The occurrence of Stenogramma interruptum in northeast Asian waters is a matter of debate. We analyzed rbcL from Korean specimens filed under the species name S. interruptum in combination with morphological observations. A partial rbcL sequence revealed that the Korean specimens were clearly distinct from other species of Stenogramma as well as S. interruptum from Europe. The morphology and anatomy of the Korean specimens were consistent with the rbcL data. Based on these results, we herein describe the new species Stenogramma guleopensis from Korea. Stenogramma guleopensis is distinguished mostly by its small size (up to 8 cm), slender thallus with narrow and parallel-sided segments, terminal branches basally constricted, two layers of cortical cells, two to three layers of medullary cells, and a gradient of two layers of smaller cells between the cortex and medulla. Further collections and careful examinations in the field will probably reveal extension of the distribution of S. guleopensis to the waters surrounding Korea.
Stenogramma Harvey is a well-defined genus of red algae in the family Phyllophoraceae. It is characterized by a medial line of procarps and carposporangia resembling an interrupted midrib on female plants, its rose-red flattened thallus, dichotomous branching, and scattered circular to irregular tetrasporangial nemathecia (Harvey 1849, Millar 1990, Lewis and Womersley 1994, Le Gall and Saunders 2010). Harvey (1841) erected the genus with the generitype S. californicum Harvey from San Francisco, California, USA. Montagne (1846) included a second species, S. interruptum (C. Agardh) Montagne, based on a specimen in Bory St. Vincent’s herbarium, PC, which was labeled Delesseria interruptum C. Agardh from Cadiz, Spain. However, Harvey (1849) considered that S. californicum might actually be identical to S. interruptum, and in his subsequent papers (Harvey 1853, 1862), he recognized S. interruptum only, following Principle III, priority of publication (McNeill et al. 2012). Harvey’s wide concept of S. interruptum from California, USA, to Cadiz, Spain, was mostly accepted in subsequent phycological works (Taylor 1945, Dawson 1961, Abbott and Hollenberg 1976, Dixon and Irvine 1977). Although several phycologists did not agree with Harvey’s synonymy (e.g., Smith 1944, Kylin 1956, Schotter 1968, Joly and Alveal 1969), S. interruptum became the most common species in the genus Stenogramma worldwide, including the southwest Atlantic Ocean (Yoneshigue-Valentin and Gestinari 2000, Wynne 2011), the eastern Atlantic and Mediterranean Sea (Dixon and Irvine 1977), and the Pacific Ocean (Okamura 1901, Dawson 1961, Millar 1990, Ramírez and Santelices 1991). The results of recent molecular studies (Lopez-Bautista et al. 2002, Le Gall and Saunders 2010) support the reinstatement of S. californicum from California and of S. rhodymenioides Joly et Alveal, which was previously considered conspecific with S. interruptum by Ramírez and Rojas (1986). Six Stenogramma species are currently listed in AlgaeBase (Guiry and Guiry 2014): S. bamfieldiense Le Gall et Saunders, S. californicum, S. interruptum, S. leptophyllum J. Agardh, S. phyllophoroides (J. Agardh) A.J.K. Millar, and S. rhodymenioides.
Although its distribution was suggested to be limited to European waters (Le Gall and Saunders 2010), Stenogramma interruptum has been reported in Korea and Japan, even from quite northern sites (Yoshida 1998, Boo and Ko 2012). In previous collections of marine algae from the west coast of Korea, we found 15 herbarium specimens that were filed under the name S. interruptum. Based on detailed anatomical observations and partial rbcL sequence analyses, we herein describe this organism as the new species Stenogrammaguleopensis sp. nov.
Materials and methods
Specimens of Stenogramma were collected in the subtidal zone from Guleopdo Isle off the western coast of Korea during a study of subtidal algal flora. Fifteen specimens were collected in August 1993. They were fixed in about 5% formalin/seawater and then pressed on herbarium sheets. Of these, nine were tetrasporophytes, five were females with cystocarps, and one was vegetative. Microscopic observations were made by sectioning with a razor and staining with 1% aqueous aniline blue acidified with 1% HCl and mounted in 70% glycerine. Photomicrographs were taken with a DP-71 Olympus camera attached to a BX51 Olympus microscope. A total of 25 replicates from five individuals were selected for measurement of quantitative characters, and means and standard deviations (SD) were calculated. Voucher specimens have been deposited in the herbarium of Chungnam National University (CNUK), Daejeon, Korea.
DNA from three herbarium specimens of Stenogramma guleopensis was extracted and amplified following the procedures described by Lindstrom et al. (2011). Because Stenogramma specimens had been previously processed in the laboratory, we followed the guidelines and precautionary methods of Hughey and Gabrielson (2012). Four negative control extractions were performed prior to extraction to determine the level of contamination. The specific rbcL primer pairs were designed by Jeffery Hughey: F753 5′-GGAAGACATGTATGAAAGAGC-3′ and R900 5′-GACGAGAGTAAGTTGAATTAC-3′. We also analyzed a fresh collection of Stenogramma from Australia (CNU047638, Kingscote, South Australia; 15 April 2003) following the methods of Boo et al. (2013). Sequencing was performed using both forward and reverse primers provided by Genotech (Daejeon, Korea). Electropherograms were edited using the program Chromas v.1.45 (Conor McCarthy, Griffith University, Australia; http://www.technelysium.com.au/chromas.html). Sequences were compiled and aligned using the MUSCLE algorithm in MEGA5 v5.2 (Tamura et al. 2011). The short rbcL sequences (125 bp) from three Korean specimens are deposited in BOLD and the rbcL from an Australian specimen in GenBank (Table 1).
|Stenogramma bamfieldiense (GQ338127)||A||A||C||A||A||T||A||T||A|
|Stenogramma californicum (U27020)||G||G|
|Stenogramma guleopensis (STENO)||G||G||C|
|Stenogramma interruptum (AY135168)||G||T||T|
|Stenogramma phyllophoroides (GQ338123)||G||G||G|
|Stenogramma rhodymenioides (AY135169)|
|Stenogramma sp. from Australia (KM262208)||G||G||G||G||C||C||G|
Bold type indicates base pair differing from the majority base pair at each site. Accession numbers in GenBank or acronym in bold indicated in parentheses.
Partial rbcL sequences (sites 774–898 of the full rbcL sequences) were successfully amplified and analyzed from Stenogramma guleopensis specimens fixed with formalin/seawater, as was an almost full-sized rbcL (1430 bp) from a specimen from Australia. All three specimens of S. guleopensis were identical in 125 rbcL sequences. However, S. guleopensis was distinct from other published species of Stenogramma, including the Australian taxon analyzed in the present study (Table 1). For example, S. guleopensis differed from S. interruptum from Europe at four sites. It also differed from S. phyllophoroides at two sites, from S. californicum and S.bamfieldiense at three sites, and from Stenogramma sp. from Australia at five sites. The Australian taxon that we analyzed here was distinct from all other Stenogramma species in rbcL sequences (Table 1).
Thalli 5–8 cm high, rose-red to darker red in color, and dichotomously or subdichotomously branched three to five times; thalli slender with narrow and parallel-sided segments, terminal branches basally constricted (Figures 1–3); surface cells (Figure 4) angular with a maximum width of 8.3±1.3 μm; structure multiaxial, thallus 238.5±16.6 μm thick, cortex two cells thick, medulla two to three cells thick, with two layers of smaller cells present between cortex and medulla (Figure 5); tetrasporophyte and female gametophyte isomorphic; tetrasporic thalli with ovate nemathecia scattered as wart-like blotches over both surfaces, formed by divisions of outer cortical cells (Figure 6) giving rows of cruciately divided tetrasporangia 24.7±2.7 μm long and 10.1±1.1 μm wide (Figure 7); three-celled carpogonial branches; cystocarps developing inwardly, forming an interrupted median thickened line containing masses of carposporangia; male thalli not observed. Plants grow irregularly in isolated patches on sand-covered rocks at a depth of 5–10 m.
CNU014891 (female thallus), subtidal zone (depth of 5–10 m), Guleopdo Isle, Dukjeokdo Islands, Korea (37°11′20″ N 125°58′45″); 4 August 1993 (Sung Min Boo, collection numbers not given). Isotypes CNU014888-9, CNU014891-1, 2.
The specific epithet refers to Guleopdo Isle, where the type specimen was collected.
Plants formed part of the drift and also grew in the sublittoral zone. The pre- and postfertilization development of the female gametophyte and carposporophyte were observed in detail. Procarps were placed in the center of the thallus. Nearby large medullary cells were replaced by smaller and stellate-shaped, irregularly arranged cells. Procarps were situated in the new thicker median cortex and comprised a large supporting cell bearing a three-celled carpogonial branch (carpogonium, hypogynous cell, and basal cell) provided with a two-celled sterile lateral branch. The supporting cell bore a four- to seven-celled sterile branch and was subtended by a two-celled vegetative branch of large and ovoid-shaped mother cells with denser content (Figure 2). The postfertilization stage was presumed to be the direct fusion of the fertilized carpogonium with the supporting cell. The auxiliary cell with a diploid nucleus formed small protuberances on the inside, which became gonimoblast filaments (Figure 9) and spread across the medulla (Figure 10). Multiple young unfertilized procarps were scattered in the cortical region (Figure 11). Fusiform conjunctor cells expanded from the gonimoblast cells and fused with vegetative cells (Figure 12). The mass of carposporangia, 407.5±23.5 μm in diameter, was surrounded by a multilayered cortex (Figure 13). Male thalli were not observed. A morphological comparison of Stenogramma guleopensis with other Stenogramma species is given in Table 2.
|S. guleopensis||S. bamfieldiensis||S. californicum||S. interruptum||S. leptophyllum||S. phyllophoroides||S. rhodymenioides|
|Habit||Narrow segments; terminal branches basally constricted||Sparse branching||Linear segments, gradually broader at tips||Broad segments||Linear segments||Segments are spirally twisted, margins are finely dentate||Segments gradually constricted above the dichotomy|
|Height (cm)||5–8||c. 8||6–20||2–10||5–20||c. 12||11–18|
|Width (mm)||3–5||c. 5||5–18||5–10||2–3||2–5||4–7|
|Number of dichotomies||3–5||2||3–5||2–4||3–9||3–9||5–9|
|Layers of cortical cells||2||1||1–2||1–2||2–3||1||2|
|Layers of medullary cells||2–3||1||1–3||2–3||2–3||1–2||3|
|Transition layer of cells||2||2–3||–||1||–||1||2|
|Type locality||Guleopdo Isle, Dukjeokdo Islands, Korea||Bamfield, British Columbia, Canada||San Francisco, CA, USA||Cadiz, Spain||Port Phillip Heads, Victoria, Australia||Richmond River, New South Wales, Australia||Valparaiso, Chile|
|Reference||Present study||Le Gall and Saunders 2010||Smith 1944, Abbott and Hollenberg 1976 (as S. interruptum)||Harvey 1849, Schotter 1968, Dixon and Irvine 1977||Lewis and Womersley 1994||Millar 1990||Joly and Alveal 1969|
Previous studies have documented the utility of using relatively short rbcL DNA sequences when the full sequence is not available from old or formalin-fixed red algal specimens (Hughey et al. 2001, 2002, Lindstrom et al. 2011). Comparison of these partial rbcL sequences and comprehensive morphological observations consistently support the description of the new species Stenogramma guleopensis from Korea and its distinctness from congeners. Stenogramma guleopensis is characterized by its small size, slender thallus, two layers of cortical cells, two to three layers of medullary cells, and two layers of smaller cells between the cortex and medulla. Thalli are characterized by parallel-sided and narrow segments becoming slightly broader upward, while the terminal branches are basally constricted. Despite our repeated collection of specimens of Stenogramma in Korea, there were no specimens referable to S. interruptum, the European species that had been reported in several papers and flora lists in Korea (Kang 1966, Lee and Kang 2001, Boo and Ko 2012). We, thus, agree with the conclusions reached by Le Gall and Saunders (2010) that different species of Stenogramma occur in different biogeographical areas.
Stenogramma is a well-defined and monophyletic genus that is characterized by a medial line of procarps and carposporangia along the female thallus (Le Gall and Saunders 2010). However, some species of Stenogramma are morphologically similar with respect to their dichotomous branching and medullas of a few layers of large cells. Stenogrammainterruptum is up to 20 cm long, dichotomously and palmately divided, and its segments are about 10 mm wide (Harvey 1849, Schotter 1968, Dixon and Irvine 1977). Furthermore, its distribution is limited to European waters (Le Gall and Saunders 2010). The Canadian S.bamfieldiensis is similar to Stenogramma guleopensis with respect to its slender fronds and small size, but the former is distinguished by its strap-like thallus with adventitious proliferations, two dichotomies, inner structure comprising one layer of small cortical cells, and a layer of large medullary cells (Le Gall and Saunders 2010). In contrast to S.bamfieldiensis, S. guleopensis is fan shaped and comprises two layers of cortical cells and two to three layers of medullary cells. The general morphology of the other four species is easily distinguishable from that of S. guleopensis.Stenogramma californicum has a long (up to 20 cm) and wide (5–18 mm) thallus (Smith 1944, Abbott and Hollenberg 1976 as S. interruptum), and S. rhodymenioides is distinguished by five to nine dichotomies and thallus size (Joly and Alveal 1969). Stenogramma leptophyllum is characterized by its linear segments and three to nine dichotomies (Lewis and Womersley 1994). Stenogramma phyllophoroides has spirally twisted segments and finely dentate margins (Millar 1990).
Relatively few studies have evaluated postfertilization and diploidization events in the genus Stenogramma (Fritsch 1945, Kylin 1956). Our observation of the female structures of S. guleopensis revealed a three-celled carpogonial branch, a feature that has also been reported in S. interruptum (Kylin 1956, Schotter 1968). However, three- to four-celled carpogonial branches were described for the so-called “S. interruptum” and S. leptophyllum from Australia (Lewis and Womersley 1994). Four-celled carpogonial branches were also reported in S. rhodymenioides from Chile (Joly and Alveal 1969). Detailed observation of the carpogonial branches is needed for other species of Stenogramma.
In conclusion, we have described Stenogramma guleopensis in the deep water off Guleopdo Isle (65 km off Incheon City, Korea). Further collection and careful examination of specimens will probably extend the known distribution of S. guleopensis to surrounding Korean waters. The finding of Stenogramma sp. from Australia in the present study highlights the fact that the number of Stenogramma species will increase with additional collection.
We thank Drs. W.J. Lee and E.C. Yang for the collection of Australian specimen and late Prof. H.B.S. Womersley for his help in the collection trip and Jeffery Hughey for designing the primers. Funding for this work was provided by the Korean Research Foundation Grant (2013-0699), and Marine Biotechnology program grants from the Ministry of Oceans and Fisheries to SMB.
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