Molecular analyses revealing that G. pacifica and G. “filamentosa” are the same species
The rbc L sequences analyzed in this study were obtained primarily from GenBank, NCBI (Table 1). Two taxa of the Scinaiaceae were used as the outgroup. The analyzed matrix included 1407 characters, but some taxa only comprised partial characters because of their incomplete rbc L sequences. The topology of the ML tree was largely identical with that of the Bayesian tree, so only the ML tree is presented (Figure 1). Four major clades (representing the genera Actinotrichia, Dichotomaria, Galaxaura, and Tricleocarpa) with statistical support were identified by molecular analysis (Figure 1). For the intergeneric relationship, Actinotrichia and Galaxaura grouped together as a monophyletic group, referred as to the Actinotrichia/Galaxaura clade, whereas Tricleocarpa and Dichotomaria grouped together as the other monophyletic group, referred to as the Tricleocarpa/Dichotomaria clade. The Actinotrichia/Galaxaura clade is the sister lineage of the Tricleocarpa/Dichotomaria clade. The intergeneric relationship among these genera received high statistical support (Figure 1). Three distinct sub-clades were observed in our phylogenetic analyses in the genus Galaxaura (Figure 1). All of them received high statistical support (99%-100%). The basal clade of the genus Galaxaura is G. divaricata from Japan and Taiwan (Figure 1). The second clade of the genus Galaxaura consists of three distinct lineages that are related to the species G. pacifica obtained from its type locality in the Bonin Islands (Ogasawarajima), Japan, and are referred to as the G. pacifica assemblage (Figure 1). Within the G. pacifica assemblage, we unexpectedly discovered that two morphologically distinct species, G. pacifica from Xiao-Liu-Qiu Island, Taiwan and G. “filamentosa” from Sorsogon, the Philippines shared identical rbc L sequences, indicating that they are a single species. The rbc L sequences of the specimens from Xiao-Liu-Qiu Island (Taiwan) and Sorsogon (the Philippines) differ by approximately 2% (27 of 1380 nucleotides) from that of G. pacifica collected from the southernmost insular Japan (the type locality of G. pacifica). Another specimen that was morphologically identified as G. “filamentosa” occupies the basal lineage of the G. pacifica assemblage (Figure 1). The last clade of the genus Galaxaura comprises the generitype species, G. rugosa, from Guadeloupe, which is geographically close to its type locality in Jamaica, and other Galaxaura rugosa-related complexes from various places around the world, collectively referred to as the G. rugosa assemblage (Figure 1). Further morphological examinations are necessary for identification of different species because only G. cuculligera was recognized within the G. rugosa assemblage (Figure 1).
Rbc L sequence analyses revealing seasonal variation of the external morphology in G. pacifica
After we determining that G. pacifica and G. “filamentosa” are the same species, this study observed that specimens from different locations in southern Taiwan showed a range of external morphological variation in different seasons. Figure 2 shows that the specimens from Xiao-Liu-Qiu Island collected in the summer often possess a tuft of larger and distinctly villous branches in the lower part of the thallus (Figure 2A-2C, 2G-2I). The overall size of the lower villous part of the thallus and the density of upper glabrous branches vary among different individuals within the population. Some individuals had few clusters of loosely dichotomous glabrous branches in the upper part of the thallus (Figure 2A) and a tuft of wider and larger villous branches in the lower part of the thallus (Figure 2G). Some individuals had few clusters of densely dichotomous glabrous branches in the upper part of the thallus (Figure 2B) and a tuft of narrower and smaller villous branches in the lower part of the thallus (Figure 2H). Interestingly, one of the specimens consisted solely of very few glabrous branches in the upper part of the thallus (Figure 2C) and some residuals of glabrous branches can still be seen attaching on the lower villous branches (arrowheads in Figure 2C, 2I). A careful examination of the glabrous branches on this particular specimen showed that most of them were old and showed numerous lesions (image not shown). This observation suggests that the glabrous branches might eventually decay or die off in summer and the villous branches might be retained for some time after the decay/die-off of the glabrous branches, often leading to its misidentification as G. filamentosa (imagine the scenario that the last cluster of glabrous branches and those residuals decay in Figure 2C). In contrast, the specimens from Small Port (Figure 2D-2E, 2J-2K) and Sail Rock (Figure 2F, 2L) collected in the winter showed a tuft of small villous branches in the lower part of the thallus. The size of the villous branches in the lower part of the thallus and the density of the glabrous branches in the upper part vary across different individuals within the overall local population. In some cases, involving presumably more mature individuals, plants show several clusters of densely glabrous branches in the upper part of the thallus (Figure 2D) and a tuft of small villous branches in the lower part (Figure 2J). In other cases, representing presumably younger individuals, plants possess few clusters of densely dichotomous glabrous branches in the upper part of the thallus (Figure 2E-2F) and a tuft of tiny (occasionally unnoticeable) villous branches (Figure 2K-2L). To better quantify the external morphological difference among the specimens from different locations, we estimated the ratio between the height of the glabrous branch (G) and the villous branch (V). The G/V ratio of the specimens from Xiao-Liu-Qiu Island in summer is significantly smaller than those from Small Port and Sail Rock collected in the winter based on our comparison (Figure 3; p < 0.05). This result supports our previous observations that the specimens in the summer have larger villous branches than those in the winter. To rule out the possibility that our observed external variation results from the comparison of two different species, we additionally obtained rbc L sequences of the specimens from Small Port and Sail Rock and then compared these with that from Xiao-Liu-Qiu Island, as well as that from Sorsogon, Philippines. Results revealed that they share 100% identical rbc L sequences, indicative of conspecificity. Phylogenetic analysis also supports the same conclusion as they are grouped together (Figure 4). However, the inter-specific relationship differs from previous results. The lack of statistical support suggests that a difference might be caused by insufficient informative sites from the smaller set of characters (669 vs. 1407) used in this analysis (Figure 4). Because the glabrous branches of G. pacifica contain considerable mucilage, it is often difficult to obtain pure DNA for further PCR reactions. The traditional CTAB method (Doyle and Dickson, 1987) with at least three rounds of chloroform: isoamyl alcohol (24:1) treatments works more effectively than the commercial DNA extraction kit. Morphological observations and molecular analyses revealed that the villous branches are small in the winter and grow larger in the summer. Considering that one of the specimens from Xiao-Liu-Qiu Island showed extremely scarce glabrous branches (Figure 2F), it is tempting to speculate that the material identified as G. filamentosa might be the remaining villous part of senescing G. pacifica. Similar gross morphology and G/V ratio among specimens obtained during similar seasons over different years (e.g., Figure 2D-2E from March in 2003 and Figure 2F from February in 2012) (Figure 3) suggests that environmental cues such as temperature might serve as the most significant factor affecting external morphological development in G. pacifica.
Morphological description
Galaxaura pacifica Tanaka, 1935: 55-57, Figures 5A, 5B, 6, pl. 17: Figure 2.
(Figures 5A-5N, 6A-6E, 7A-7C, 8A-8H, 9A-9E and 10A-10G).
Putative synonym
Galaxaura filamentosa Chou 1945: 39-41, pl. I: Figures 1, 2, 3, 4, 5 and 6; pl. VI: Figure 1 (type locality: Sulphur Bay, Clarion Island, Revillagigedo Islands, Mexico).
Type locality
Bonin Islands, Japan.
Distribution: predominately distributed in the warm temperate, subtropical, and tropical regions of the Pacific Ocean, including Japan, Taiwan, the Philippines, and possibly the Revillagigedo Islands of Mexico.
Specimens examined
Xiao-Liu-Qiu Island, southern Taiwan: (1) Wukeuitung, coll. S.L. Liu and C.S. Lin, 15.viii.2002 (#TU_GaPa2002.08.15.01 ~ #TU_GaPa2002.08.15.08, female gametophyte); Kenting National Park, southern Taiwan: (1) Small Port, coll. S.L. Liu, 13.iii.2003 (#TU_GaPa2003.03.13.01 ~ #TU_GaPa2003.03.13.03, female gametophyte); (2) Small Port, coll. S.L. Liu, 13.iii.2003 (#TU_GaPa2003.03.13.04 ~ #TU_GaPa2003.0312.07, male gametophyte); (3) Sail Rock, coll. S.L. Liu, 19.ii.2012 (#TU_GaPa2012.02.19.01 ~ #TU_GaPa2012.02.19.07, female gametophyte); Sorsogon, the Philippines: (1) Bulusan, coll. L.M. Liao and S.L. Liu, 19.ii.2003 (#TU_GaPa2003.02.19.01, possible remnants of the villous parts of senescing gametophyte or tetrasporophyte).
Habitat and seasonality
Collections were made seasonally in February, March, and August. Plants often grew on rocky (or coral reef) substrates at depths of 1-3 m.
Habit and vegetative structure
The thalli comprise two distinct forms: 1) glabrous-type individual (Figure 5A, 5C) and 2) villous-type individuals (Figure 6A). Glabrous-type thalli are light-red or pink in color and up to 6 cm high at full maturity. There are no gross morphological differences between female (Figure 5A) and male plants (Figure 5C). Villous-type thalli are dark-red in color and up to 2.5 cm in height. Both types of thalli initially consist of a primary cylindrical axis that originated from a discoid holdfast. The holdfast diameter is approximately 1-3 mm. The initial primary terete axis continuously develops several terete branches. These branches are produced in a dichotomous or subdichotomous manner. The length of internodes is 5-15 mm, and the width of branches is 1-2 mm. Branches in the glabrous-type thallus are smooth in the upper portion (Figure 5A, 5C) and villous in the lower portion of the thalli (Figure 5B, 5H), whereas the branches in the villous-type thallus are hairy throughout (Figure 6A). Both types of thalli show light to heavy calcification in the cortical and medullary parts. Different cortical sections on the terminal branches of the glabrous area show different degrees of calcification that subsequently lead to the appearance of annulations (Figure 5D). The cross section of the branches from the glabrous-type plants (i.e., gametophytes) and that from the villous-type plants display two different cortical structures. The first type can be observed from the smooth portion of the glabrous-type thallus (Figure 5E-5F) wherein growth is apical with a sunken growing point (Figure 5E). Young cortical initials on the apex of glabrous branches are slender (Figure 5E) and then develop into a three cell-layers (Figures 5F, 7B). The outermost layer consists of highly pigmented epidermal cells 12-18 (20) μm in diameter (Figures 5F, 7B). The middle layer is composed of slightly larger cells that are 25-38 μm in width and 30-40 μm in length (Figures 5F, 7B). The innermost layer consists of the largest cells which are 25-50 μm in width and 38-105 μm in length (Figures 5F, 7B). In the surface view the outermost cortical cells show 4-6 sided and angular cells. Each cell contains one well-developed stellate chromatophore with a large central pyrenoid (Figures 5G, 7A). The second cortical type can be observed from the villous branches of the lower section of the glabrous-type thalli (Figure 5B, 5H, 5L) and the villous-type thalli (Figures 6A-6B). The cross-sectioned inner part of the branch is the medulla (Figures 5I, 6C, 7C), which comprises heavily calcified dense medullary filaments (Figure 5I). The inner layer of the cortex shows a mixture of medullary and assimilatory filaments (Figures 5J, 6D, 7C). The outer layer of the cortex is composed of 10- to 50-celled, long assimilatory filaments (Figures 5I-5J, 6C-6D) that arise from an undifferentiated, non-swollen basal cell (Figures 5K, 6E). These long assimilatory filaments are dense and up to 1 mm long at distal portions of villous branches, but are scarce at the lower part of villous branches (Figures 5H, 6B). Overall, there are no obvious differences in the cortical structures between the villous branches in the lower part of the glabrous-type thalli and the villous-type thalli. The assimilatory filament growth pattern of G. pacifica differs from that in the villous-type thalli of G. rugosa, which possessed two different kinds of assimilatory filaments: long (5- to 12-celled) and short (2- to 3-celled) assimilatory filaments (for details, see Figure 6d in Wang et al. 2005). The swollen basal cell from which the assimilatory filaments of G. rugosa are derived was not observed in the villous branches of G. pacifica. For the glabrous-type thalli, young glabrous branches are issued from the tip of villous branches (Figure 5L). The cortical structure transforms from the early production of assimilatory filaments to the subsequent production of a 3-celled cortex (Figure 5M-5N). Following transformation, the medullary filaments become less compact in the glabrous branch compared to those in the villous branch (Figure 5M-5N).
Reproductive structure
Tetrasporangia were not observed in our materials. Plants are dioecious. All reproductive structures are scattered throughout the glabrous branches (Figure 5D) and located in the boundary between the cortex and the medulla. Both female (i.e., cystocarp) and male structures develop to form a conceptacle at maturity.
Cystocarps are scattered over the fertile thallus except for the basal part of the glabrous branches (Figure 5D). During cystocarp development, young carpogonial branches are often 3-celled and consist of the carpogonium, the hypogynous cell, and the basal cell (Figures 8A, 9A). A 4-celled carpogonial branch with one additional basal cell is occasionally observed in our materials (Figure 9D). Three different types of carpogonial branch initiation were observed near the branch tip. The first type replaces an ordinary vegetative filament (Figures 8A, 9A). The second type arises between the dichotomous ordinary filaments (Figures 8B, 9B). The third type is a combination of the first and the second types of growth patterns (Figures 8C, 9C). When carpogonial branches of the third type becomes fertilized, neighboring carpogonial branches stop developing. However, fate of these abortive carpogonial branches remains unclear. Before fertilization, three to four sterile branches arise from the hypogynous cell (Figures 8C, 9D-9E). As the carpogonial branch matures, the hypogynous cell and its derived sterile branches enlarge and become darkly stained (Figures 8D, 9D-9E). Their single nuclei become extremely dark. The basal cell cuts off several involucral filaments from the cells, the nuclei of the basal cell and its derived filaments do not enlarge (Figures 8D-8F, 9D-9E). Fertilization was not observed, but the gonimoblast initial is presumably produced from the fertilized carpogonium. Pit plugs linking the inner gonimoblast cells to the gonimoblast initial break down at an early stage of carposporophyte development (Figure 8E-8F), but the hypogynous cell, the cells of the sterile branches, and the basal cell still remain distinct and retain their relative positions throughout cystocarp development (Figure 8E-8F). The involucral filaments from the basal cell do not form the pericarp and are restricted to the base throughout the development of the cystocarp (Figure 8D-8F). Ultimately, seven to ten inner cells of the gonimoblast filaments and the basal gonimoblast cell are incorporated into a multinucleate fusion cell in the mature carposporophyte (Figure 8E-8G). Mature cystocarps reach 400-800 μm in diameter (Figure 8H). The secondary gonimoblast filaments derived from the primary gonimoblast filaments produce terminal oval to obovate carposporangia, 12-45 μm by 38-80 μm (Figure 8G-8H). New carpospores sometimes arise from the remnant walls of previously shed carposporangia.
During development of male structure, the spermatangial branch initial arises in place of one of the cortical filaments (Figure 10A). Subsequently, the young spermantangial branch divides laterally or transversely to give rise to primary spermantangial filaments (Figure 10B). These primary spermatangial filaments further divide to produce numerous spermantangial filaments (Figure 10C) that grow into a cluster of highly branched spermantangial filaments (Figure 10D). Eventually, they form a hemispherical conceptacle (Figure 10E), 180-240μm in diameter at maturity (Figure 10F). Numerous secondary spermatangial filaments are issued from the inner side of the conceptacle and produce spermatangial mother cells terminally (Figure 10F). One spermatangium, 4-6 μm in width and 8-10 μm in length, is cut off terminally from the secondary spermatangial filament (Figure 10G).
Remarks
Tanaka (1936) and Tseng (1941) reported the occurrence of Galaxaura rudis Kjellman in Taiwan (as Formosa) and Hainan Island, respectively. Based on their descriptions, the specimens from these two localities showed undifferentiated non-tumid basal cells (i.e., supporting cells of assimilatory filaments). Thus, both authors argued that these specimens were morphologically distinct from G. rudis. Afterward, when Chou (1945) proposed the new species, G. filamentosa, she suggested that the specimens from Taiwan and Hainan Island should be treated as synonyms of G. filamentosa because their cortical structures are extremely similar. This study shows that the cortical structure of the specimen from the Philippines is highly similar to that of G. filamentosa from Taiwan and Hainan Island. The size of G. filamentosa from these three locations is small and ranges between 2 and 4 cm (more often less than 2.5 cm) in height. It is also noteworthy that Svedelius (1953) showed that the gross morphology of G. filamentosa from Hawaii comprises two different types, the tufted type and the freely growing type. Most tufted-type specimens were 1.5 cm high and crowded together. In contrast, the freely growing type could grow up to 3 cm in height. Consistent with the observations on G. filamentosa (as G. rudis) by Tanaka (1936) and Tseng (1941), most G. filamentosa specimens from Hawaii examined by Svedelius (1953) are “obtuse at the apex”. Svedelius (1953) also reported that G. filamentosa lacks the sunken growing point at the tip of branch and lacks fertile structures. This study shows that these unique morphological features may not be surprising when considering that G. filamentosa might be the remaining part of the tufted villous branches of G. pacifica. It will be interesting to test this hypothesis if G. filamentosa-type plants are more common in the tropical region of the Pacific Ocean while G. pacifica-type plants are more common in the subtropical or temperate region of the Pacific Ocean.
Although the specimen from the Philippines had a cortical structure similar to that of G. filamentosa, the type locality of G. filamentosa in Mexico (southeastern Pacific coast), is far from Taiwan, Hainan, and the Philippines (northwestern Pacific coast). Furthermore, the thallus size of G. filamentosa recorded in Chou (1945) [3.5-5 cm] is generally larger than our specimen from the Philippines, which was only 2.5 cm in height. Such a variation of plant size might be caused by ambient environmental factors such as light or temperature. However, without verification based on molecular analyses of G. filamentosa specimens obtained from its type locality, we can at best only propose that G. filamentosa might be the same species as G. pacifica from the northwestern Pacific Ocean.