Portrait of a Species

Polymerase chain reactions and sequencing:

DNA to serve as template in PCR reactions was extracted from ∼0.1 mg wet weight of cells, using InstaGene Matrix (Bio-Rad, Hercules, CA). The standard protocol used to obtain PCR products encompassing the entire ITS1, 5.8S, and ITS2 regions was 95° for 5 min; five repetitions of 90° for 1 min, 50° for 2 min, and 72° for 1 min; and then 30 cycles of 90° for 1 min, 60° for 1 min, 72° for 1 min, ending with a final 72° for 10 min. Taq polymerase was added after the reaction reached 95°.

Initial studies involved purifying the PCR products from agarose gels (QIAquick gel extraction kit; QIAGEN, Valencia, CA), subcloning them into pT7 Blue T-vector (Novagen, Madison, WI), infecting Escherichia coli, and preparing DNA by miniprep (Wizard Plus miniprep kit; Promega, Madison, WI). Later studies utilized direct sequencing of the gel-purified mixture of PCR products. Primers included the standard pair (derived from White et al. 1990, ITS5 and ITS4) that we call here Gfor and Grev, priming, respectively, in the 3′ end of the small subunit (SSU) rDNA and the 5′ end of the large subunit (LSU) rDNA and producing the full ITS1-5.8S-ITS2 as the PCR product. Additional special-purpose forward primers, each paired with Grev, are shown in Figure 1. Sequencing in both directions was done earlier manually [United States Biochemicals (Cleveland) 2.0 kit] and later using an automatic sequencing system (ABI dye systems and ABI Prism 377 automated sequencer; Applied Biosystems, Foster City, CA). Primers for sequencing were those used for the initial PCR plus a pair in the 5.8S (ITS3 and ITS2 of White et al. 1990).

Open in a separate window

Figure 1.—

Alignment of ITS2, hairpin loop I, sequences of standard C. reinhardtii “short” and “long,” plus that of C. smithii + and of the closely related Chlamydomonas strains. Paired nucleotide positions in standard C. reinhardtii are overlain with a line. An X marks the nucleotide diagnostic for standard C. reinhardtii vs. C. smithii +. The strains CC-1952, CC-2342, CC-2931, and CC-2935 are newly collected (sources given) and interfertile with standard C. reinhardtii. No long variant was obtained among the few subclones of CC-2931 sequenced. At the bottom are listed the five special primers used (paired with Grev) for analysis of the ITS2 helix I indel.

Alignment of sequences utilized MacVector and AssemblyLIGN software (Kodak, International Biotechnologies, New Haven, CT) and took into account the known secondary structure of the ITS1 and ITS2 RNA transcripts (Figure 2). Phylogenetic comparison utilized PAUP* version 4.0b10 (Swofford 2002). The evolutionary model for the data sets was calculated by Modeltest 3.06 (Posada and Crandall 1998). The ITS sequences have been deposited in GenBank as listed in supplementary Table S2 (http://www.genetics.org/supplemental/).

Open in a separate window

Figure 2.—

Secondary-structure diagram of Chlamydomonas reinhardtii ITS2 RNA transcripts. In the ITS2 diagram, the relatively conserved positions (Mai and Coleman 1997) are presented in boldface type. All nucleotide variants of the standard C. reinhardtii and of C. smithii + are indicated by arrows. (A) ITS1. Four nucleotide variants were each found in only one subcloned sequence of the standard C. reinhardtii strains (in order, CC-278, CC124J, CC-620, and UTEX 2247); the nucleotide in parentheses was a variant in one subclone of C. smithii +. (B) ITS2 had no variant nucleotide positions among all the standard C. reinhardtii subclones sequenced; as indicated by the arrows, in C smithii +, one subclone had a variant position, compensatory for pairing, in helix II, while all C. smithii + differed from all standard C. reinhardtii at the circled position in helix I. Shown in addition to the “long” form of helix I is the alternative “short” form of helix I, found in differing proportions in the standard C. reinhardtii strains. In the short form, the AflIII site is highlighted in boldface type. Only the short form, with the C substitution, has been recovered so far from C. smithii +, which is otherwise identical to C. reinhardtii in both ITS1 and ITS2.

Background information:

What we refer to here as the “standard C. reinhardtii” strains are those in use widely, all allegedly derived from the meiotic products of a single zygote isolated in 1945 by G. M. Smith from a Massachusetts site (Harris 1989). A laboratory history of the standard strains, modified from Harris (1989) and from Kubo et al. (2002), is provided in Figure 3. Three basic sublines, I, II, and III, are reconstructed from the literature and culture collection records.

Open in a separate window

Figure 3.—

Genealogy of standard C. reinhardtii laboratory strains, modified from Harris (1989). Note that CC-1690 (thick box) was used for creation of EST libraries for cDNA sequencing in the Chlamydomonas Genome Project. The three major sublines each are uniform for the genetic loci cited, with certain striking exceptions. In subline 1, two strains that Sager had by 1980 (UTEX 2246 and 2247) are unlike the remainder of subline 1 and probably came from the Gillham laboratory in an exchange. In subline 3, the CC-124J in Matsuda's laboratory, although obtained from Harris in 1983, is almost pure for the “long” ITS2 form, in contrast with the other subline 3 strains. Likewise, all the strains derived from the cell-wall-less mutants of Davies and Plaskitt (1971) are almost pure “shorts,” while the remainder of subline 3 are clearly mixed for ITS2 type. Data for mmp1-mmp2 are from Kubo et al. (2002). In sublines I and II, the + mating type has the RFLP allele called A for the mmp1-mmp2 region of linkage group XIX, while the − mating type has the B allele. In subline III, both mating types carry the A allele. The final metalloprotease gene (mmp3) is not yet mapped but segregates independently of all the other loci and also has two alleles by RFLP analysis. Again, in lines I and II, the + mating types have the A allele and the − mating type the B, while in subline III both mating types carry the A allele for this gene.

The genetic constitution characterizing the three major sublines of the standard C. reinhardtii strains for five unlinked loci is shown in the Figure 3 diagram. One locus is mating type, found on linkage group VI (Harris 1989). Two, nit1 and nit2 (respectively, linkage group IX and III), singly or together, prevent growth on nitrate; organisms utilize ammonia or urea as a nitrogen source instead, and Smith used a medium containing ammonia, so would have been unaware of any genetic variation in this trait. Essentially all subline I (the only exception is UTEX 2247, which we believe to be a late exchange between the Duke and Sager laboratories) and subline II strains can grow on nitrate, while subline III strains cannot (Harris 1989; Saito et al. 1998). Where analyzed, they bear both the nit1 and nit2 alleles. The next locus is the nucleolar organizer repeats, as yet unmapped. The final locus contains genes for “autolysins,” concerned with wall digestion, metalloproteases surveyed by Matsuda's laboratory (Kubo et al. 2001, 2002). Not shown in Figure 3 are other known genetic differences that have led to some confusion among different laboratories in the past. These are documented in Harris (1998) and in Saito et al. (1998) and include green vs. yellow colony color when growing in the dark and the requirement for light to evoke and maintain gamete activity.

As made obvious from Figures 3 and ​and4,4, the current strains of standard C. reinhardtii cannot all be the immediate products of a single zygote as previously assumed (see Harris 1989), a point made clearly by Matsuda's laboratory (Kubo et al. 2002). Two other possible sources of participants exist. At the time Smith was dispersing samples, at least by February of 1950, he had in his laboratory literally hundreds of C. reinhardtii F₁ clones, generated by the doctoral research of Regnery (Smith and Regnery 1950). In addition, a single strain designated C. smithii (Hoshaw and Ettl 1966) that is mating-type plus, also collected from Massachusetts in 1945, was present in Smith's laboratory and is fully interfertile with the standard strains (Harris 1989). C. smithii + shares with the standard C. reinhardtii most of the alleles shown in Figure 3. It can use nitrate and has the B allele of the mmp1-mmp2 locus, but a variant allele of the mmp3 locus (Kubo et al. 2002).

Open in a separate window

Figure 4.—

Possible origin of standard Chlamydomonas reinhardtii strains. The distribution of alleles in sublines I, II, and III cannot be accommodated in a single tetrad, but could be generated if at least one F₁ is included.

The ITS2 data (below) rule out participation of C. smithii + in the standard C. reinhardtii lineages. C. smithii + has only “short” versions of the ITS2, whereas all standard C. reinhardtii strains are mixed for this character. More importantly, C. smithii + is uniform for a C residue where all standard C. reinhardtii have a T in the ITS2 sequences (Figure 1).

This leaves us with the single zygote germinated by Smith. Is it possible to derive all the strains listed in Figure 3 from a single zygote? No it is not (not directly), even if all four zygote products were isolated. All the strains could, however, be derived from three of the four products of a single zygote, plus one or more F₁ products of their intercross with one another (possibilities are illustrated in Figure 4). Perhaps the earliest pair of strains Smith gave out was the pair in lines I and II that might be two of the original zygote tetrad. Line III appears later with strains brought by Ebersold to Levine's laboratory; these are two new genotypes, perhaps an additional one of the originals and one F₁.

Heterogeneity among nuclear ribosomal repeats in standard C. reinhardtii:

Almost all eukaryotes have multiple copies of their nuclear ribosomal RNA cistrons, arranged in a long tandem array. Each cistron contains one gene for SSU, one for 5.8S, and one for LSU ribosomal RNA. These are transcribed as one long RNA; “RNA processing” then removes the transcribed spacers (ITS1 and ITS2—see supplementary Figure S1 at http://www.genetics.org/supplemental/) lying between the genic regions, salvaging only the rRNAs. The genic sequences are relatively stable, evolutionarily. By contrast the ITS regions combine stable and less stable regions and have gained wide usage in phylogenetics at lower taxonomic levels (Coleman 2003). Within a single organism (with the exception of hybrids), the nuclear ribosomal ITS sequences are typically essentially identical among all the repeats.

C. reinhardtii is known to have at least 200 copies of the nuclear ribosomal repeats (Howell and Walker 1976). We have sequenced the internal transcribed spacer regions flanking the 5.8S gene and concentrated on the second of these, the ITS2. Our initial sequencing by subcloning of the ITS2 region revealed a 10-nucleotide INDEL, found in some cistrons but not others, of the same clonal strain of cells. The position of the INDEL in ITS2 is shown in Figures 1 and ​and22 in its two alternative forms. Thirty-three standard C. reinhardtii strains tested all have both short and long forms of this region, but in varying proportions, as assessed by direct sequencing of the PCR product or sequencing of multiple subclones. Where subcloning frequency had suggested a near equal proportion of “longs” and “shorts” in a genome, direct sequencing of the mixture of PCR products gave an unreadable series starting at just the INDEL position. Where subcloning suggested a predominance of, for example, the short version, direct sequencing of the PCR product gave a clear read, but close examination revealed minor peaks starting at the INDEL position. Only CC-124J, UTEX 2246, and the strains associated with the cell-wall-less subgroup appeared to lack two forms. Likewise, the isolate known as C. smithii + appeared to have only the short form, and its sequence was identical in all three extent samples of the strain (UTEX 1062, SAG 54.72, and CC-1373).

To check these exceptional standard C. reinhardtii strains, we turned to a further method of assessment. The short version of the ITS has an AflIII site (ACPuPyGT) in the terminus of the first hairpin loop in the ITS2 transcript secondary structure; this cut site is absent from the long version. The AflIII site in the short version of ITS2 is shown in Figure 2, and it and the additional AflIII site in the adjacent LSU are mapped in supplementary Figure S1 (http://www.genetics.org/supplemental/). After BamHI/AflIII endonuclease digestion of genomic DNA, short cistrons should have two bands, of 870 and 1650 nucleotides in length, where long cistrons should retain a single band of 2500 nucleotides.

To ascertain directly what the genomic DNA contained, we digested total DNA with the diagnostic restriction endonucleases, separated the products on an agarose gel, and prepared a Southern blot. This was probed with a radioactively labeled plasmid containing a complete C. reinhardtii ITS1-5.8S-ITS2 sequence (supplementary Figure S2 at http://www.genetics.org/supplemental/).

Of 13 different standard C. reinhardtii strains examined this way, most showed autoradiography patterns on Southern blots indicating the presence of both long and short versions of the ITS within a genome, and the proportions of each roughly corresponded with what had been observed either by direct PCR sequencing or by frequency among multiple subclones (supplementary Table S1 at http://www.genetics.org/supplemental/). There were still, however, strains that appeared to be pure types, from the diagnostic bands on the autoradiogram. CC-124J, the strain from Matsuda in Japan, appeared to be lacking any short version; this was not true of the CC-124 we obtained directly from the CC collection. Several strains collectively associated with the cell-wall-less mutation work of Davies and Plaskitt (1971) appeared to be lacking longs. The same is true of two other strains, UTEX 2246 shown in Figure 3, subline I, a strain deposited by Sager in UTEX in 1980, and C. smithii +.

To discern finally whether these exceptional strains were indeed homogeneous for only one version of the ITS2, forward primers designed to discriminate between shorts and longs were used, with Grev, for PCR. Using the J short primer, a band resulted from the Japanese sample CC-124J mating-type minus, and its sequence was the expected short. Likewise, use of the rein-long and smith-long special primers on the cell-wall-less strains and on UTEX 2246 produced a band that, when sequenced, was the expected long version. Thus specific primers were capable of detecting a minority cistron type not detectable by genomic digestion and probing, and all standard C. reinhardtii organisms contain both long and short versions of ITS2, albeit in very different proportions in different strains.

However, no long band was obtained with C. smithii + DNA, using either of the special long primers. C. smithii + seems to be uniform for the short version, in agreement with the absence of any indication of variation in this region on direct sequencing of mixtures of PCR products of the Gfor-Grev primer pair, and from analysis of genomic DNA. More important to the genealogy study, C. smithii + appeared to differ from all standard C. reinhardtii at one nucleotide site, marked X in Figure 1. For a more stringent examination of this nucleotide position we designed two relatively short forward primers differing only by C/T at the 3′ end (Figure 1, 17-T and 17-C), and paired each with Grev for PCR to test whether this nucleotide position was indeed homogenized completely to T in C. reinhardtii and to C in C. smithii +. With the use of higher annealing temperatures, as seen in supplementary Figure S3 (http://www.genetics.org/supplemental/), 17-T succeeded in priming a PCR product only with standard C. reinhardtii DNA, while 17-C succeeded only with C. smithii + DNA, indicating that this nucleotide position is uniformly T in standard C. reinhardtii and C in C. smithii +.

It then seems most likely that there was no contribution of C. smithii + to the standard strains of C. reinhardtii unless there were absolutely no crossover events in the entire set of ribosomal repeats (8.5 kb × 200 = 1700 kb) in a hypothetical reinhardtii × smithii zygote germination. In a final effort to examine the possibility that a cross between C. reinhardtii and C. smithii + had been made, but only C. reinhardtii cistrons were present in the product because there had been no crossover in the region of the ribosomal repeats, we examined known products of such a cross. We obtained 10 randomly selected F₁ products of a cross of standard C. reinhardtii (CC-29) × C. smithii + (Ranum et al. 1988) and determined the sequence of their Gfor-Grev PCR products as well as whether DNA from each could be primed successfully by 17-T and by 17-C. The CC-29 parent was examined as well and proved to contain predominantly shorts; as expected, these primed with 17-T but failed to prime with 17-C. Two of the F₁ products had the same phenotype and might well be uninterrupted standard C. reinhardtii parental cistrons. The other eight F₁ clones, however, displayed a wide variety of sequence disturbances starting at the position equivalent to the end of helix I in ITS2 and primed successfully with both 17-T and 17-C at discriminatory reannealling temperatures. We conclude then that in the region of ribosomal repeats the average frequency of crossover per zygote is about two; that is, the vast majority of zygote products would have a mixture of C. reinhardtii and C. smithii + repeats. With the additional information that C. smithii + has a plastid DNA restriction pattern with a number of differences from that of standard C. reinhardtii, but no C. reinhardtii examined has the C. smithii + plastid DNA pattern (Harris et al. 1991), we conclude that C. smithii + has not contributed genetically to the standard C. reinhardtii strains.

Algal strain confusions:

Not only is our knowledge of past strain exchanges among laboratories incomplete, but also in the course of the 60 years since the C. reinhardtii standard strains were first isolated and dispersed to other laboratories, some laboratories subsequently have deposited strains in culture collections as C. reinhardtii from different collection sites. The older strains in supplementary Table S1 (http://www.genetics.org/supplemental/) purporting to be C. reinhardtii from various different places are all actually standard C. reinhardtii strains, since their ITS sequences are identical to those of the standard strains, their plastid DNA shows identical endonuclease restriction fragment patterns (Harris 1998), and their distribution of copies of the Gulliver transposon matches those of the standard strains (Ferris 1989). These include the strains ostensibly from the Caroline Islands (SAG 11-32c), Florida (SAG18.79), France (SAG 77.81), Japan (SAG 73.72), and Pringsheim's strain SAG 11-31. In addition, the short ITS2 sequence of C. reinhardtii in GenBank ({"type":"entrez-nucleotide","attrs":{"text":"AF156601","term_id":"5031531","term_text":"AF156601"}}AF156601), sequenced in China, is actually from a strain obtained from a Denmark laboratory, one of the standard C. reinhardtii lines.

The C. smithii story is a cautionary tale for control reactions in mating experiments. Culture collections actually contain two cultures labeled C. smithii, one designated a (+) mating type (UTEX 1062, SAG 54.72, and CC-1373) and the other a (−) mating type (UTEX 1061 and SAG 53.72). This latter strain was isolated in California by Smith. Several laboratories used this mating-type minus strain and its presumed partner C. smithii + in pairings with standard C. reinhardtii, but Harris (1989) noted that the (−) strain did not seem to mate with C. reinhardtii while the (+) strain did. Subsequently, the ITS sequence of this minus strain was found to be very different from that of C. reinhardtii, more like C. culleus, and it was found to be homothallic, making zygotes in clonal culture (Coleman and Mai 1997).

With respect to C. incerta (SAG 7.73) from Cuba, as recognized by ITS2 sequence identity two other strains are also identical, SAG 81.72 from Holland (originally submitted as C. globosa) and two equivalent strains, NIVA Chl13 = NIVA Chl21, from Norway. The Norway strains were originally labeled C. reinhardtii. Their putative SAG duplicate (SAG23.90) is not the same, but is rather a C. noctigama. The culture called C. incerta, SAG 23.72, from France is morphologically very different, a large cell with four flagella (Schlösser 1994).

How homogeneous are the ribosomal cistrons of an organism?

In the course of this study, some 73 subclones of ITS were sequenced, and an additional 63 sequences were obtained by direct PCR sequencing (supplementary Table S1 at http://www.genetics.org/supplemental/). For many of these, the ITS1 was largely ignored, but the ITS2 was completed. Among the ITS1 sequences of all strains of standard C. reinhardtii, four examples of a single-nucleotide variant were found. For each of these four cases, either the nucleotide is unpaired in the RNA transcript secondary structure or the substituted nucleotide is compensatory and preserves the pairing at that position (see Coleman and Mai 1997). For ITS2, except at the tip of the first helix of ITS2, no nucleotide position variants were encountered among the standard C. reinhardtii strains. A single variant nucleotide was found in one subclone of C. smithii +, in helix II of ITS2, a compensatory change preserving the pairing, as indicated in Figure 2.

Some additional strains called C. reinhardtii, interfertile with the standard strains, have subsequently been isolated from other areas (see supplementary Table S2 at http://www.genetics.org/supplemental/). At least three of the newly collected C. reinhardtii strains (from Minnesota, Pennsylvania, and Quebec) have both long and short versions of ITS2 helix I (Figure 1), as revealed by sequencing subclones of the ITS. Each long version is slightly different in sequence. Except for the INDEL and the C/T position differentiating standard C. reinhardtii and C. smithii +, there is essentially no other difference in either ITS1 or ITS2 of all these strains capable of interbreeding.

It remains unknown how rapidly ITS is homogenized within a genome. Homogenization of ITS repeats is apparent in all the standard C. reinhardtii, even to the crucial nucleotide that distinguishes it from its near relative C. smithii +, with the sole exception of the loop at the end of helix I in ITS2. This remains unhomogenized in the standard C. reinhardtii and also in the more recently collected interfertile C. reinhardtii strains. This is a region that is relatively poorly conserved between species evolutionarily, but is almost universally homogenized within an organism. Why should this exception exist? We offer no suggestion, except that these Chlamydomonas strains would seem to provide ideal material for further study, given the apparent frequency of crossover events among the ribosomal cistrons.

Phylogenetics:

Prompted by the studies of Goodenough and Ferris (Ferris et al. 1997, 2002) on the structure of the C. reinhardtii mating-type locus, we obtained and sequenced the ITS region of all available chlamydomonads of grossly similar morphology, as well as the ITS of newly collected strains and others examined in the literature (e.g., Nozaki et al. 2002). At the same time we made pairings of these organisms with standard plus and minus mating types of C. reinhardtii to assess mating potential.

These latter pairings include the one C. smithii + isolate from Massachusetts and isolates from Eastern Canada (Sack et al. 1994), Minnesota (Gross et al. 1988), Pennsylvania and Florida (Spanier et al. 1992), and North Carolina (Harris 1998). All show high interfertility, zygote formation, and zygote germination with viable progeny when crossed with the standard C. reinhardtii. All the strains found capable of interbreeding with C. reinhardtii originate from the east coast of North America, extending as far west as Minnesota.

Figure 5 presents the phylogenetic analysis of organisms found to be most similar to C. reinhardtii in ITS sequence. Also included are Volvox carteri, several Gonium isolates, and an array of Chlamydomonas species, omitting many groups of even more distant chlamydomonads (e.g., C. moewusii/eugametos). Since the group encompasses considerable phylogenetic depth we first utilized the relatively conserved nucleotide positions of ITS2 (Figure 2, boldface type). As indicated by the asterisk in Figure 5, tree A, there is a major evolutionary dichotomy in the backbone. Of the six most conserved pairings in the whole Volvocacean ITS2 (Mai and Coleman 1997), one has undergone a compensatory base change (CBC) that separates the top half of the tree from the bottom; U-A has changed to Pu-Py. The only other CBCs involving these conserved pairings are two; the U-A change to G-U supporting the clade of SAG 5.93 with SAG 62.72 and the A-U change to a G-C, a CBC supporting the association of SAG19.90 with SAG 18.90 and C. asymmetrica. The second marker of the major dichotomy concerns the most conserved DNA sequence in all of ITS2, a marker found on the 5′ side of helix III. The top half of the tree is uniform for GGCCTCTACTGGGTAGGCA at that position, except for one transition in a secondary structure bulge in V. carteri, while several variants appear among these sequences in the bottom half of the tree.

Open in a separate window

Figure 5.—

Phylogenetic analyses of Chlamydomonas reinhardtii strains and putative relatives. (A) Tree, based on comparisons of the relatively conserved positions of ITS-2 of 36 taxa (116 positions marked in boldface type in Figure 2B), representing a maximum-likelihood analysis using the model according to Tamura and Nei (1993) with equal base frequencies and Gamma distribution shape parameter (G = 0.3853; TrNef + G). The model was calculated as the best model with Modeltest 3.06 (Posada and Crandall, 1998). The upper bootstrap values are neighbor joining (boldface type; 1000 replicates), using the same model criteria; the lower bootstrap values are maximum parsimony (boldface italic type; 1000 replicates). The tree was rooted using the basal clade, marked with a bracket. (B) The smaller tree presents the further analysis of the 23 nearest neighbors of C. reinhardtii (boxed in A, a branch marked with an asterisk), using the entire ITS-1 and ITS-2 sequence (851 positions) and strains UTEX 1341 and SAG 73.81 as outgroup. The tree was obtained using maximum-likelihood analysis, the Tamura and Nei model (TrN + I + G), with the proportion of invariable sites (I = 0.2182) and Gamma distribution shape parameter (G = 0.5983) calculated as best model with Modeltest. The bootstrap values are neighbor joining (boldface type; 1000 replicates), using the same model above and maximum parsimony (boldface italic type; 1000 replicates) below. Only bootstrap values >50% are presented. Taxon names in boldface type are newly sequenced here. The brace on the tree in B indicates interfertile Chlamydomonas strains.

We then selected the strains of the top half of the tree and examined their relationships using all positions of ITS1 and ITS2, including gaps as a fifth character. This produces a slightly more subdivided branching, at least in distance analysis, but did not change the relationships of the C. reinhardtii and their closest relatives, as shown in Figure 5B. As expected, all the interbreeding strains of C. reinhardtii/smithii + group together. Their ITS1 and ITS2 sequences are identical (except the tip of helix I, ITS2). The C. reinhardtii/smithii + strains are barely separated from C. incerta and Chlamydomonas sp. from Kenya; all are identical for nucleotide positions in ITS2 found to be relatively conserved (Figure 2).

The next closest chlamydomonads are Chlamydomonas sp. (from Kenya) and C. incerta (from Cuba). Strains isolated from the same site in Kenya several times, and identical in ITS sequence, fail to interact sexually with each other or with C. reinhardtii. C. incerta from Cuba is a single strain that does not interact sexually with either C. reinhardtii mating type or the Kenya strains. The second C. incerta (NIVA Chl13/21) from Norway has an identical ITS sequence to that from Cuba, as does the C. incerta (SAG 81.72) from the Netherlands. All are equally intractable for mating. Since we never obtained a sexual pair of the Kenya material, and the C. incerta strains have been in culture for years, all might possibly be sterile. C. incerta from Cuba is genetically mating-type minus, on the basis of the presence of the MID gene in the mating-type locus (Ferris et al. 1997), and so also is the Netherlands strain, but this latter strain differs by RFLP profile from the Cuba strain (T. Pröschold, unpublished results).

The next closest relative, by DNA comparison, is a 16-celled colonial green alga, Gonium pectorale. With respect to the set of relatively conserved nucleotide positions in ITS2, G. pectorale Alaska differs from the taxa above it in the tree at only two positions (one in a single-stranded region and one transition at the distal-most pairing of helix II), and G. pectorale Africa differs at one additional nucleotide, a bulge in helix II. A number of G. pectorale mating-type pairs capable of interbreeding are available (Fabry et al. 1999); none crosses with C. reinhardtii, but the Alaska pair of strains is most similar to C. reinhardtii by ITS comparison.

A relatively close evolutionary relationship between colonial greens and the C. reinhardtii grouping is supported by other evidence. In a study of their hatching enzymes, autolysins, enzymes that act to release daughter cells from the mother cell wall, both Schlösser (1976)(1984) and Matsuda et al. (1987) found that chlamydomonads fall into subgroups, on the basis of their autolysin sensitivity. The autolysin enzyme isolated from one member of a subgroup would digest the mother wall of all the members of the same subgroup. The chlamydomonads found to be in the same autolysin subgroup as C. reinhardtii included C. incerta, C. smithii +, and C. globosa SAG 81.72 (known now to be C. incerta from the Netherlands). C. reinhardtii, C. smithii +, and C. incerta also share recognizably similar ypt 2 and actin gene exons (Liss et al. 1997).

The gametangium autolysin of C. reinhardtii can also digest the gametangium stage of six species of Gonium (Matsuda et al. 1987), an indication of genetic relatedness further supported here since the next closest relatives by ITS analysis are a pair of G. pectorale strains and V. carteri—in fact, this is roughly the position of all the Volvocaceae. The next most similar taxon for these relatively conserved nucleotide positions is G. sacculiferum, which differs at 5 positions. C. parallestriata differs at 18 positions.

The discovery that the colonial alga G. pectorale is more similar on a DNA basis to C. reinhardtii than are many other chlamydomonads is not entirely unexpected. In the asexual reproduction of these algae, the mother cell undergoes 2ⁿ mitoses before release of the daughter cells. Morphologically, the colonial forms appear to derive from the delay of completion of cytokinesis of the multiple daughter cells that the chlamydomonad mother cell makes.

Biogeography:

All the interbreeding strains of C. reinhardtii and the one interfertile strain of C. smithii occur naturally in eastern North America. We have not found any organisms capable of interbreeding with it, or sharing ITS sequence, anywhere outside this area, despite considerable collecting and also sequencing of all available germane organisms. The studies of unicellular green soil algae by Bold's laboratory (Deason and Bold 1960) in Texas also failed to isolate any organism interfertile with C. reinhardtii, although they report finding at least seven different chlamydomonads of similar morphology.

One additional insight from the recent successful rediscoveries of C. reinhardtii lends support to the idea of its relatively localized distribution. Four separate laboratories instituted a search for chlamydomonads interfertile with the standard C. reinhardtii. None mentions any excessive numbers of natural samples tried before attaining success. Gross et al. (1988) mention sampling 24 sites, obtaining chlamydomonad-like isolates from each, but only one (a minus mating type from Minnesota) that interacted with C. reinhardtii. Spanier et al. (1992) found three clones (two from Pennsylvania and one from Florida) interfertile with standard C. reinhardtii from ∼300 sampled sites in Pennsylvania and Florida. Sack et al. (1994) examined 352 samples from 22 localities in Quebec and Ontario, Canada, and the midwestern United States, differing in their soil type. Nineteen of the agricultural soil types, and these only, yielded strains interfertile with C. reinhardtii, and all of these came from Quebec. Finally, Harris (1998) isolated + and − mating types of a strain interfertile with C. reinhardtii from a single sample of garden soil taken in North Carolina. Together with C. smithii +, all these strains consititute a clear biological species, since their interfertility and survival rates of intercross zygote progeny are high. They also constitute a single Z clade (Coleman 2000). These organisms also share an essentially identical ITS sequence, with the exception of the tip of ITS2 helix I (Figure 1), which lies in a region that is relatively nonconserved evolutionarily and which is known to vary in other algae capable of interbreeding.

We are very grateful to L. Krienitz (Institute of Aquatic Ecology, Neuglobsow) for dedicated collecting in Kenya, to C. Forest (Brooklyn College) for C. reinhardtii strains, and to Y. Matsuda (Kobe University) for calling our attention to the unusual ITS repeat structure of his CC-24J strain and providing it to us. We are also grateful for support provided to T.P. by Deutsche Forschungsgemeinschaft grant PR 682/1-1&2.