New “missing link” genus of the colonial volvocine green algae gives insights into the evolution of oogamy
© Nozaki et al.; licensee BioMed Central Ltd. 2014
Received: 18 December 2013
Accepted: 25 February 2014
Published: 3 March 2014
The evolution of oogamy from isogamy, an important biological event, can be summarized as follows: morphologically similar gametes (isogametes) differentiated into small “male” and large “female” motile gametes during anisogamy, from which immotile female gametes (eggs) evolved. The volvocine green algae represent a model lineage to study this type of sex evolution and show two types of gametic unions: conjugation between isogametes outside the parental colonies (external fertilization during isogamy) and fertilization between small motile gametes (sperm) and large gametes (eggs) inside the female colony (internal fertilization during anisogamy and oogamy). Although recent cultural studies on volvocine algae revealed morphological diversity and molecular genetic data of sexual reproduction, an intermediate type of union between these two gametic unions has not been identified.
We identified a novel colonial volvocine genus, Colemanosphaera, which produces bundles of spindle-shaped male gametes through successive divisions of colonial cells. Obligately anisogamous conjugation between male and female motile gametes occurred outside the female colony (external fertilization during anisogamy). This new genus contains 16- or 32-celled spheroidal colonies similar to those of the volvocine genera Yamagishiella and Eudorina. However, Colemanosphaera can be clearly distinguished from these two genera based on its sister phylogenetic position to the enigmatic flattened colonial volvocine Platydorina and external fertilization during anisogamy. Two species of Colemanosphaera were found in a Japanese lake; these species are also distributed in European freshwaters based on a published sequence of an Austrian strain and the original description of Pandorina charkowiensis from Ukraine.
Based on phylogeny and morphological data, this novel genus exhibits a missing link between Platydorina and the typical spheroidal colonial volvocine members such as Pandorina or Yamagishiella. Considering the external obligate anisogamy, oogamy evolution may have been preceded by the transition from external to internal fertilization during anisogamy within the volvocine green algae.
The volvocine algae from unicellular Chlamydomonas to multicellular Volvox represent an “evolutionary time machine” model lineage to study the evolution of female–male sexual dimorphism and multicellularity because they encompass the evolutionary ranges of vegetative and reproductive morphologies between these two extremes [1, 2]. Based on chloroplast multigene phylogeny, a major evolutionary scenario within the lineage was resolved ranging gradually from primitive unicellular organisms with conjugating gametes of identical size (isogamy) to the advanced multicellular spheroid with differentiation of male–female sexes (oogamy) such as Volvox[3–5]. The colonial volvocine algae show two types of gametic union: conjugation between isogametes outside the parental colonies (external fertilization during isogamy) and fertilization between small motile male gametes (sperm) and large female gametes (eggs) inside the female colony (internal fertilization during anisogamy and oogamy). Although recent cultural studies on the volvocine algae revealed morphological diversity and molecular genetic data of sexual reproduction [6–9], an intermediate between these two gametic unions has not been demonstrated.
The colonial volvocine algae include two morphologically unique organisms: Volvox sect. Volvox with thick cytoplasmic bridges between cells and Platydorina with flattened vegetative colonies developing via unique morphogenesis “intercalation” . However, their phylogenetic positions are ambiguous even using multiple chloroplast genes [4, 5], possibly due to the lack of closely related sister lineages that may represent their ancestral morphological traits. Thus, identifying the missing links may resolve these problems. Although Coleman  confirmed that the Eudorina cylindrica strain ASW05157 (originating from Regelsbrunn, Austria ) is sister to Platydorina, its morphological details were not reported; however, the internal transcriber spacer (ITS) of nuclear ribosomal DNA (rDNA) of this strain is now available (AF182439).
During our field surveys of freshwater algae in Japan, two colonial volvocalean green algae were collected from a lake. Although these algae are closely related to Platydorina in molecular phylogeny, their vegetative colonies are spheroidal in shape, resembling those of the colonial volvocine genera Yamagishiella and Eudorina (Volvocaceae) under a light microscope . Notably, this new genus showed “external fertilization” between male and female gametes. Morphology, sexual reproduction, taxonomy, and phylogenetic significance of the two species of Colemanosphaera gen. nov. are described in this report.
Results and discussion
Morphology and taxonomy
The Austrian strain ASW05157 and C. angeleri 2010-0126-1 formed a small clade robustly separated from C. charkowiensis in the phylogenetic analysis of the ITS region of rDNA (Figure 4), and showed only one nucleotide difference in the ITS region (Additional file 2: Information S1). The ITS region of nuclear rDNA is rapidly evolving and shows nucleotide variation within a single biological species . Thus, ASW05157 unambiguously belongs to C. angeleri, although its morphological data are lacking.
Sexual reproduction was observed in C. charkowiensis (Figure 3) and generally occurred 2 days after possible male and female cultures were mixed in the nitrogen-deficient mating medium . Cells of the colony divided to form a bundle of 16 or 32 male gametes or sperm packets. The packet was almost spherical in shape, measured 15–17 μm in diameter (Figure 3A, B), and swam to the female colony, which was almost indistinguishable from the vegetative colony. The packet on the female colony eventually dissociated into individual, biflagellate male gametes. The male gametes were spindle-shaped to elongate-ellipsoidal in shape and measured 5–10 μm in length. A tubular cytoplasmic protrusion was observed near the base of the flagella of the male gametes (Figure 3C, D), similar to male gametes of Eudorina elegans. During dissociation of the packet, biflagellate protoplasts or female gametes escaped from the gelatinous matrix of the female colony (Figure 3E-G). Female gametes were biflagellate and spherical in shape, measuring 13–17 μm in diameter. Female and male gametes fused outside the female colony (Figure 3H–K). The anterior end of the male gamete connected the anterior region of the female gamete, and the plasmogamy proceeded to form a quadriflagellate zygote. The zygotes then lost their flagella and secreted a reticulate cell wall (Figure 3L). The zygotes eventually turned reddish brown in color after about 1 week. The mature aplanozygotes were 14–18 μm in diameter (excluding reticulation).
The present multigene phylogeny demonstrated that Colemanosphaera is robustly sister to the unique flattened volvocacean genus Platydorina (Figure 2). Although these genera differ in the form of their vegetative colonies (spheroidal vs. flattened, respectively), they share fundamental morphological characteristics during the vegetative phase and sexual reproduction. The vegetative colonies of both genera are composed of 16 or 32 cells of identical size [7, 22, 24] (Figure 1). Two or three contractile vacuoles are limited to the anterior end of the vegetative cells in both genera (Figure 1; Additional file 1: Figure S7). Vegetative ultrastructures of these two genera were used to observe colonial boundaries and cellular envelopes  (Additional file 1: Figure S2). In addition, both genera show sperm packet formation and “external fertilization” between male and female motile gametes  (Figure 3). Thus, Colemanosphaera may exhibit ancestral morphological attributes of Platydorina, and a Colemanosphaera-like spheroidal colonial ancestor might have evolved to Platydorina by acquiring intercalation to form flattened vegetative colonies during embryogenesis.
Taxonomic consideration of species
Although small contractile vacuoles are distributed on the surface of the vegetative cells of Eudorina, Huber-Pestalozzi  and Ettl  characterized the genus Eudorina as having two anterior contractile vacuoles. Thus, some Eudorina species in which contractile vacuoles have not been studied carefully may actually belong to Colemanosphaera. Among the described species of Eudorina[19, 27], E. echidna and E. interconnexa have not been studied in culture. In addition, the strain of E. conradii is not available from the UTEX Culture Collection (http://web.biosci.utexas.edu/utex/). However, these three species of Eudorina can be clearly distinguished from the present two species of Colemanosphaera based on differences in vegetative morphology. These three Eudorina species have almost spherical vegetative cells [19, 29, 30]. In contrast, cells of two species of Colemanosphaera are ovoid, subspheroidal, or lenticular in shape with a broad anterior face that seems more or less angular by mutual compression (Figure 1). In addition, the three species of Eudorina are characterized by distinctive vegetative colony structures . E. conradii and E. interconnexa have species-specific individual gelatinous sheaths surrounding vegetative cells [19, 30], while E. echidna shows short conical projections covering the vegetative colony .
Thompson  observed conjugation between small male and large female gametes outside the female colony in “Pandorina charkowiensis.” However, male gametes are not elongate or spindle-shaped in this alga. Although P. charkowiensis contains multiple pyrenoids in the cup-shaped chloroplast in each vegetative cell  (Figure 1), information on pyrenoids of the alga observed by Thompson  is lacking in his description and figures. Therefore, the alga may be Yamagishiella unicocca, in which small and large gametes can fuse [15, 18].
Korshikov’s description of the alga from the Ukraine that he called Pandorina charkowiensis indicates that it is highly similar in vegetative morphology to the Japanese C. charkowiensis strains described here, in that it has subspheroidal cells with a broad anterior face, two contractile vacuoles, and multiple (up to seven) pyrenoids in a cup-shaped chloroplast with prominent longitudinal striations on its surface . He did not evaluate its sexual reproduction , but if he had, there is little doubt that he would have found that it was not isogamous like Pandorina. On the other hand, the other Colemanosphaera species (C. angeleri) contains more flattened vegetative cells and less prominent chloroplast striations than those of C. charkowiensis (Figure 1). Thus, C. angeleri represents a previously undescribed morphological species.
Two species of Colemanosphaera were found in a Japanese lake (Additional file 2: Table S1). As discussed above, comparison of ITS sequences demonstrated that the Austrian strain ASW05157 unambiguously belongs to C. angeleri (Figure 4). C. charkowiensis was originally described based on material originating from Ukraine . Thus, Colemanosphaera may be distributed in worldwide freshwater habitats. Although P. charkowiensis was reported from the United States [31, 33, 34], it may be Y. unicocca or Eudorina species because information on contractile vacuoles and/or pyrenoids is limited. Further detailed molecular and morphological studies of the Eudorina- and Yamagishiella-like colonial volvocine algae will resolve actual worldwide distribution and diversity of the new genus Colemanosphaera.
The present study unambiguously demonstrated external fertilization between male and female gametes using Colemanosphaera cultures. When such a type of gametic union is considered with previously published data , sexual reproduction of the volvocine green algae can be clearly distinguished into four types (with the exception of monoecism) (Figure 5): 1) External fertilization during isogamy with gametes directly released from colonial cells, 2) external fertilization during anisogamy with the formation of bundles of spindle-shaped male gametes (sperm packet formation), 3) internal fertilization during anisogamy (with flagellated female gametes retained within the parental female colony), and 4) internal fertilization during oogamy (with flagella-lacking female gametes formed within the parental female colony). Thus, oogamy evolution may have been preceded by the transition from external to internal fertilization during anisogamy within the volvocine green algae.
Colemanosphaera Nozaki gen. nov.
Vegetative colonies spheroidal in shape, composed of 16 or 32 cells of approximately identical sizes embedded at the peripheral regions of the gelatinous matrix forming a hollow colonial structure. The extracellular matrix of vegetative colonies exhibiting colonial boundaries and cellular envelopes. Each cell biflagellate, more or less angular in front view, containing a massive cup-shaped chloroplast, a stigma, and a nucleus. Two or three contractile vacuoles distributed near the base of the flagella. Chloroplasts containing longitudinal striations on the surface and multiple pyrenoids in mature cells. A gradual decrease in stigma size occurring from the anterior to posterior pole of the colony. Asexual reproduction accomplished by successive divisions of each vegetative cell, forming a square plakea undergoing typical inversion to produce a spheroidal daughter colony.
Type species: Colemanosphaera charkowiensis (Korshikov) Nozaki, T. K. Yamada, F. Takahashi, Matsuzaki et Nakada comb. nov.
Etymology: The generic name “Colemanosphaera” honoring Dr. Annette W. Coleman who contributed much to the species systematics of microalgae .
Colemanosphaera charkowiensis (Korshikov) Nozaki, T. K. Yamada, F. Takahashi, Matsuzaki et Nakada comb. nov.
Synonym: Eudorina charkowiensis (Korshikov) Pascher 1927 : 441.
Epitype here designated: Resin-embedded vegetative spheroids of 2013-0615-IC-7 (= NIES-3388) deposited in the herbarium of the Department of Botany, National Museum of Nature and Science (TNS), Tsukuba, Japan (TNS-AL-56994).
Strains examined: 2013-0615-IC-7, 2013-0615-IC-4, 2013-0615-IC-3, Isa 7–1, 2010-0713-E2, and 2010-0713-E5 (Additional file 2: Table S1).
Distribution: Ukraine , Japan.
Colemanosphaera angeleri Nozaki sp. nov.
Vegetative colonies 16- or 32-celled, cylindrical to elongate-ovoid in shape, measuring up to 95 μm long and up to 70 μm wide. Cells subspheroidal to lenticular in shape, measuring up to 20 μm in surface diameter. Chloroplasts in mature cells containing a large pyrenoid and one to five small pyrenoids. Two flagella in each cell of a newly formed daughter colony growing equally. Sexual reproduction not observed.
Holotype: Resin-embedded vegetative spheroids of 2010-0126-1 (= NIES-3382) deposited in the herbarium of the Department of Botany, National Museum of Nature and Science (TNS), Tsukuba, Japan (TNS-AL-56995).
Strain examined: 2010-0126-1 (Additional file 2: Table S1).
Etymology: The species epithet “angeleri” honoring Dr. David Angeler who studied the Austrian strain ASW05157 of this species .
Type locality: Lake Isanuma, Isanuma, Kawagoe-shi, Saitama, Japan (Additional file 2: Table S1).
Distribution: Austria , Japan.
Samples of Colemanosphaera were collected four times during the last several years from Lake Isanuma and its adjoining small pond, Isanuma, Kawagoe-shi, Saitama Prefecture, Japan (Additional file 2: Table S1). Clonal cultures were established using the pipette-washing method  either directly from water samples or from media obtained by rewetting a small amount of dried soil in a 90- × 20-mm Petri dish. The cultures were grown in screw-cap tubes (18 × 150 mm) containing ~11 mL of AF-6 medium  modified by Kasai et al.  or VTAC medium (containing 200 mg L−1 sodium acetate 4H2O [18, 38]) at 23-25°C on a 14-h light:10-h dark schedule under cool-white fluorescent lamps (color temperature = 4000–5000 K) at an intensity of 110–150 μmol∙m−2∙s−1. Vegetative colonies and asexual reproduction were observed by examining a small aliquot of cells grown continuously by inoculating ~0.5–1.0 mL of actively growing cells into fresh medium every 3–10 days.
To induce sexual reproduction, cultures grown in the modified AF-6 medium at about 25°C were concentrated from 11 to 0.5 mL by centrifugation. The concentrated possible male and female cultures were mixed with 10 mL of nitrogen-deficient “mating medium”  in a 60 × 10-mm Petri dish.
To examine the light microscopic details and the ITS region of nuclear rDNA, Platydorina caudata UTEX 1661 and NIES-728 (= UTEX 1658) were obtained from the Culture Collection of Algae at the University of Texas at Austin  (http://web.biosci.utexas.edu/utex/) and Microbial Culture Collection at the Institute for National Environmental Studies  (http://mcc.nies.go.jp/), respectively, and they were cultured in the modified AF-6 medium as described above. Light microscopy was performed using a BX60 microscope (Olympus, Tokyo, Japan) equipped with Nomarski optics. TEM was performed using 0.8% glutaraldehyde during pre-fixation as described previously .
Five chloroplast genes (atpB, rbcL, psaA, psaB, and psbC) and ITS region of rDNA (ITS-1, 5.8S rDNA, and ITS-2) of two species of Colemanosphaera and the ITS region of P. caudata UTEX 1661 and NIES-728 were sequenced as described previously [3, 40]. For phylogenetic analysis of chloroplast genes, the combined coding regions of the five chloroplast genes  from 58 operational taxonomic units (OTUs) of the colonial volvocine algae (Additional file 2: Table S2) were subjected to Bayesian inference using MrBayes 3.2.1.  as described previously . In addition, 1,000 replicates of bootstrap analyses  were performed by maximum-likelihood (ML) analysis with RAxML ver. 7.0.4  using a GTR model of each codon position in the five concatenated genes unlinked and by the maximum-parsimony (MP) method using PAUP 4.0b10 , as described previously . Because the third codon positions of the combined five genes showed heterogeneous base compositions among OTUs (p < 0.01) based on chi-square tests of PAUP 4.0, the third codon positions of psaB genes, which seemed to have the greatest effect on the heterogeneity among the five genes, were excluded to minimize heterogeneity (p > 0.05). The alignment (5,525 positions and 38 OTUs) used for the present phylogenetic analyses (Figure 2) is available from TreeBASE (http://treebase.org/treebase-web/home.html; study ID: S15331).
Goniacean and tetrabaenacean OTUs were designated as the outgroup because Volvocaceae is monophyletic and sister to the Goniaceae, and these two families and the Tetrabaenaceae form a large monophyletic group [3–5]. Because the colonial volvocine Astrephomene species (Goniaceae) showed possible saturation of the chloroplast genes  and were positioned outside the Volvocaceae [3, 5], we excluded this genus.
Nucleotide sequences for the ITS region of rDNA from Colemanosphaera (Additional file 2: Table S1), ASW05157, and the outgroup P. caudata UTEX 1661 and NIES-728 (accession numbers AB905587 and AB905588) were aligned using ClustalX . After refining the alignment based on the secondary structures of ITS-1 and ITS-2 [10, 48] and reducing identical sequences to a single OTU, the alignment (676 positions and five OTUs; study ID: S15331 available from TreeBASE) were subjected to MP and ML (based on HKY85 model) methods based on a branch-and-bound search by PAUP 4.0 with bootstrap analysis based on 1,000 replicates. The secondary structures of ITS-2 were predicted based on that of Platydorina and refined as described previously  using Centroidfold  and RNAfold at the RNAfold WebServer  (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi).
Compensatory base changes
Internal transcriber spacer
Operational taxonomic unit
Transmission electron microscopy.
This work was supported by Grants-in-Aid for Scientific Research on Innovative Areas “Allogeneic Authentication” (grant number 24112707 to HN), and Scientific Research (B) (grant number 25304012 to HN) from MEXT/JSPS KAKENHI.
All new sequences have been deposited to DDBJ/EMBL/GenBank (accession numbers: AB905580 - AB905598). Sequence alignments of the five chloroplast genes and the ITS region of nuclear rDNA have been deposited in TreeBASE (study ID: S15331).
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