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Systematics and Phytogeography |
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island 02912 USA
Received for publication December 4, 2001. Accepted for publication April 25, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: algae • allopolyploid • DNA sequence • Eudorina • internal transcribed spacer • ITS • Pleodorina • Volvox
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Volvocaceae are haplonts, with the only diploid cell in the life cycle being the zygote. In the genera examined here, Eudorina, Pleodorina, and Volvox, most isolates are heterothallic, i.e., clonal cultures are either exclusively female or exclusively male. A few isolates are homothallic, i.e., make both sperm and eggs and form zygotes within a clonal culture. Thanks to the relative ease of maintaining Volvocales in vegetative culture, the original two heterothallic pairs of parental clones (Eudorina elegans UTEX 1192 and 1193 and Pleodorina illinoisensis UTEX 807 and 808) as well as four of the F1 products studied by Goldstein (1964)
were still available for study. The F1 clones were known to display anomalous mixtures of reproductive characters, and all four studied were reported to contain twice the number of chromosomes as their parents. They are effectively "allopolyploids," apparently the product of unreduced zygotes (Goldstein, 1964
).
All the available isolates of the genus Pleodorina, except P. californica and P. japonica (see Coleman, 1999
), have been included in this study, as well as two species of Volvox for which molecular data (Coleman, 1999
) indicated an affinity to Eudorina. The primary difference between Pleodorina and Eudorina is in the consistent presence in Pleodorina of a proportion of anterior cells in the colony that never undergo daughter colony formation. For a history of the nomenclature of these genera and species, see Nozaki (1989
, Table 2); a summary of known chromosome numbers is given in Coleman (1979)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
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Polymerase chain reactions (PCR) were performed using standard primers from the 3' end of the small subunit ribosomal DNA and the 5' end of large subunit ribosomal DNA (primers "a" and "b" of Coleman, Suarez, and Goff, 1994
) and 2 µL DNA extracted from the organisms as template in a total reaction volume of 50 µL. Taq polymerase was added after the reaction reached 95°C. The PCR protocol consisted of 95°C for 5 min, five repetitions of 90°C for 1 min, 50°C for 2 min, then 72°C for 1 min, followed by 30 cycles of 90°C for 1 min, 60°C for 1 min, 72°C for 1 min, ending with a final 72°C for 10 min. Those reactions of which a subsample showed a band on agarose were combined and incubated at 72°C for a further 30 min to ensure that all strand synthesis was complete. After purification from an agarose gel (QIAquick Gel Extraction Kit, QIAgen, Valencia, California, USA), the mix of PCR products was cloned into pT7 Blue T-vector (Novagen, Madison, Wisconsin, USA), and transformed into E. coli JM109. Transformants were grown for minipreps (Wizard Plus Miniprep Kit, Promega, Madison, Wisconsin, USA) which were run on an agarose gel. For each algal isolate, at least two minipreps showing vector with insert were sequenced. Sequences were obtained manually for both strands using the US Biochemicals 2.0 kit (USBiochemicals, Cleveland, Ohio, USA) and primer regions in the adjacent vector sequence, but more recently sequencing was carried out using ABI dye terminators and run on an ABI Prism 377 automated sequencer (Applied Biosystems, Foster City, California, USA), using the same primers as for PCR.
Sequence alignment was done manually, with the aid of MacVector and AssemblyLIGN software (Kodak, Int. Biotechnologies, New Haven, Connecticut, USA), guided by the homologies revealed by the secondary structure of the ITS1 and ITS2 RNA transcripts, derived with the help of the mfold software available at for use on the website http://bioinfo.math.rpi.edu/
mfold/rna/form1.cgi, a website offering mfold version 3.0 (Zucker, Matthews, and Turner, 1999
). Where extremely long hairpin loops were present in one or a few sequences in the alignment, the terminal region of the helix was truncated to shorten the total alignment (in ITS1, helix #1, 47 nucleotides of PleoRT; in ITS1, helix #2, 24 nucleotides of ASW05130 112 nucleotides of UTEX 240 and ASW05146 and 11 nucleotides of UTEX737; in ITS2, helix #I, 9 nucleotides of ASW05144. The total alignment was further shortened by removing the 5.8S region because it contributed no phylogenetic information. Where variant sites were found in separately cloned cistrons from the same clonal organism, the appropriate IUPAC symbol was used in the alignment (IUPAC-IUB Commission in Biological Nomenclature, 1968
).
Analyses of the entire set of Eudorina, Pleodorina, and two Volvox isolates, plus Chlamydopmonas and Yamagishiella as outgroups, utilized the parsimony (maximum parsimony heuristic search with all positions weighted equally, with and without gaps = fifth character) and distance-based neighbor joining algorithms (applied using a Kimura two-parameter model, with minimum evolution objective) of PAUP* version 4.0b4a (Swofford, 1999
). The ITS1 and ITS2 were assessed both separately and together to check their consistency. Support for individual clades was examined by running 500 bootstrap iterations. The ITS sequences have been deposited in GenBank (Table 1), and the alignment is available from the author.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Mai and Coleman, 1997
; Coleman, 1999
), approximately 300 base pairs (bp) each. Their base composition likewise is approximately 50% guanine plus cytosine. The 5.8S RNA gene is 159 bp in all sequences, and only four sites showed variation, transitions that carried no phylogenetic information. Except in the Eudorina x Pleodorina hybrids (see Hybrids below) both cloned repeats gave essentially identical sequences, subject only to one or a few transitions (no transversions) in single stranded regions of the ITS1 and ITS2 secondary structure. Where six cloned repeats were sequenced from a single isolate and also from its complementary mating type isolated at the same time and place, the level of variation was similar. For the 807/8 mating pair, there is one transition in ITS1, in a single-stranded region of the RNA transcript secondary structure, and three transitions in ITS2 of which two are in single-stranded regions and one preserves a pairing. For the 1192/3 mating pair, there are two variant nucleotide positions in ITS1, one a transversion in a single-stranded region and one in a secondary structure helix, a transition preserving pairing potential; there are six variant nucleotide positions in ITS2, one of which is a transversion in a single-stranded region, and five are transitions in single-stranded positions.
Phylogenetic analyses
A phylogenetic tree is presented in Fig. 1, one obtained using the parsimony algorithm of PAUP* with an heuristic search for the most parsimonious set of trees. Of the total of 721 positions of combined ITS1 and ITS2, 357 were parsimony informative. There are four major clades of Eudorina/Pleodorina taxa, labelled clades alpha, beta, gamma, and delta, plus two additional small clades, a Pleodorina indica grouping and the clade of two Volvox species. The ingroup topology of the tree does not change if Chlamydomonas alone or Chlamydomonas plus Yamagishiella (perhaps the closest clade of colonial organisms) is used as outgroup. The same six clades with essentially identical branching pattern are found by all methods of analysis, whether gaps are defined as a fifth character or not, and are supported by bootstrap values >74% in both parsimony and distance analyses. They also appear unchanged in similar analyses including additional Volvox, Platydorina, and Pleodorina species (Coleman, 1999
); likewise, use of only the ITS2 alignment, or even ITS2 relatively conserved positions, gives the same set of clades. The only one of these six clades that changes position in the tree is that of the two Volvox species; the same parsimony analysis that produced the tree depicted in Fig. 1 yielded a group of equally parsimonious trees where the Volvox gigas/powersii clade is associated with clade delta, albeit with low bootstrap support. However, this same positioning appears consistently in distance analyses, with bootstrap support as high as 86%.
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The insets in Fig. 1 present the putative secondary structure of the ITS1 and ITS2 RNA transcripts, a structure common to all the isolates as determined using mfold and sequence comparisons. The regions of sequence characteristically most conserved among species of a genus and genera in a family (Coleman and Mai, 1997
; Mai and Coleman, 1997
; Coleman et al., 1998
) include certain pairing regions in the secondary structure, highlighted in Fig. 1. In ITS1 they are limited to the basal three pairings of the first three helices and all the pairings of helix 4. In ITS2 they are the pairings of the first 13 nucleotides in hairpin loop II (the one that contains the characteristic pyrimidine-pyrimidine pairing) and the pairing involving the very highly conserved sequence lying 5' to the tip of loop III. The "proof" of secondary structure by phylogenetic comparisons is the presence of compensating base changes that preserve the helical stems. The strongest "proof" is the finding of compensating base changes (CBCs) where one example has, for instance, a G-C pairing at its position in a helix and another taxon has at that same pairing position an A-U; both nucleotides have changed, and changed in such a way as to preserve the pairing potential. Hemi-CBCs, where only one nucleotide of the pair is altered and retains the pairing potential (e.g., A-U to G-U) are numerous among the Eudorina/Pleodorina/Volvox representatives, and there are a number of CBCs supporting the secondary structure among this set of organisms.
Parallelism of phylogenetic position and mating affinity
In all cases where mating potential between taxa is known (Table 2 of Goldstein, 1964
), organisms capable of intercrossing to form zygotes are nearest neighbors on the tree. From this results of his attempts to obtain crosses, Goldstein divided his Eudorina taxa into five groups, called syngens. Organisms in the same syngen are capable of forming zygotes when paired, but fail to form any zygotes when paired with any isolate of another syngen, with rare exceptions (Sonneborn, 1957
). Goldstein's syngen designation is included in Table 1. In the phylogenetic tree of Fig. 1, Goldstein's syngen I and II examples are found in the upper half and syngen III in the lower half of clade gamma. His syngen IV strains all fall within clade beta. Syngen V is confined to clade alpha. Clade delta is comprised primarily of true homothallic isolates (see below), among which at least two produce zygotes within a clonal culture that are capable of germination and production of large numbers of viable, fertile products. None of the "homothallic" clones studied by Goldstein yielded viable zygotes (see DISCUSSION).
Hybrids
Table 2 summarizes the lack of variation found among cloned sequences of the parents, and the types of variation among cloned sequences of the four hybrids. There are two methods of obtaining sequences. The first is to sequence the mixture of PCR products directly, and the second is to first subclone each ITS repeat from the PCR product mixture. Direct sequencing of the mixture of PCR products from each parental isolate, 1192, 1193, 807, and 808, revealed no ambiguity or difference in sequence from that of the cloned repeats for the parental strains. However direct sequencing of the PCR products from each of the four hybrid strains gave clear sequence only until the first region differing between the two parents was reached, at which point "N"s began to appear (automatic sequencing) or shadow bands on the autoradiograms generated from manual sequencing. Clearly there is a mixture of ITS types in the hybrids that results in ambiguities when the mixture of PCR products is subjected to direct sequencing.
When we inserted individual ITS PCR products into T-vector and sequenced the cloned ITS cistrons individually, the sequence of a clone was unambiguous. As shown in Table 2, there was no variation among the subclones from parental strains except one clone of 808 that was missing a CAA of the CAA repeats in the low G region of ITS1 (Fig. 1A). Among the first six ITS subclones of each hybrid isolate sequenced we obtained some ITS examples indistinguishable from one parent and some indistinguishable from the other. The exception was hybrid 1225, where six subcloned ITS sequences were all identical to the 1192/3 parent. Perhaps further subclones would yield the sequence of the other parent. Furthermore, though only six clones were sequenced for each of the algal isolates in question, we obtained from two of the hybrids a few ITS clones that were distinctly different from either parent. Figure 2 shows the alignment of the regions where the two parental types differ in sequence significantly, regions (boxed in the Fig. 1 insets) that are found in the terminal loops of helices 1, 2, and 3 in ITS1 and the terminal loops of helices I and II in ITS2. One cloned sequence (1224-63) was identical in ITS1 to one parent and identical in ITS2 to the other parent, identical not only for the sequences shown aligned in Fig. 2 but also for all five of the single nucleotide positions that distinguish unambiguously between the parental types, as well as the four sites of distinctive single or double nucleotide insertion/deletions. Another (1226-1) was identical to one parent from the 5' end until loop II of ITS2 but loop II typified the opposite parent. A final variant (1226–61) had an altered loop 3 in ITS1, and its loop II of ITS2 differed by one transition from the other parental type, a change that alters the predicted transcript folding. These results suggest that, although the hybrids had not undergone a normal meiosis since they retained two sets of chromosomes, there had been some close association between the two homologues carrying the ribosomal repeats, such that crossover had occurred.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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contains an analysis of the morphological characters utilized in classical species taxonomy, the range of chromosome numbers found, and the sexual behavior of the clones. With respect to morphological characters, the most revealing summary is that forms recognizable as E. elegans were found among four of the five sexually isolated groups (syngens) established by Goldstein. In his syngen IV there are three taxonomic types, E. elegans, E. cylindrica, and Pleodorina illinoisensis.
The species names cited in Table 1 are those used by the authority who isolated the culture initially. The species criteria deal largely with three aspects, sexual characteristics, overall colony shape, and the tendency for the anterior-most cells in the colony to fail to undergo daughter colony formation—they merely die. A set of such anterior cells was the major original taxonomic characteristic of the genus Pleodorina. The dispersion of the various species of Eudorina in the tree of Fig. 1 results in part from the utilization of aspects of the gelatinous matrix as primary species characters for taxonomy. The amount and disposition of the colony gel matrix seems quite variable both here and in other Volvocacean groups (Coleman, 2001
). For example, the species of the genus Volvulina are, by DNA sequence, derivatives of various Pandorina morum clades, and the 16-celled Gonium species other than Gonium pectorale [eight isolates of G. quadratum and two isolates of G. multicoccum (GenBank entries AF182430, AF182431, AF504058–AF504060, U66970, U66971, U66979, U66966, and U66967)] have ITS sequence relationships that do not conform with their species determination (Fabry, Kohler, and Coleman, 1999
; Coleman, unpublished data). There is probably no way to define Eudorina species by morphology in a way that matches their phylogenetic affinity by DNA sequence. Even the character of single versus multiple pyrenoids per cell fails. This latter designation characterizes the isolates where pyrenoid replication precedes cell division significantly, resulting in multiple young pyrenoids per cell.
With respect to the classification of clones as male, female, or additional categories, the situation appears more complex in Eudorina than in such isogamous relatives as Pandorina or Gonium. In Goldstein's Table 2, a summary of all the mating results from the strains he tested, he designates five subgroups, syngens, within each of which all or most pairings are successful in producing zygotes but between which there is essentially total reproductive isolation. There are some exceptions. Two male strains (17m = UTEX1209 and 62m = UTEX1201) were equally compatible with females of syngens I and II; a close relationship between syngens I and II is reflected in Fig. 1. For his mating studies, Goldstein used not only clones that behaved exclusively as females, or as males, but also a number of "selfing male" clones (5 "sm" of a total of 24 males) where "zygotes" were formed in small numbers intraclonally while large numbers of zygotes were formed when such males were paired with an appropriate female clone. The few zygote-like bodies formed intraclonally were never observed to germinate, and there is no observation of their being formed by fusion of two cells. Their nature remains undiscovered. However, at least two of the more recently collected Eudorina strains are fertile homothallics (Table 1), and these all fall in Clade delta. Unfortunately, it was not possible to assess the potential for mating of newly collected strains with those of Goldstein's study, for his Eudorinas fail to express their sexual cycle after years in culture, a problem typical of Volvocaceae (Coleman, 1975
, 2001
). However, the sexual affinity, as reported by Goldstein, parallels perfectly the genetic affinity as established by DNA sequence comparison.
ITS results
As has been found previously in studying Volvocaceae (Coleman, 1999
, 2001
), homogenization of ribosomal cistrons seems sufficiently complete within an organism as to offer no hindrance to utilizing either direct or subclone sequencing results. Furthermore, the clades resulting from the ITS sequence analysis here are in full agreement with prior DNA sequencing studies of some of the same entities, using the rDNA small subunit (Larson, Kirk, and Kirk, 1992
), rbcL (Nozaki et al., 1997
), nuclear ITS (Angeler, Schagerl, and Coleman, 1999
; Coleman, 1999
), and five plastid genes, rbcL, atpB, psaA, psaB, and psbC (Nozaki et al., 2000
). The most inclusive plastid DNA study (Nozaki et al., 1997
), although omitting the two Volvox species utilized here, revealed the same five Eudorina/Pleodorina clades found, with the proviso that our clade delta is probably the same as group C in Nozaki et al. (1997)
. We have not used any of the same clonal cultures as presented in his group C, but in both cases, this clade contains a high proportion of true homothallic clones. Nozaki et al. (1997)
included Pleodorina illinoisensis, with the same result as here; the species is polyphyletic. Pleodorina indica appears in the same position (their group A and our clade alpha) within the greater Eudorina group. Their group B equates to clade beta, and their group D equates to the upper subclade of clade gamma; Nozaki et al. (1997)
did not include any representative of Goldstein's syngen III.
The inclusion here of two Volvox species resulted from the findings of an earlier more limited study of ITS in all genera of the family (Coleman, 1999
). This revealed the sequence similarity of the two Volvox species, and only these two, with Eudorina. The result is not totally unanticipated for three reasons. Shaw (1916)
, who isolated the organisms that came to be named V. powersii, considered it a form intermediate between Pleodorina and Volvox. Pocock, who isolated V. gigas (Pocock, 1933
), stated (Cave and Pocock, 1951
) that these two Volvox species were "primitive" and different from other Volvox representatives. The two species, though producing very large colonies with excessive extracellular matrix, have actually the smallest number of cells per colony among Volvox species, at most 1000–2000. Finally, VandeBerg and Starr (1971)
reported obtaining a stable morphological variant clone of V. powersii that had typically 128 cells with the anterior quarter nonreproductive. In essentially all its features, it resembled a Pleodorina. This variant subclone was lost before it could be analyzed genetically.
ITS transcript secondary structure
A knowledge of the ITS1 and ITS2 RNA transcript secondary structure provides not only a criterion for alignment but also an explanation for the relative level of primary sequence conservation of different subregions (Coleman and Mai, 1997
; Mai and Coleman, 1997
). Studies of rRNA processing have revealed the importance of secondary structure of the ITS RNA transcript to provide guidance for the processing necessary to form ribosome components (reviewed in Venema and Tollervey, 1999
). The significant subregions for ITS1 are still not fully clear, but for ITS2 the folding depicted in Fig. 1B now seems to be nearly universal among eukaryotes, with its pyrimidine-pyrimidine bulge in helix II and its most highly conserved primary sequence on the 5' side of helix III (Joseph et al., 1999
; Coleman, 2001
). Thus, in the regions of relative conservation there are only two CBCs (one in arm II and one in arm III of ITS2) among all the sequences in the tree of Fig. 1. The only other Eudorina clone (by morphology) available that we have sequenced is ASW05157from Austria. As reported earlier (Coleman, 1999
), this organism clades with Platydorina and it differs from all the other Eudorinas by two additional CBCs in the relatively conserved pairings of ITS2 (Coleman, 2000
).
In turn, this information provides an approach to comparisons at higher taxonomic levels among genera of the family. If genetic distance (p value) or the number of CBCs (in the relatively conserved pairings of ITS2 transcript secondary structure) can each be considered a measure of the passage of evolutionary time among Volvocaceae, then the Eudorina/Pleodorina clade (p = 0.333) is intermediate in age between the Gonium pectorale clade (ITS p value = 0.115) and the Pandorina/Volvulina clade (p = 0.450), the latter having by far the longest evolutionary history (Coleman, 2000
). The G. pectorale clade, though spread worldwide, has no CBCs in relatively conserved pairings of ITS2, the Eudorina/Pleodorina clade has two, while the Pandorina/Volvulina clade encompasses 13 CBCs. Finally, the G. pectorale clade has 1–2 of the mating subgroups, akin to syngens, called Z species (Coleman, 2000
); the Eudorina/Pleodorina clade has >5; and the Pandorina/Volvulina clade has >25.
The hybrids
For most of the clones examined by Goldstein, the haploid chromosome number was 14–15, but a few contained 5, 6, or 7 chromosomes. The parental strains of the E. elegans x P. illinoisensis hybrid had 14 chromosomes, while the four F1 strains he examined had a polyploid or aneuploid number of 24–28. Normally, a single product survives from meiosis in a Eudorina or Pleodorina zygote, forming a small colony that engenders a new clonal culture. For both the parental pairings (1192 x 1193 and 807 x 808), zygotes showed >50% survival of offspring and 1:1 segregation for mating type, while the hybrids between Eudorina elegans x Pleodorina illinoisensis gave low germination and very poor survival of the zygote products, even of those capable of forming an initial colony. Mating type ratios were skewed heavily to apparent males. Backcrosses of the F1 hybrid clones to the parentals failed to produce any viable colonies; however intercrossing of F1's showed limited success and produced an array of female, male, selfing male, selfing female, and homothallic clones, all with chromosomes numbers in the 20s. Thus the F1 hybrids appear to have arisen from unreduced zygotes, as are "diploid" strains of Chlamydomonas obtained experimentally (Harris, 1989
).
The Pleodorina x Eudorina cross that Goldstein studied is found in syngen IV (Clade beta on the tree in Fig. 1). Both of the UTEX 1192/3 strains have been sequenced and do not differ, and likewise both of the UTEX 807/8 pair were sequenced, with only a trivial difference found in one ITS subclone. However, these two mating pairs are not the most closely allied by ITS comparisons; another pair of syngen IV strains, UTEX 1196/7, is more similar by ITS sequence (p = 0.03) to UTEX 807/8, but Goldstein did not study the zygotes from the cross of these. The greater genetic distance (p = 0.08) between UTEX 1192/3 versus UTEX 807/8 may help to explain why the resultant hybrid zygotes germinated so poorly, and the products rarely survived, as well as the unreduced chromosome number of the surviving products.
Typically, there are >100 copies of the rDNA cistrons in a eukaryote genome, arranged in tandem on one chromosome, the "nucleolar organizer" region. The multiple copies in the tandem array are identical or nearly so, a consequence of a complicated and as yet poorly understood process called concerted evolution (Dover, 1982
; Elder and Turner, 1995
). The means by which the many tandem copies maintain their identity in the face of presumably accumulating mutations is a subject of continuing research in parallel with the search for exactly what subcharacteristics of the ITS sequences are required for accurate processing of the transcripts to yield ribosomal RNA (Hillis et al., 1991
).
The essential homogeneity of the tandem repeat sequences in an organism's genome proves true here, with the exceptions of the four hybrid organisms. Sequencing individually cloned copies of the cistrons found in the hybrid organisms revealed a totally unexpected range of variation. From analysis of only six subcloned repeats of the four hybrid strains (thus a total of 24 subclones), three non-parental ITS rearrangements were found. In Fig. 2, 1226-1 is explainable simply by a crossover between helix I and helix II of ITS2, within 2–3 nucleotides following ITS2 helix I. This was the sequence utilized for the PAUP alignment, with the result that its placement on the tree is intermediate between the two parental types, as would be the 1224-63 and 1226-61 sequences (tree not shown). The 1224-63 clone likewise could be a crossover somewhere between helix 3 of ITS1 and helix I of ITS2, more specifically, somewhere between the low G region in ITS1 and the first helix in ITS2. Clone 1226-61 shows ITS1 helices 1 and 2 and ITS2 helix I identical to 1192/3, as well as all the rest of the ITS1 and ITS2 sequence, but helix 3 of ITS1 and helix II of ITS2 are anomalous, the latter differing from 807/8 by a single nucleotide position. Simple crossover cannot explain the sequence of this clone.
The variety of sequences found among the individual ITS repeats in the hybrids, not just both parental types but also recombinant types and novelties, is truly unusual, compared to all the other volvocacean isolates studied, and suggests that such variety, when found, reflects a recent hybridization event. Recognition of such a hallmark may be useful in analyses of other organisms.
The organisms here are the only haplonts in which ITS sequence has been studied in a known hybrid. Understanding of the homogenization process affecting ITS repeats might benefit from further use of appropriate haplonts. Studies of ITS in diplont hybrids, particularly plants (e.g., Sang, Crawford, and Stuessy, 1995
; Wendel, Schnabel, and Seelanan, 1995
; Campbell et al., 1997
; Alice et al., 2001
; Mayol and Rossello, 2001
; Muir, Fleming, and Schlotterer, 2001
) also suggest that ITS sequences resulting from crossover or other events have occurred, but none has been analyzed using multiple clones and knowledge of ITS transcript secondary structure, supported by comparative analyses of CBCs, to understand the locale of the variation. We can say from the data here that 40 yr of vegetative propagation has not produced ITS cistron homogenization; what we cannot say is the state of the repeats 40 yr ago and whether years of vegetative reproduction might have contributed to ITS diversity when disparate homologues are present in the same nucleus.
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FOOTNOTES |
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2 Annette_Coleman{at}brown.edu
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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