BACKGROUND
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  <cit> .
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  to the advanced multicellular spheroid with differentiation of male–female sexes  such as volvox <cit> .
the colonial volvocine algae show two types of gametic union: conjugation between isogametes outside the parental colonies  and fertilization between small motile male gametes  and large female gametes  inside the female colony .
although recent cultural studies on the volvocine algae revealed morphological diversity and molecular genetic data of sexual reproduction  <cit> , 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”  <cit> .
however, their phylogenetic positions are ambiguous even using multiple chloroplast genes  <cit> , 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  <cit>  confirmed that the eudorina cylindrica strain asw <dig>  is sister to platydorina, its morphological details were not reported; however, the internal transcriber spacer  of nuclear ribosomal dna  of this strain is now available .
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  under a light microscope  <cit> .
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
morphology and taxonomy
two species of the new colonial volvocine genus colemanosphaera showed essentially the same colonial organization during the vegetative phase .
vegetative colonies were ovoid to ellipsoidal in shape in c. charkowiensis comb.
nov.  or cylindrical to elongate-ovoid in c. angeleri sp.
nov. .
they contained  <dig> or  <dig> cells of approximately identical size embedded at the peripheral regions of the gelatinous matrix, forming a hollow colonial structure.
the 32-celled colonies of c. charkowiensis measured up to 85 μm long and 67 μm wide, whereas those of c. angeleri were up to 95 μm long and 70 μm wide.
the cells were ovoid to subspheroidal in c. charkowiensis or subspheroidal to lenticular in c. angeleri, having a broad anterior face that was more or less angular by mutual compression, measuring up to 20 μm in surface diameter in both species .
each cell had two equal flagella, a massive cup-shaped chloroplast, a stigma, and two or three contractile vacuoles near the base of the flagella .
no other contractile vacuoles were distributed on the surface of the protoplast.
a gradual decrease in stigma size occurred from the anterior to posterior pole of the colony.
the chloroplast contained longitudinal striations on the surface , which were prominent in c. charkowiensis .
although the chloroplast of both species contained only a single pyrenoid in the immature cells, three to eight pyrenoids of almost identical size were distributed throughout the chloroplast of mature vegetative cells in c. charkowiensis , whereas the cup-shaped chloroplast in mature cells of c. angeleri contained a large single pyrenoid in the bottom, as well as one to five small pyrenoids .
the present two species of colemanosphaera represent vegetative and reproductive morphological attributes that are characteristic of the volvocaceae  <cit>  , including 1) cruciate eight-celled plakea , 2) typical inversion during daughter colony formation , and 3) tripartite colonial boundary of the extracellular matrix in vegetative colonies under transmission electron microscopy  .
in addition, the present molecular phylogeny confirmed that this new genus is robustly positioned within the volvocaceae .
within the volvocaceae, colemanosphaera is morphologically similar to yamagishiella and eudorina in having 16- or 32-celled spheroidal vegetative colonies without differentiation of obligative somatic cells, but with a cellular envelope that encompasses each vegetative cell tightly inside the colonial boundary under tem  <cit>  .
however, colemanosphaera and yamagishiella can be clearly distinguished from eudorina based on differences in contractile vacuoles in vegetative cells.
small contractile vacuoles are distributed on the surface of the vegetative cells of eudorina, whereas vegetative cells of yamagishiella and colemanosphaera contain only two or three contractile vacuoles near the base of the flagella  <cit>  .
furthermore, colemanosphaera differed from yamagishiella in pyrenoid characteristics and sexual reproduction.
colemanosphaera contained multiple pyrenoids in the chloroplast of mature vegetative cells and showed anisogamous sexual reproduction with sperm packets .
yamagishiella unicocca, the only species of the genus, was described as having a single basal pyrenoid in the chloroplast and isogamous sexual reproduction  <cit> .
therefore, colemanosphaera is a previously undescribed morphological genus within the volvocaceae.
the present two species of colemanosphaera can be clearly distinguished based on differences in chloroplast morphology, development of flagellar elongation in newly formed daughter colonies, and molecular data.
the chloroplast in c. charkowiensis shows prominent longitudinal striations on the surface and has more than two pyrenoids of almost identical size in mature cells, whereas c. angeleri has chloroplast striations that are not as prominent and contains a large basal pyrenoid and small pyrenoids in the cup-shaped chloroplast in mature cells .
during asexual reproduction, two new flagella in each cell of a newly formed daughter colony are markedly different in length in c. charkowiensis, whereas the two flagella of c. angeleri grow to an equal length .
in addition, c. charkowiensis and c. angeleri were robustly separated from each other in the phylogenetic analysis of the its region of rdna , and two compensatory base changes  were detected in helix i and helix iii of its- <dig> of the rdna between these two species .
the latter cbc is positioned within the most conserved region of its- <dig> of rdna   <cit> .
according to coleman  <cit> , organisms that differ by even one cbc in this region also are completely unable to cross.
thus, the two species represent two genetically and morphologically distinct entities.
the austrian strain asw <dig> and c. angeleri 2010-0126- <dig> formed a small clade robustly separated from c. charkowiensis in the phylogenetic analysis of the its region of rdna , and showed only one nucleotide difference in the its region .
the its region of nuclear rdna is rapidly evolving and shows nucleotide variation within a single biological species  <cit> .
thus, asw <dig> unambiguously belongs to c. angeleri, although its morphological data are lacking.
sexual reproduction
sexual reproduction was observed in c. charkowiensis  and generally occurred 2 days after possible male and female cultures were mixed in the nitrogen-deficient mating medium  <cit> .
cells of the colony divided to form a bundle of  <dig> or  <dig> male gametes or sperm packets.
the packet was almost spherical in shape, measured 15–17 μm in diameter , 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 , similar to male gametes of eudorina elegans <cit> .
during dissociation of the packet, biflagellate protoplasts or female gametes escaped from the gelatinous matrix of the female colony .
female gametes were biflagellate and spherical in shape, measuring 13–17 μm in diameter.
female and male gametes fused outside the female colony .
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 .
the zygotes eventually turned reddish brown in color after about 1 week.
the mature aplanozygotes were 14–18 μm in diameter .
in c. charkowiensis, male colonial cells underwent successive cell divisions to form bundles of spindle-shaped male gametes , whereas female gametes formed without successive cell divisions of colonial cells .
these characteristics are essentially the same as in other members of the anisogamous/oogamous volvocacean genera eudorina, pleodorina, platydorina, and volvox <cit> .
however, c. charkowiensis differed from dioecious members of eudorina, pleodorina, and volvox during conjugation between male and female gametes.
male and female gametic union in the latter genera takes place within the female colony after penetration by the male gametes  <cit> .
in contrast, female gametes are released from the colonial matrix, and male and female gametes fuse outside the female colony in c. charkowiensis .
a similar “external fertilization” between male and female motile gametes was observed in natural-collected samples of platydorina <cit> .
this type of anisogamous conjugation is similar to isogamous conjugation in pandorina, volvulina, and yamagishiella in that the gametes of both sexes are released from the parental colonies  <cit> .
in addition, the present multigene phylogeny demonstrated that colemanosphaera and platydorina form a robust clade that is sister to the lineage of volvox sect.
volvox , which shows “internal fertilization during oogamy”  <cit> .
thus, “external fertilization during anisogamy,” as found in colemanosphaera and platydorina, may represent an evolutionary intermediate between “external fertilization during isogamy” and “internal fertilization during anisogamy/oogamy” in the colonial volvocine green algae .
phylogenetic significance
the present multigene phylogeny demonstrated that colemanosphaera is robustly sister to the unique flattened volvocacean genus platydorina .
although these genera differ in the form of their vegetative colonies , they share fundamental morphological characteristics during the vegetative phase and sexual reproduction.
the vegetative colonies of both genera are composed of  <dig> or  <dig> cells of identical size  <cit>  .
two or three contractile vacuoles are limited to the anterior end of the vegetative cells in both genera .
vegetative ultrastructures of these two genera were used to observe colonial boundaries and cellular envelopes  <cit>  .
in addition, both genera show sperm packet formation and “external fertilization” between male and female motile gametes  <cit>  .
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 <cit> , huber-pestalozzi  <cit>  and ettl  <cit>  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 <cit> , e. echidna and e. interconnexa have not been studied in culture.
in addition, the strain of e. conradii <cit>  is not available from the utex culture collection .
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  <cit> .
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 .
in addition, the three species of eudorina are characterized by distinctive vegetative colony structures  <cit> .
e. conradii and e. interconnexa have species-specific individual gelatinous sheaths surrounding vegetative cells  <cit> , while e. echidna shows short conical projections covering the vegetative colony  <cit> .
thompson  <cit>  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  <cit>  , information on pyrenoids of the alga observed by thompson  <cit>  is lacking in his description and figures.
therefore, the alga may be yamagishiella unicocca, in which small and large gametes can fuse  <cit> .
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  pyrenoids in a cup-shaped chloroplast with prominent longitudinal striations on its surface  <cit> .
he did not evaluate its sexual reproduction  <cit> , 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  contains more flattened vegetative cells and less prominent chloroplast striations than those of c. charkowiensis .
thus, c. angeleri represents a previously undescribed morphological species.
CONCLUSIONS
two species of colemanosphaera were found in a japanese lake .
as discussed above, comparison of its sequences demonstrated that the austrian strain asw <dig> unambiguously belongs to c. angeleri .
c. charkowiensis was originally described based on material originating from ukraine  <cit> .
thus, colemanosphaera may be distributed in worldwide freshwater habitats.
although p. charkowiensis was reported from the united states  <cit> , 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  <cit> , sexual reproduction of the volvocine green algae can be clearly distinguished into four types  : 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 , 3) internal fertilization during anisogamy , and 4) internal fertilization during oogamy .
thus, oogamy evolution may have been preceded by the transition from external to internal fertilization during anisogamy within the volvocine green algae.
taxonomic treatments
colemanosphaera nozaki gen. nov.
vegetative colonies spheroidal in shape, composed of  <dig> or  <dig> 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  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  <cit> .
colemanosphaera charkowiensis  nozaki, t. k. yamada, f. takahashi, matsuzaki et nakada comb.
nov.
basionym: pandorina charkowiensis korshikov  <dig>  <cit> :  <dig>  pl.
<dig>  figure  <dig>
synonym: eudorina charkowiensis  pascher  <dig>  <cit> :  <dig>
holotype: figure  <dig> of pl.
<dig> in korshikov  <cit> .
epitype here designated: resin-embedded vegetative spheroids of 2013-0615-ic- <dig>  deposited in the herbarium of the department of botany, national museum of nature and science , tsukuba, japan .
strains examined: 2013-0615-ic- <dig>  2013-0615-ic- <dig>  2013-0615-ic- <dig>  isa 7– <dig>  2010-0713-e <dig>  and 2010-0713-e <dig> .
distribution: ukraine  <cit> , 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- <dig>  deposited in the herbarium of the department of botany, national museum of nature and science , tsukuba, japan .
strain examined: 2010-0126- <dig> .
etymology: the species epithet “angeleri” honoring dr. david angeler who studied the austrian strain asw <dig> of this species  <cit> .
type locality: lake isanuma, isanuma, kawagoe-shi, saitama, japan .
distribution: austria  <cit> , japan.

 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. 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 to the advanced multicellular spheroid with differentiation of male–female sexes such as volvox. the colonial volvocine algae show two types of gametic union: conjugation between isogametes outside the parental colonies and fertilization between small motile male gametes and large female gametes inside the female colony. although recent cultural studies on the volvocine algae revealed morphological diversity and molecular genetic data of sexual reproduction, 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, 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 asw is sister to platydorina, its morphological details were not reported; however, the internal transcriber spacer of nuclear ribosomal dna of this strain is now available. 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 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.  morphology and taxonomy two species of the new colonial volvocine genus colemanosphaera showed essentially the same colonial organization during the vegetative phase. vegetative colonies were ovoid to ellipsoidal in shape in c. charkowiensis comb. nov. or cylindrical to elongate-ovoid in c. angeleri sp. nov.. they contained or cells of approximately identical size embedded at the peripheral regions of the gelatinous matrix, forming a hollow colonial structure. the 32-celled colonies of c. charkowiensis measured up to 85 μm long and 67 μm wide, whereas those of c. angeleri were up to 95 μm long and 70 μm wide. the cells were ovoid to subspheroidal in c. charkowiensis or subspheroidal to lenticular in c. angeleri, having a broad anterior face that was more or less angular by mutual compression, measuring up to 20 μm in surface diameter in both species. each cell had two equal flagella, a massive cup-shaped chloroplast, a stigma, and two or three contractile vacuoles near the base of the flagella. no other contractile vacuoles were distributed on the surface of the protoplast. a gradual decrease in stigma size occurred from the anterior to posterior pole of the colony. the chloroplast contained longitudinal striations on the surface, which were prominent in c. charkowiensis. although the chloroplast of both species contained only a single pyrenoid in the immature cells, three to eight pyrenoids of almost identical size were distributed throughout the chloroplast of mature vegetative cells in c. charkowiensis, whereas the cup-shaped chloroplast in mature cells of c. angeleri contained a large single pyrenoid in the bottom, as well as one to five small pyrenoids. the present two species of colemanosphaera represent vegetative and reproductive morphological attributes that are characteristic of the volvocaceae, including 1) cruciate eight-celled plakea, 2) typical inversion during daughter colony formation, and 3) tripartite colonial boundary of the extracellular matrix in vegetative colonies under transmission electron microscopy. in addition, the present molecular phylogeny confirmed that this new genus is robustly positioned within the volvocaceae. within the volvocaceae, colemanosphaera is morphologically similar to yamagishiella and eudorina in having 16- or 32-celled spheroidal vegetative colonies without differentiation of obligative somatic cells, but with a cellular envelope that encompasses each vegetative cell tightly inside the colonial boundary under tem. however, colemanosphaera and yamagishiella can be clearly distinguished from eudorina based on differences in contractile vacuoles in vegetative cells. small contractile vacuoles are distributed on the surface of the vegetative cells of eudorina, whereas vegetative cells of yamagishiella and colemanosphaera contain only two or three contractile vacuoles near the base of the flagella. furthermore, colemanosphaera differed from yamagishiella in pyrenoid characteristics and sexual reproduction. colemanosphaera contained multiple pyrenoids in the chloroplast of mature vegetative cells and showed anisogamous sexual reproduction with sperm packets. yamagishiella unicocca, the only species of the genus, was described as having a single basal pyrenoid in the chloroplast and isogamous sexual reproduction. therefore, colemanosphaera is a previously undescribed morphological genus within the volvocaceae. the present two species of colemanosphaera can be clearly distinguished based on differences in chloroplast morphology, development of flagellar elongation in newly formed daughter colonies, and molecular data. the chloroplast in c. charkowiensis shows prominent longitudinal striations on the surface and has more than two pyrenoids of almost identical size in mature cells, whereas c. angeleri has chloroplast striations that are not as prominent and contains a large basal pyrenoid and small pyrenoids in the cup-shaped chloroplast in mature cells. during asexual reproduction, two new flagella in each cell of a newly formed daughter colony are markedly different in length in c. charkowiensis, whereas the two flagella of c. angeleri grow to an equal length. in addition, c. charkowiensis and c. angeleri were robustly separated from each other in the phylogenetic analysis of the its region of rdna, and two compensatory base changes were detected in helix i and helix iii of its- of the rdna between these two species. the latter cbc is positioned within the most conserved region of its- of rdna. according to coleman, organisms that differ by even one cbc in this region also are completely unable to cross. thus, the two species represent two genetically and morphologically distinct entities. the austrian strain asw and c. angeleri 2010-0126- formed a small clade robustly separated from c. charkowiensis in the phylogenetic analysis of the its region of rdna, and showed only one nucleotide difference in the its region. the its region of nuclear rdna is rapidly evolving and shows nucleotide variation within a single biological species. thus, asw unambiguously belongs to c. angeleri, although its morphological data are lacking. sexual reproduction sexual reproduction was observed in c. charkowiensis 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 or male gametes or sperm packets. the packet was almost spherical in shape, measured 15–17 μm in diameter, 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, 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. female gametes were biflagellate and spherical in shape, measuring 13–17 μm in diameter. female and male gametes fused outside the female colony. 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. the zygotes eventually turned reddish brown in color after about 1 week. the mature aplanozygotes were 14–18 μm in diameter. in c. charkowiensis, male colonial cells underwent successive cell divisions to form bundles of spindle-shaped male gametes, whereas female gametes formed without successive cell divisions of colonial cells. these characteristics are essentially the same as in other members of the anisogamous/oogamous volvocacean genera eudorina, pleodorina, platydorina, and volvox. however, c. charkowiensis differed from dioecious members of eudorina, pleodorina, and volvox during conjugation between male and female gametes. male and female gametic union in the latter genera takes place within the female colony after penetration by the male gametes. in contrast, female gametes are released from the colonial matrix, and male and female gametes fuse outside the female colony in c. charkowiensis. a similar “external fertilization” between male and female motile gametes was observed in natural-collected samples of platydorina. this type of anisogamous conjugation is similar to isogamous conjugation in pandorina, volvulina, and yamagishiella in that the gametes of both sexes are released from the parental colonies. in addition, the present multigene phylogeny demonstrated that colemanosphaera and platydorina form a robust clade that is sister to the lineage of volvox sect. volvox, which shows “internal fertilization during oogamy”. thus, “external fertilization during anisogamy,” as found in colemanosphaera and platydorina, may represent an evolutionary intermediate between “external fertilization during isogamy” and “internal fertilization during anisogamy/oogamy” in the colonial volvocine green algae. phylogenetic significance the present multigene phylogeny demonstrated that colemanosphaera is robustly sister to the unique flattened volvocacean genus platydorina. although these genera differ in the form of their vegetative colonies, they share fundamental morphological characteristics during the vegetative phase and sexual reproduction. the vegetative colonies of both genera are composed of or cells of identical size. two or three contractile vacuoles are limited to the anterior end of the vegetative cells in both genera. vegetative ultrastructures of these two genera were used to observe colonial boundaries and cellular envelopes. in addition, both genera show sperm packet formation and “external fertilization” between male and female motile gametes. 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, 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. 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. 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. 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, 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, 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. 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 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 contains more flattened vegetative cells and less prominent chloroplast striations than those of c. charkowiensis. thus, c. angeleri represents a previously undescribed morphological species.  two species of colemanosphaera were found in a japanese lake. as discussed above, comparison of its sequences demonstrated that the austrian strain asw unambiguously belongs to c. angeleri. 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, 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: 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, 3) internal fertilization during anisogamy, and 4) internal fertilization during oogamy. thus, oogamy evolution may have been preceded by the transition from external to internal fertilization during anisogamy within the volvocine green algae. taxonomic treatments colemanosphaera nozaki gen. nov. vegetative colonies spheroidal in shape, composed of or 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 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 nozaki, t. k. yamada, f. takahashi, matsuzaki et nakada comb. nov. basionym: pandorina charkowiensis korshikov: pl. figure  synonym: eudorina charkowiensis pascher: holotype: figure  of pl. in korshikov. epitype here designated: resin-embedded vegetative spheroids of 2013-0615-ic- deposited in the herbarium of the department of botany, national museum of nature and science, tsukuba, japan. strains examined: 2013-0615-ic- 2013-0615-ic- 2013-0615-ic- isa 7– 2010-0713-e and 2010-0713-e. 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- deposited in the herbarium of the department of botany, national museum of nature and science, tsukuba, japan. strain examined: 2010-0126-. etymology: the species epithet “angeleri” honoring dr. david angeler who studied the austrian strain asw of this species. type locality: lake isanuma, isanuma, kawagoe-shi, saitama, japan. distribution: austria, japan. 
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. 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. 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 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 and fertilization between small motile gametes and large gametes inside the female colony. 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. 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. 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. the evolution of oogamy from isogamy, an important biological event, can be summarized as follows: morphologically similar gametes differentiated into small “male” and large “female” motile gametes during anisogamy, from which immotile female gametes evolved.
 the evolution of oogamy from isogamy, an important biological event, can be summarized as follows: morphologically similar gametes differentiated into small “male” and large “female” motile gametes during anisogamy, from which immotile female gametes 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 and fertilization between small motile gametes and large gametes inside the female colony. 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. 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. 