Genetic and morphological analyses of Gracilaria firma and G. changii (Gracilariaceae, Rhodophyta), the commercially important agarophytes in western Pacific

Many studies classifying Gracilaria species for the exploitation of agarophytes and the development of the agar industry were conducted before the prevalence of molecular tools, resulting in the description of many species based solely on their morphology. Gracilaria firma and G. changii are among the commercially important agarophytes from the western Pacific; both feature branches with basal constrictions that taper toward acute apices. In this study, we contrasted the morpho-anatomical circumscriptions of the two traditionally described species with molecular data from samples that included representatives of G. changii collected from its type locality. Concerted molecular analyses using the rbcL and cox1 gene sequences, coupled with morphological observations of the collections from the western Pacific, revealed no inherent differences to support the treatment of the two entities as distinct taxa. We propose merging G. changii (a later synonym) into G. firma and recognize G. firma based on thallus branches with abrupt basal constrictions that gradually taper toward acute (or sometimes broken) apices, cystocarps consisting of small gonimoblast cells and inconspicuous multinucleate tubular nutritive cells issuing from gonimoblasts extending into the inner pericarp at the cystocarp floor, as well as deep spermatangial conceptacles of the verrucosatype. The validation of specimens under different names as a single genetic species is useful to allow communication and knowledge transfer among groups from different fields. This study also revealed considerably low number of haplotypes and nucleotide diversity with apparent phylogeographic patterns for G. firma in the region. Populations from the Philippines and Taiwan were divergent from each other as well as from the populations from Malaysia, Thailand, Singapore and Vietnam. Establishment of baseline data on the genetic diversity of this commercially important agarophyte is relevant in the context of cultivation, as limited genetic diversity may jeopardize the potential for its genetic improvement over time

Phylogenetic studies of the red algal parasite Congracilaria babae (Gracilariaceae, rhodophyta) / Ng Poh Kheng

Since the first description of red algal parasites in the last century, phylogenetic relationships between most of the red algal host-parasite associations have only been inferred based on the degree of morpho-anatomical similarity between the host and parasite. The limited morphological characters available for taxonomic inference had driven the use of molecular analyses to unravel the evolutionary relationships between red algal parasites and their host. Furthermore, the broad host range of certain red algal parasitic taxa and the possible occurrence of host-switching event can only be revealed using molecular data. In view of the lack of a clear classification scheme supported by molecular data that take into consideration Congracilaria babae, a parasitic alga found on Gracilaria salicornia, this study represents an initiative aimed to clarify the classification of the parasite, particularly the Malaysian samples. It also served to provide insights into the phylogenetic relationships between the parasites with their hosts by integrating the comparative DNA sequence analyses with morphological and anatomical observations. The possibility of these red algal parasitic taxa extending the host range from G. salicornia to other members of Gracilariaceae was also explored. Specimens of G. salicornia bearing red algal parasites were collected from various localities in Malaysia, Singapore, Indonesia, Thailand and Japan for examination. A similar parasitic taxon observed on a Hydropuntia species was also collected. The parasites found on both G. salicornia and Hydropuntia sp. have similar morphology and anatomy – they form pigmented pustules devoid of rhizoids, with spermatangia borne in deep conceptacles and projecting cystocarps with tubular filaments connecting to the pericarp scattering over surface of the pustules. Comparative phylogenetic analyses based on the DNA sequences of nuclear markers recovered a monophyletic clade comprising the red algal parasites found on G. salicornia and Hydropuntia sp.. Morpho-anatomical observations in concert with molecular analyses revealed that these parasites are conspecific, regardless of their host species. Staining reactions from the histological study, as well as the molecular phylogenies inferred from the DNA sequences of the genetic markers belonging to three different genomes (plastid rbcL gene, mitochondrial cox1 gene and nuclear LSU rRNA gene and ITS region), indicated that C. babae most likely have directly evolved from G. salicornia, and later extended its host range to a distantly related Hydropuntia species. Congracilaria babae featured DNA sequences characteristic of G. salicornia, of which the parasite found on G. salicornia had rbcL and cox1 gene sequences identical to those of the host it originated from, while retaining its unique nuclear identity. The position of C. babae in the same genetic species group as G. salicornia supports its transfer to Gracilaria. To conclude, molecular tools are useful in elucidating the phylogenetic relationships between red algal parasites, as well as that between the parasites and their hosts. The use of the plastid rbcL gene and mitochondrial cox1 gene complementary to the nuclear markers is advocated to trace the original host of a red algal parasite

Correction: Radiation of the Red Algal Parasite Congracilaria babae onto a Secondary Host Species, Hydropuntia sp. (Gracilariaceae, Rhodophyta).

[This corrects the article DOI: 10.1371/journal.pone.0097450.]

Radiation of the Red Algal Parasite <i>Congracilaria babae</i> onto a Secondary Host Species, <i>Hydropuntia</i> sp. (Gracilariaceae, Rhodophyta)

<div><p><i>Congracilaria babae</i> was first reported as a red alga parasitic on the thallus of <i>Gracilaria salicornia</i> based on Japanese materials. It was circumscribed to have deep spermatangial cavities, coloration similar to its host and the absence of rhizoids. We observed a parasitic red alga with morphological and anatomical features suggestive of <i>C. babae</i> on a <i>Hydropuntia</i> species collected from Sabah, East Malaysia. We addressed the taxonomic affinities of the parasite growing on <i>Hydropuntia</i> sp. based on the DNA sequence of molecular markers from the nuclear, mitochondrial and plastid genomes (nuclear ITS region, mitochondrial <i>cox</i>1 gene and plastid <i>rbc</i>L gene). Phylogenetic analyses based on all genetic markers also implied the monophyly of the parasite from <i>Hydropuntia</i> sp. and <i>C. babae</i>, suggesting their conspecificity. The parasite from <i>Hydropuntia</i> sp. has a DNA signature characteristic to <i>C. babae</i> in having plastid <i>rbc</i>L gene sequence identical to <i>G. salicornia</i>. <i>C. babae</i> is likely to have evolved directly from <i>G. salicornia</i> and subsequently radiated onto a secondary host <i>Hydropuntia</i> sp. We also recommend the transfer of <i>C</i>. <i>babae</i> to the genus <i>Gracilaria</i> and propose a new combination, <i>G. babae</i>, based on the anatomical observations and molecular data.</p></div

Phylogenetic relationships for host-parasite associations of <i>Congracilaria babae</i> from <i>Gracilaria salicornia</i> and <i>Hydropuntia</i> sp. inferred from ITS region.

<p>The –Ln likelihood was 16,797.503. Numbers above or below branches denote MP (left) and ML (middle) bootstrap values, and Bayesian posterior probability (right). Dashes indicate percentages<50% or that the node did not occur in the MP or BI tree. Asterisks indicate maximum bootstrap support or posterior probabilities. Brackets after species names indicate sample origins and sometimes isolate number: MR = Morib, PB = Pulau Besar, TP = Teluk Pelanduk, TS = Teluk Sari, and PBB = Pulau Bum Bum. Arrows indicate host-parasite associations; arrowheads indicate hosts.</p

Phylogeny of <i>Congracilaria babae</i> from <i>Gracilaria salicornia</i> and <i>Hydropuntia</i> sp. inferred from <i>rbc</i>L gene.

<p>The –Ln likelihood was 9,974.033.</p

<i>Congracilaria babae</i> Yamamoto on <i>Hydropuntia</i> sp.

<p>A: Habit of parasite on host thallus in herbarium press (PSM 12754), inset, a close-up of a parasite pustule (arrow). B: Habit of a female gametophyte preserved in formalin. C: Habit of a tetrasporophyte preserved in formalin. D: Transverse section of the host-parasite association, in which the parasite was lightly stained and the host, including the stalk-like structure was darkly stained. E: Transverse section showing abrupt transition of cell size from cortex to medulla of a vegetative parasite pustule. F: Transverse section showing densely staining fusion cell at the base of the developing pericarp. G: Transverse section showing a mature cystocarp with tubular filaments penetrating into the pericarp. H: Transverse section showing the <i>verrucosa</i> type of spermatangial conceptacles at the periphery of the thallus. I: Transverse section of a tetrasporangium. [A: scale bar = 1 cm, inset, scale bar = 1 mm; B, C: scale bar = 1 mm; D: scale bar = 500 µm; E, F, I: scale bar = 50 µm; G, H: scale bar = 100 µm].</p

Comparison of <i>Congracilaria babae</i> and its morphotypes reported from the Southeast Asian countries.

a<p>From the figures in the references; m. d., missing data.</p