Fig 1.
Map showing distribution of Gracilaria firma.
The white star denotes the type locality at Xuwen County in the Guangdong Province of China; black stars denote the general collection locations and do not correspond to sample size.
Fig 2.
Gracilaria firma Chang & Xia, G. changii (Xia & Abbott) Abbott, Zhang & Xia and G. fisheri (Xia & Abbott) Abbott, Zhang & Xia.
(A) Holotype of G. firma, AST 66–540, tetrasporophyte, collected on 2 April 1966 from Xindi, Xuwen County, Guangdong Province, China. Scale bar = 1 cm. (B) Isotype of G. firma, AST 66-540a, cystocarpic plant, collected on 2 April 1966 from Xindi, Xuwen County, Guangdong Province, China. Scale bar = 1 cm. (C) Holotype of G. changii, Fisher # 1022, tetrasporic and male plants, collected on 10 March 1983 from Middle Bank, Penang, Malaysia. (D) Holotype of G. fisheri, Fisher # 1087, cystocarpic plant, collected on 28 June 1983 near Pattani, Thailand.
Fig 3.
Phylogeny and haplotype network for Gracilaria firma based on the rbcL gene.
(A) Phylogeny of Gracilaria inferred from the rbcL gene alignment consisting of 1070 base pairs (bp) using the ML method (-lnL = 6707.49073). Numbers at the nodes denote ML bootstrap support and Bayesian posterior probabilities. Dashes denote ML bootstrap support or Bayesian posterior probabilities of less than 50% or 0.50, respectively. The numbers in parentheses after the taxon denote the number of specimens with identical sequences from each country. (B) Haplotype network for G. firma based on 1070 bp of rbcL. Circle sizes are proportional to haplotype frequency. Lines connecting the haplotypes represent single base pair mutations. Small black circles represent undetected or hypothetical haplotypes. Haplotypes are colored according to the geographic origin as shown in the key.
Table 1.
Variation sites in DNA sequences of Gracilaria firma for haplotypes based on the rbcL gene.
Fig 4.
Phylogeny and haplotype network for Gracilaria firma based on the cox1 gene.
(A) Phylogeny of Gracilaria inferred from the cox1 gene alignment consisting of 1118 base pairs (bp) using the ML method (-lnL = 5827.26951). Numbers at the nodes denote ML bootstrap support and Bayesian posterior probabilities. Dashes denote ML bootstrap support or Bayesian posterior probability less than 50% or 0.50, respectively. Numbers in parentheses after taxa denote the number of specimen with identical sequences from each country. (B) Haplotype network for G. firma based on the alignment of partial cox1 genes consisting of 834 bp. Circle size is proportional to haplotype frequency. Lines connecting the haplotypes represent single base pair mutations. Small black circles represent undetected or hypothetical haplotypes. Haplotypes are colored according to the geographic origin as shown in the key.
Table 2.
Variation sites in DNA sequences of Gracilaria firma for haplotypes based on the cox1 gene.
Fig 5.
Thalli habit and cross sections of liquid-preserved Gracilaria firma from different localities.
Note the common basal constrictions (arrowheads) of branches that taper to either acute apices or truncated tips that tend to regenerate new branchlets (arrows). (A) Tetrasporic plant from the Philippines (#00013). Scale bar = 2 cm. (B) Another tetrasporic plant with thick axes and dense branching from the Philippines (#00045). Scale bar = 2 cm. (C) Sterile plant from a cultivation farm in Taiwan showing intense branching with many newly regenerated branchlets (NTOU-KH-5i2016-Gf). Scale bar = 2 cm. (D) Sterile plant from a wild population in Malaysia as G. changii showing several branches with less robust branching arising from a single holdfast (PSM12881). Scale bar = 2 cm. (E) Transverse section through young vegetative branch of a tetrasporic plant from the Philippines (#00013). Scale bar = 0.25 mm. (F) Transverse section through young thallus of G. firma from Taiwan (NTOU-KH-5i2016-Gf). Scale bar = 0.25 mm. (G) Transverse section through young thallus of G. changii from Malaysia (PSM12881). Scale bar = 0.25 mm. (H) Transverse section through older thallus of G. firma from Taiwan (NTOU-KH-5i2016-Gf). The transition of cell size from the cortex to medulla is gradual in young branches and abrupt in older branches. Scale bar = 0.25 mm.
Table 3.
Morphological comparison of Gracilaria firma, G. changii and G. fisheri.
Fig 6.
Reproductive morphology of Gracilaria firma from the Philippines.
(A) Early stage of cystocarp development showing fusion cell (fc) and gonimoblast initial (arrowhead). Scale bar = 25 μm. (B) Young cystocarp section showing the fusion cell (fc) bearing gonimoblast cell clusters (arrowheads). Note the development of multinucleate tubular nutritive cells at the inner pericarp (arrow). Scale bar = 50 μm. (C) Mature cystocarp section showing multinucleate tubular nutritive cells (arrows) distributed around the floor. Scale bar = 200 μm. (D) Close-up of (C) showing the inconspicuous tubular nutritive cells (arrow) connecting the gonimoblast cells and inner pericarp cells at the floor. Scale bar = 100 μm. (E) Mature cystocarp showing a persistent fusion cell (arrowhead) and tightly packed small gonimoblast cells terminally bearing carpospores in straight chains. Scale bar = 200 μm. (F) Aborted cystocarp where the fusion cell (fc) has failed to produce gonimoblasts. Scale bar = 100 μm. (G) A young spermatangial conceptacle showing spermatangial parent cells (arrows) and spermatangia (arrowheads). Scale bar = 20 μm. (H) A developing spermatangial conceptacle showing spermatangial parent cells (arrows) and spermatangia (arrowheads). Scale bar = 20 μm. (I) Mature spermatangial conceptacle of the verrucosa-type lined by spermatangia (arrowheads) cut off from spermatangial parent cells (arrows). Scale bar = 20 μm. (J) An immature tetrasporangium embedded in the cortical layer of elongated cells showing pit-connection (arrowhead) to a subcortical cell. Scale bar = 20 μm.