with hair (arrowhead) and siphons with an H-shaped septum (arrows) inside relating utricles. (D) Gametangia dangling from a short stalk (arrow). Scale bars represent: A C & D 100 ㎛; B 150 ㎛.
sheet-like, and velvety texture.
The thallus is composed of numerous concatenate utricles with entangled siphons, creating the medullary filaments. Two or three siphons are linked to each utricle base, and extend towards nearby utricle bases (
Fig. 3
A & B). The siphon diameter is 28-41 ㎛, and typically has an H-shaped septum (
Fig. 3
A & B) on one side. The utricle is cylindrical, sub-pyriform and clavate, and the utricle base is not as thick as the apex. The utricle body is approximately 85 ㎛ in diameter, and the apex is 110-160 ㎛. The utricle apex is flat and rarely concave, and becomes slightly more tumid and swollen compared to the basal and middle sections (
Fig. 3
A-C). Utricles are 260-410 ㎛ long, and the length is three times longer than the width. Utricles are generally hairless, but hairs up to 700-1,000 ㎛ long can grow on the apical section of the utricle (
Fig. 3
C). Globular gametangia dangle from the short pedicel, and are tinged with a dark green color. They develop irregularly on the siphons, following initiation at the utricle bases (
Fig. 3
D).
- Molecular studies
We sequenced six carpet-like
Codium
specimens from Jeju for
rbc
L, and aligned these with four Japanese
C. tenuifolium
specimens, and nine
Codium
species from GenBank. In total, 602 bp of
rbc
L were aligned: 143 sites (23.8%) were variable and 108 sites (17.9%) were phylo-
Phylogenetic tree of Codium species inferred from maximum likelihood analysis of chloroplast encoded rbcL sequences. The bootstrap values shown above the branches are from 1000 bootstrap resamplings.
genetically conserved. There were excesses of adenine (28.9%) and thymine (30.9%) at all codon positions. Six carpet-like
Codium
specimens from Jeju were identical to four
C. tenuifolium
specimens from Japan. The
C. tenuifolium
sequence divergence differed by 2.82% from
C. latum
, 5.0% from
C. intricatum
, 5.3% from
C. subtubulosum
, 5.3% from
C. contractum
, and 9.1% from
C. minus.
In the phylogenetic tree (
Fig. 4
), all carpet-like
Codium
specimens from Jeju and C. tenuifolium from Japan were associated on the same node. This clade had strong support (99%) and formed a sister group with
C. latum
from Japan with lower support (77%). The
C. tenuifolium
and
C. latum
clades were grouped with C. intricatum,
C. contractum,
and
C. subtubulosum
, with bootstrap values of 89% (
Fig. 4
).
DISCUSSION
Based on the analysis of
rbc
L gene and a comprehen sive morphological study, we confirmed the name of the carpet-like
Codium
species from Jeju as
Codium tenuifolium
Shimada, Tadano and J. Tanaka (2007), instead of
C. tapetum
Y. Lee or
C. latum
Suringar.
C. tenuifolium
has the following characteristics: (1) tenuous, flat and broadly expanded erect thallus with a fragile and velvety surface, (2) simple, bi- or trifurcate fronds, (3) sub-pyriform utricles with a swollen and tumid head, and (4) globular gametangia borne from a short pedicel attached to a utricle base. Although
C. tenuifolium
, as originally described (Shimada et al. 2007), has variable type gametangia, we observed only globular shaped gametangia. Phylogenetic analysis of
rbc
L gene sequences of carpet-like specimens from Jeju indicated that they were identical to
C. tenuifolium
from Japan.
C. tenuifolium
is distinct from Japanese
C. latum
and the two are sister species.
C. tenuifolium
occurs in the low intertidal and shallow subtidal zones on Jeju Island, with erect fronds individually attached to a rock or shell. In Korea, carpet-like
Codium
were previously identified as
C. latum
after Kang’s description (1968). Lee (2008) reported a new carpet-like species,
C. tapetum
Y. Lee. He suggested that the new species has been known in Korea as
C. latum
. Comparing morphological features of
C. tapetum
with
C. tenuifolium
, we confirmed that both species have the same morphological characteristics (
Table 1
). Thus,
C. tapetum
Y. Lee (2008) should be regarded as a synonym of
C. tenuifolium
Shimada, Tadano and J. Tanaka (2007).
According to Okamura’s description in 1915,
C. latum
has a broad thallus that is flat, expanded, and lanceolate or band-like externally. Because
C. latum
has similar morphological features to
C. tenuifolium
, most carpet-like
Codium
species had been recognized as
C. latum
in Korea. However, we identified two species based on our comprehensive morphological observations. The diagnostic features distinguishing the two species are the shapes of the utricles and gametangia. The utricles of
C. latum
are cylindrical and slightly enlarged at the top, forming a roundish knob with thin apical walls (Okamura 1915). In
C. tenuifolium
, utricles have a tumid head that is more swollen than the basal and middle sections. The gametangia of
C. latum
extend from the middle portion of the utricle (Okamura 1915, Schmidt 1923), but those of
C. tenuifolium
develop at the utricle base.
C. latum
gametangia are oblong or elongate-ovate (Okamura 1915, Schmidt 1923), but
C. tenuifolium
are variable and may
Comparison of morphological characteristics among similar species of Codium
Comparison of morphological characteristics among similar species of Codium
be globular, subglobular, or oval (Shimada et al. 2007, Lee 2008).
The molecular phylogenetic analysis using
rbc
L gene sequences demonstrated that the carpet-like specimens from Korea corresponded with
C. tenuifolium
from Japan and revealed sufficient sequence divergence to separate
C. tenuifolium
from other species. The sequence divergences of six
C. tenuifolium
samples were identical to each other, and they had 2.82% sequence divergence with
C. latum
from Japan. Although
C. tenuifolium
is superficially similar to
C. latum
in having a thin and broad thallus,
rbc
L sequence data identified a sister relationship between the two. Shimada et al. (2007) confirmed that
C. tenuifolium
specimen is separate from
C. latum
specimen through analysis of
rbc
L gene sequences with 2.6% pair-wise distance.
Silva (1954) characterized
Codium
with two subgenera: the subgenus
Tylecodium
has prostrate or erect thalli showing applanate, pulvinate, or compressed cylindrical fronds without true branching; the subgenus
Schizocodium
has erect thalli consisting of cylindrical or compressed fronds that are dichotomously or polytomously branched. Silva (1962) raised the subsection
Lata
, which was established by Schmidt (1923), to the section
Lata
for
C. latum,C. latum
subsp. palmeri,
C. laminarioides
, and
C. platylobium
, and these taxa have applanate, thin
and broad erect thalli. Verbruggen et al. (2007) studied the phylogenetic relationships of
Codium
using
rbc
L and rps3-rpl6 sequences, and demonstrated that
C. latum
formed a distant clade with
C. laminarioides
and
C. platylobium
. According to the results of our study,
C. tenuifolium
would belong to the section
lata
based on morphological features and a molecular analysis that
C. tenuifolium
is tied with
C. latum
as a sister taxon (
Fig. 4
). Therefore, it is necessary to demonstrate whether or not
C. tenuifolium
belongs in the section
Lata
by re-examining morphological features and DNA sequences.
In conclusion, we confirmed that the morphology and
rbc
L gene sequences of a carpet-like
Codium
from Jeju, Korea were identical to those of
C. tenuifolium
from Japan. We propose that carpet-like
Codium
from Jeju is
C. tenuifolium,
and that
C. tapetum
should be regarded as a synonym of
C. tenuifolium. C. tenuifolium
is not only clearly distinguished from
C. latum
based on morphological characteristics of the utricles and gametangia, but plastid
rbc
L gene sequences do provide sufficient pair-wise differences to separate the two species. Since it remains to be demonstrated whether or not
C. latum
occurs in Korea, more studies are required on the entity of
C. latum
from Korea.
Acknowledgements
We thank Mr. J. C. Kang, Mr. E. G. Han and Mr. Y. D. Koh for collecting samples from Jeju Island. This work was supported by a National Research Foundation of Korea Grant, funded by the Korean Government (2010-0015429) and The Survey of Indigenous Biology of Korea from the National Institute of Biological Resources (NIBR).
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