The Ceramiales is the most varied order among red algae (ca. 420 genera, 2,660 species) (Guiry and Guiry 2015), but only a few genera have been found from freshwater habitats. The genus
The closely related
We have investigated the
Collection details are shown in Fig. 1A map and Table 1. The Chalakkudy River general habitat and the collectors, L. Jose and E. K. Ganesan, are shown in Fig. 1B and the typical habit of a field collected plant is in Fig. 1C. Herbarium specimens of
[Fig. 1.] (A) Map of Kerala, India showing Periyar and Chalakkudy River collecting sites. (B) Ganesan and Jose collecting at Ezhattumugham, Chalakkudy River in 2013, showing solid rock substrate and topography. (C) Habit of field specimens from Ezhattumugham, Chalakkudy River.
Caloglossa beccarii collection locations, substrates, temperatures, salinities, reproduction, collectors, and dates, Kerala, India
Isolation and culture methods are similar to those used in earlier work (West et al. 2001, West 2005). Irradiance was 10 : 14 h diurnal light-dark, cool white LEDs or cool white fluorescent lamps (3-12 μmol photons m-2 s-1) and temperature 18-22℃ air-conditioning. Although the field specimens were collected in very low salinity (< 0.5) we cultured them in salinities of 2 or 5 with standard modified Provasoli’s medium (MPM) supplemented with peat extract to mimic tannin levels in tropical freshwater rivers. Sterile peat extract was prepared as 500-mL MilliQ water, 15 g peat moss (commercial garden package), steam sterilized for 30 min, cooled, vacuum filtered with two sheets of Whatman No. 1 (9 cm), re-sterilized and stored at 4℃. The extract fluid was clear and dark brown. One litre of culture medium was prepared as 975-mL MilliQ water, 5 mL peat extract, 10 mL seawater (salinity of 30), and 10 mL MPM nutrient enrichment. This has been useful for culturing many red algae from freshwater / low salinity habitats.
The DNA extraction method, polymerase chain reaction (PCR) conditions, and sequencing procedure followed Hayakawa et al. (2012); specifically PCR and sequencing were carried out on the large subunit of rRNA (LSU) gene and the large subunit of RuBiSCO (
The information of primers for PCR and sequencing
The methods for alignment of sequence data and construction of phylogenetic trees were the same as those described by Kamiya and West (2014). Adjustments of the resulting alignments were performed manually, resulting in 1,329 bp for LSU and 1,417 bp for
Thalli collected in the Periyar River on February 14, 2014 were growing on shallow rocks, dark red to brown, forming densely branched tufts up to 2 cm high (Figs 1C & 2A). The blades branched subapically and were linear to narrowly lanceolate, slightly constricted at the nodes, up to 4.5 mm long (Fig. 2B). Blade width was 50-160 μm at the upper part, 90-320 μm at the middle part, and 40-120 μm at the lower part. Initials of lateral axes were frequently seen not only at the first but at the second or third nodes because of their slower elongation than the main axes (Fig. 2C). The numbers of cell rows around the node, which are the important characters for identifying
[Fig. 2.] Field specimens collected from Chalakkudy River, Kerala, India on November 24, 2012. (A) Whole thallus. Several rhizoidal filaments are produced from the ventral side of the nodes (arrowheads). (B) Slightly constricted node. (C) Part of thallus at second node from apex. Lateral axis (large arrow) is still diminutive. Nodal cell (diamond) produces one cell row (small arrow) toward main axis side. First axial cell of lateral axis (triangle) forms one cell row on adaxial side. First axial cell of the main axis (star) produces two cell rows (arrowheads) opposite lateral blade. (D) Rhizoidal filaments (arrowheads) produced from pericentral cells at node. (E) Adventitious blade initials (arrows) developed from lateral pericentral cells of first axial cell above node. (F) An adventitious blade initial (arrow) derived from internodal marginal cell. (G) Immature tetrasporangia (arrows). Scale bars represent: A, 500 μm; B, 200 μm; C, E & G, 50 μm; D & F, 100 μm.
Both strains (4840 from Chalakkudy River and 4841 from Periyar River) were cultured, initially in a salinity of 5 at low photon fluence rates (3-5 μmol photons m-2 s-1) for 2-3 months and then shifted to >10 μmol photons m-2 s-1 for 4 months. Isolate 4840 grew more slowly than 4841 and did not reproduce in over one year of culture. By contrast 4841 grew robustly, plants reaching >20 mm in 3 months (Fig. 3A). After four months robust tetrasporangial sori developed (Fig. 3A & B) and many spores were discharged but germination was poor. However surviving sporelings (Fig. 3C) grew well and after 7 months male gametophytes developed spermatangial sori (Fig. 3D & E) with normal spermatia released. As in field specimens, adventitious blades of cultured blades were derived from internodal marginal wing cells of vegetative blade (Fig. 3F). Female gametophytes developed carpogonial branches along the midrib (Fig. 4A & C). Numerous carposporophytes developed in about one month (Fig. 4B & D) and discharged some carpospores from which a few sporelings developed. Also some ‘pseudocystocarps’ developed that did not form carpospores (Fig. 4E). Although at a salinity of 5 the thalli grew well and reproduced, the overall viability of spores was low. We attempted culturing field collected plants initially at a salinity of 1-2 but survival and growth were not as good as in a salinity of 5. It is puzzling that tetraspore and carpospore survival and germination were low at a salinity of 5.
[Fig. 3.] (A) Habit of culture 4841 showing branching, nodes and tetrasporangial sori. (B) Tetrasporangial sori (single row of sporangia on each side of midrib) showing developing, mature and discharged sporangia. (C) Young tetraspore germling with upper blade and rhizoids at base. (D) Mature male with spermatangial sori on many blades. (E) Blade tip with elongate spermatangial sori. (F) Adventitious blades from intermodal marginal wing cells of vegetative blade like that of field specimen, Fig. 2F. Scale bars represent: A & D, 1 mm; B, C, E & F, 100 μm.
[Fig. 4.] (A) Female trichogyne with numerous attached spermatia. (B) Female with numerous developing and mature carposporophytes. (C) Female with supporting cell (sc), carpogonial branch (prc) and trichogyne (arrowhead) on blade midrib. (D) Lateral view of young cystocarp with normal pericarp and gonimoblast with developing carposporangia visible underneath. Lightly stained with anilin blue. (E) Face view of pseudocystocarp with pericarp enclosing ostiole in center. Panels C, D, and E were treated with 10 s. 800 W microwave to shrink cells. Scale bars represent: A & D, 100 μm; B, 500 μm; C, 25 μm; E, 50 μm.
The two Indian specimens (India1 from Chalakkudy River and India2 from Periyar River) showed only one bp substitution in the LSU sequences. The LSU analyses revealed that these Indian specimens made a clade with
[Fig. 5.] Maximum-likelihood (ML) phylogeny of Caloglossa species inferred from the partial large subunit of rRNA gene sequences. Taenioma perpusillum (J. Agardh) J. Agardh was used as an outgroup. The bootstrap values for ML (>50%; left) and posterior probabilities for Bayesian inference (≥0.80; right) are presented for each branch. Accession number, strain number or sample name is shown in each parenthesis.
Although the freshwater
In both LSU and
This alga is similar to
A monophyletic relationship of