The mangrove algal flora of Guam and the Federated States of Micronesia has been poorly explored. We add to our knowledge of this region by observations of collections from these regions. This paper presents new and additional records of: Rhodophyta-
The region comprising Guam and the Federated States of Micronesia (including the islands of Chuuk, Kosrae, and Pohnpei) has abundant mangroves. Reports on the marine algae of the region focus mostly on algae from coral reefs (Hodgson and McDermid 2000, McDermid et al. 2002, Lobban and Tsuda 2003, Lobban and N’Yeurt 2006, Tsuda 2006, Tsuda et al. 2012). Consequently, algae associated with mangroves are not well recorded.
The two most well-known red algal genera associated with mangroves are
To better explore the biodiversity of these poorly explored algal habitats we investigated the mangrove-associated algae from Guam, Chuuk, Kosrae, and Pohnpei including the collection of live specimens. Most collections were placed in laboratory culture to investigate their reproductive biology (patterns of sexual and asexual reproduction) and molecular phylogeny.
In some of our algal cultures microscopic brown deposits formed on the algae and glass. The preliminary findings on the chemical constitution on these brown deposits are reported here. The low molecular weight carbohydrates (LMWCs) of
Methods for collection, isolation and maintenance have been presented previously (West and Zuccarello 1999, West 2005). Collection information on the specimens is given in Table 1.
DNA was extracted using a Chelex extraction method 242of Goff and Moon (1993). For
The LMWCs dulcitol, sorbitol and digeneaside were qualitatively and quantitatively analyzed in the
In some cultures microscopic brown deposits were observed on the algae and glass surfaces. These specimens (air-dried algae with brown deposits and glass coverslips placed in culture dishes for 4-8 weeks also with brown deposits) were mounted on double sided carbon impregnated conducting tape prior to a conducting film of carbon (approx. 400A in thickness) being applied to the surface of the samples in a vacuum evaporative coating unit to avoid charging effects. The JEOL JSM35 scanning electron microscope (Jeol Ltd., Tokyo, Japan) is equipped with an EDAX windowless energy dispersive lithium drifted silicon detector capable of semi-quantitative analysis of elements. The image capture system is the “Image Slave” designed specifically for scanning electron microscopy (SEM) operation. Spectra are collected using an accelerating voltage ranging from 15 to 25 kV. The higher kV enables reliable identification of elements present by exciting associated higher energy peaks (Kα and Kβ). Specimens were viewed in secondary and backscattered electrons and elemental X-ray data collected in specific areas of interest using an area scan mode for an average composition of a given area or spot mode that collects X-rays from approximately 1 μm2. A complete explanation for X-ray microanalysis is in Heinrich (1981).
In Guam, Chuuk, Kosrae, and Pohnpei we collected a total of 15 taxa. These are presented below taxonomically and in Table 1.
Preliminary molecular analysis showed this to be a
Identification was based on the morphological description of the species in Børgesen (1915), but molecular evidence is clearly needed to verify its identification.
Growth of all phases of isolates was good in culture with typical alternate or unilateral branching. All plants
[Fig. 1.] Colaconema sp. 4651. (A) Original spore (arrowhead) with basal system shoot and erect shoot bearing fan shaped branches and some terminal sporangia (arrows). (B) Terminal sporangial cluster. Mature sporangium (arrowhead). (C) Habit view of thallus showing elongate branched horizontal filaments with erect flabelliform shoots. (D) Young flabelliform branches with terminal sporangia (arrows) on short laterals. (E) Well-developed mature flabelliform erect shoot separated from horizontal filament. Scale bars represent: A-E, 10 μm
[Fig. 2.] Acrochaetium globosum 4626 (A-D) and Caulacanthus indicus 4625 (E-G). (A) Mature thallus with basal system and erect shoots usually bearing unilateral branches and sporangia. (B) Cells showing single parietal chloroplast with a centrally projecting pyrenoid. (C) Small clusters of sporangia (8 μm diam. × 12 μm long) borne on short unilateral branches. (D) Six-day-old sporelings, 3 cells long with empty spores at base. (E) Habit showing the horizontal stolons and short erect shoots. (F) Erect shoot apex with single apical cell, spermatangial sorus surrounding the branch. (G) Small polygonal cortical cells of mature branch. Scale bars represent: A, 80 μm; B & C, 40 μm; D, 10 μm; E, 1 mm; F & G, 50 μm.
[Fig. 3.] Bostrychia kelanensis 3075 (A-C), 4589 (D), and 4632 (E-G). (A) Habit of non-reproductive tetrasporophyte with alternate lateral branching and an attachment disc (arrow). (B) Multiple new shoots arising from cladohaptera attachment disc. (C) Microwave-treated branch showing 3-tier cells per pericentral cell. (D) Male gametophyte with continuous spermatangial sorus on upper branches. (E) Female gametophyte with short lateral branches bearing whorls of procarps. Spermatium attached to tip of carpogonium (arrow). (F) Tetrasporangial stichidia. (G) Tetraspore germlings with an initial single rhizoid developing into multiple branched rhizoids. Scale bars represent: A, 2 mm; B, 100 μm; C, 50 μm; D, 75 μm; E, 85 μm; F, 120 μm; G, 60 μm.
[Fig. 4.] Maximum-likelihood phylogeny of the RuBisCo spacer data of select Bostrychia moritziana / B. radicans isolates. Seven lineages marked (after Zuccarello and West 2003). Isolates from Micronesia (FSM) found in lineages 2, 6, and 7. * indicates PP values ≥0.95 and maximum likelihood bootstrap (ML BP) values ≥95%. Otherwise values presented as PP/ML BP.
grew to 2-3 cm overall and only occasionally producing cladohaptera (Fig. 3A & B). Three tier cells were produced per axial cell in all stages (Fig. 3C). Males of isolate 4589 produced compoundly branched spermatangial stichidia, 70-75 μm wide, along the upper shoots (Fig. 3D). The female shoots of 4632 were 85-100 μm diameter. Branching was usually alternate at 0.5-1 mm intervals (Fig. 3A). Procarps were borne in irregular whorls along upper shoots and laterals (Fig. 3E) with trichogynes up to 220 μm long and about 8 μm wide.
Tetrasporangial stichidia of 4632 were variable in length and had irregular numbers of tetrasporangia (Fig. 3F). Sporelings were about 60-70 μm wide reaching about 0.5-1.0 mm in length before branching (Fig. 3G). As sporelings grew their bases produced numerous coherent, branched, and uniseriate rhizoids (Fig. 3G).
Males had lateral branches with single spermatangial stichidia (Fig. 5C), or some had compound-branched lateral stichidia. Females bore procarps in series along the apices of polysiphonous lateral branches (Fig. 5D). Selffertilization was typical in culture with many spermatia attaching to trichogynes (Fig. 5D, arrow in center) and well developed cystocarps (Fig. 5D) releasing carpospores that germinated to form tetrasporophytes.
Cladohaptera were typical on most isolates in culture, although in 4607 the cladohaptera usually had free rhizoids and new shoot also arising from the tips of the cladohaptera (Fig. 5E). We do not know if this occurs only in culture.
Another isolate of lineage 2 (4619) also had typical polysiphonous shoots, monosiphonous secondary laterals and procarps with elongate trichogynes (Fig. 6A).
By contrast, isolate 3001, also from lineage 2, has produced for 24 years successive generations of asexual tetrasporophytes with monosiphonous filaments that begin to form short polysiphonous terminal tetrasporangial stichidia (Fig. 6B). These stichidia developing from monosiphonous shoots are similar to tetrasporangial stichidia of
Another lineage 2 isolate (4591) produced successive generations of branched monosiphonous filaments bearing terminal segments with asexual tetrasporangial stichidia (Fig. 6C) for seven years. Isolate 4596 from lineage 2 was also primarily monosiphonous, but when reproductive, developed terminal polysiphonous segments with procarps bearing elongate trichogynes (arrows in Fig. 6D). Isolate 3003 from lineage 6 produced successive polysiphonous generations of asexual tetrasporophytes for 19 years.
Two isolates from lineage 7 were quite different from each other in morphology and reproduction. Isolate 4611 was a polysiphonous tetrasporophyte from which spores produced polysiphonous females, whereas 4631 was primarily monosiphonous, producing polysiphonous shoot tips with tetrasporangial stichidia and spores resulting in successive generations of tetrasporophytes. All these isolates are identical in RuBisCo spacer sequences.
Reproductive patterns of the remaining isolates are shown in Table 1. Some isolates (e.g., 4597) lacked any form of sporic reproduction, producing new thalli by fragmentation.
In all isolates the LMWC analyses showed sorbitol and digeneaside, but no dulcitol (Table 2).
Alternate lateral branches arose at variable intervals of 2-10 axial cells (Fig. 8A). Pericentral cells and tier cells arose at the 5th-6th axial cell (Fig. 8B). Plants were ecorticate with whorls of 4-7 pericentral cells each having 2 tier cells. Peripherohaptera usually developed at the nodes of branches or without opposite branches (Fig. 8C). The lateral branches were polysiphonous or partially monosiphonous in field specimens.
The H3 lineage of
[Fig. 5.] Bostrychia moritziana / B. radicans, lineage 2, isolate 4607. (A) Habit of cultured specimen. (B) Tetrasporophyte with sporangial stichidia on a cultured specimen. (C) Male gametophyte with spermatangial stichidia, lower one releasing spermatia. (D) Female gametophytes. On the left are unfertilized procarps with trichogynes (arrows). Center branch has fertilized procarp with spermatia around the trichogyne, 2-week-old cystocarp (arrowhead) developing a pericarp. On the right is branch with two 5-week-old cystocarps showing the darkened carposporangial masses. (E) Cladohaptera often developed differently from most B. moritziana / B. radicans isolates. Individual pericentral cells at tip become dissociated instead of remaining firmly coalesced. The lateral branch tip has a new indeterminate compound branch (arrow). Scale bars represent: A, 5 mm; B, D & E, 80 μm; C, 70 μm.
[Fig. 6.] Bostrychia moritziana / B. radicans, lineage 2. (A) Isolate 4619. Female with typical polysiphonous branches and procarps (arrows show trichogynes). (B) Isolate 3001. Monosiphonous and polysiphonous branches of asexual tetrasporophyte. (C) Isolate 4591. Asexual tetrasporophyte, apical branching of monosiphonous shoots. Arrowhead indicates transition from mono- to poly-siphonous sector, arrow indicates apical meristem of branched monosiphonous shoot. (D) Isolate 4596. Monosiphonous branches and terminal polysiphonous sectors with procarps (arrows show trichogynes). Scale bars represent: A & B, 75 μm; C & D, 70 μm.
[Fig. 7.] Bostrychia radicosa. (A) Habit view of isolate 4663 with lax branching of long horizontal filaments. (B) Isolate 4614 node with 3 rhizoids and single erect shoot. (C) Isolate 4614. Rhizoid clearly showing central nucleus and peripheral plastids. (D) Isolate 4621 terminal tetrasporangial stichidium. (E) Isolate 4621 male with terminal spermatangial stichidium and discharged spermatia. (F) Isolate 4621 female with terminal cystocarp and released carpospores. Procarps with trichogynes (arrows) along a lateral branch. (G) Isolate 4621. Carpospore germlings with rhizoid and erect shoot. Scale bars represent: A, 3 mm; B, 42 μm; C, 24 μm; D, 50 μm; E, 40 μm; F & G, 45 μm.
[Fig. 8.] (A-D) Bostrychia simpliciuscula isolate 4636. (E-H) B. tenella Isolate 4622 (E & F), isolate 4662 (G & H). Isolate 4622 (E & F), isolate 4662 (G & H). (A) Habit showing alternate / bifurcate branching and nodal and intercalary peripherohaptera. (B) Branch apex and subapical branching. Pericentral and tier cells developing at the fifth-sixth axial cell. (C) Nodal and intercalary peripherohaptera both showing coalescent cells from several tier cells becoming free as individual rhizoids elongate. (D) Manganese deposits (arrows) on cell surfaces. (E) Field specimen, heavily corticated and branched, some monosiphonous laterals, large peripherohaptera (arrowhead). (F) Field specimen, narrow, reduced branching, light cortication, no peripherohaptera. (G) Grown on shaker with bright light, showing good cortication, monosiphonous laterals, peripherohaptera (arrows). (H) Small manganese deposits on branches. Scale bars represent: A, 1 mm; B & C, 60 μm; D, 20 μm; E & F, 100 μm; G, 24 μm; H, 18 μm.
and digeneaside (Zuccarello et al. 1999
Most isolates remained vegetative in culture. Only two (4595 and 4603) developed tetrasporangial stichidia in which most sporangia were abortive. A few spores were released, and sporelings developed normally, but their final reproductive status was not determined.
SEM elemental analyses were done on microscopic dark brown deposits that frequently occurred on the alga and on glass surfaces in cultures of 4636 (Fig. 8D). These dark brown bodies contained manganese, sulphur, magnesium, aluminium, chlorine, potassium, and bromine (Fig. 9B). Branch surfaces of
corticated main axes and partly monosiphonous laterals, 5-8 pericentral cells per axial cell, two tier cells per pericentral cell and conspicuous peripherohaptera at some branch nodes. Molecular evidence showed
Field specimens of 4622 showed two distinct morphologies: 1) wide, densely branched with some monosiphonous laterals, heavy cortication and frequent peripherohaptera (Fig. 8E), and 2) narrow, reduced branching, light cortication and absence of peripherohaptera (Fig. 8F). The more lax branching and reduced cortication may be due to lower light levels from self-shading and sediment cover, but this needs to be investigated further.
A similar pattern of variable morphology was visible in other specimens (e.g., 4662). Plants grown in stationary culture with low light (<3 μmol photons m-2 s-1) had reduced and somewhat irregular branching, light cortication and sparse small peripherophaptera. In shaker culture (50-60 rpm) and brighter light (6-8 μmol photons m-2 s-1) more frequent branching with heavier cortication, monosiphonous terminal segments, robust peripherohaptera (Fig. 8G) and occasional procarps were observed. No other reproduction was seen. The other four isolates were not reproductive in culture.
Microscopic brown deposits similar to those on
[Fig. 9.] Bostrychia simpliciuscula 4636. (A) Scanning electron microscopy (SEM) elemental analyses of algal cell surface. Sulphur and potassium levels very high, manganese is not evident. (B) SEM elemental analyses of dark brown deposit with highest peak of manganese and somewhat lower peaks of sulphur, aluminium, magnesium, potassium, chlorine, and bromine. Note the comparative levels of the different elements on the algal cell surface and the brown deposit. Kα and Kβ designate different energy states for each element.
Analyses showed results similar to those of
The blades (0.9-3.0 mm long and 50-250 μm wide) of cultured specimens were usually not constricted at the nodes (Fig. 10A). Secondary adventitious branches frequently arose from the marginal cells in the thallus plane, and additional adventitious branches developed from these secondary branches in the same way (Fig. 10B). Endogenous branches were not observed. Single rhizoidal filaments, 20-30 μm in diameter and up to 800 μm long, were derived from the nodal pericentral cells or the adjacent pericentral cells immediately above and below the node. Although the number of internodal cell rows was up to six in the field-collected specimens, the cultured specimens tended to be more slender and usually had one or two internodal cell rows (Fig. 10B). The axial cells around the node produced one cell row to both sides (Fig. 10B).
Reproductive structures were observed in culture. Formation of tetrasporangia took place in acropetal succession and from the lateral pericentral cells toward the margins of the blade (Fig. 10C). Sporangial sori (150-800 μm long, 110-150 μm wide) were produced on
[Fig. 10.] Caloglossa ogasawaraensis. (A) Habit photograph of cultured specimen (4628). (B) Enlarged view of node (4628) showing secondary adventitious branches (arrows) derived from lateral pericentral cells. (C) Branches of tetrasporophyte (4604) bearing long rows of tetrasporangia along the central axis. (D) Branches of male gametophyte (4604) bearing spermatangial sorus. (E) Pseudocystocarp (arrow) on a female (4623). Many trichogynes are visible (arrowheads). (F) Mature cystocarp on a female (4604). Scale bars represent: A, 200 μm; B, C & F, 100 μm; D & E, 50 μm.
both sides of the midrib in the upper part of the blades. Mature tetrasporangia, 40 to 55 μm in diameter, were divided cruciate-decussately or tetrahedrally. Tetraspores germinated into male and female gametophytes, and several females developed carposporophytes. Spermatangial sori were 200-550 μm long and 140-220 μm wide and were found on both sides of the midrib at the upper and middle parts of the blades (Fig. 10D). Spermatangial mother cells (3.8-7.5 μm in diameter) cut off three to five spherical spermatangia (2.5-5.0 μm in diameter) toward the outer surface by anticlinal divisions. Many carpogonial branches with elongate trichogynes were produced in a line along a central axis (Fig. 10E). Cystocarps were oblate-ovate, 230-350 μm in height, 230- 390 μm in diameter with a narrow ostiole and contained many carposporangia, 50-110 μm in diameter (Fig. 10F). Sometimes pseudocystocarps with a pericarp but lacking a gonimoblast occurred in the absence of males (Fig. 10E).
In the large subunit (LSU) rRNA gene tree, the entities of
[Fig. 11.] Maximum-likelihood (ML) phylogeny of Caloglossa inferred from the partial large subunit rRNA gene sequences using Taenioma perpusillum as an outgroup. * ML bootstrap (BP) values ≥95% and posterior probabilities (PP) for Bayesian inference ≥0.95. Otherwise ML BP (>50%; left) and PP (≥0.80; right) are presented for each branch. Strain numbers and localities are shown with species epithets.
Ulvophyceae, Bryopsidales, Udoteaceae.
Numerous algal records for Micronesia by Hodgson and McDermid (2000), Lobban and Tsuda (2003), Lobban and N’Yeurt (2006), McDermid et al. (2002) and Tsuda (2006) include various
[Fig. 12.] (A) Boodleopsis carolinensis isolate 4605. Habit photo showing dichotomous to polychotomous branching and scattered sporangia. (B) Boodleopsis carolinensis isolate 4624. Sporangium (130 × 200 μm) with partially collapsed stalk and some spores visible around the periphery. Colorless refractive starch containing amyloplasts (arrow) visible in filament. (C) Dictyotopsis propagulifera, Lehn Mesi River, Pohnpei. Habit photo showing typical branching. Scale bars represent: A & C, 1 mm; B, 25 μm.
This report expands information on species diversity in the islands of Chuuk, Pohnpei, and Kosrae) in Micronesia and neighbouring Guam. While extensive field work has been done in these areas (e.g., Lobban and Tsuda 2003, Tsuda 2006), the small algae associated with mangroves are often overlooked. These algae are common in mangroves around the world and add to the diversity and ecology of these ecosystems. While some of these algae are probably opportunistic epiphytes (
The taxonomy and molecular phylogeny of
This is the first report of a
Culturing isolates from the field collections is necessary for most critical observations on growth and reproduction. Even though standard culture conditions are usually satisfactory and we have tested various levels of light, temperature, salinity, water motion and nutrients it is not possible to simulate the daily tidal and salinity patterns of typical mangrove habitats. It is quite possible that these ecological factors affect growth, reproduction and many biochemical patterns.
Thirty-one isolates of
In the six
Of the seventy-eight
We have thirty-eight
We obtained seven isolates of
Microscopic dark brown deposits are seen in some cultures from their initial establishment and could not be removed with bacterial antibiotics (Penicillin G, Ciprofloxacin, Rifampin, and Rocephin). These brown deposits are found in greater abundance in cultures that are slow growing. Occasionally they appear to impair the growth of the alga but usually do not. We used electron-scatter SEM microscopy to determine the elemental composition of these brown deposits that have high levels of manganese as well as sulphur, magnesium, aluminium, chlorine, potassium, and bromine in lesser amounts, whereas branch surfaces of
Manganese deposits were reported on the basal stalks of the freshwater Eustigmatophycean alga