SEXUAL AND GENETIC ISOLATION IN THE COSMOPOLITAN ALGAL SPECIES PANDORINA MORUM

In an effort to analyze the nature of a taxonomic species in the microalgae, the major biological characters of various isolates of Pandorina morum have been studied. Seventy heterothallic clones from 31 collecting sites on three continents were found to represent 20 reproductively isolated groups, based on their ability to form zygotes when paired in all possible combinations. Successful germination of the zygotes and demonstrated fertility of their products suggest that each reproductively isolated group represents a syngen—a group of organisms sharing a common gene pool. A further indication of the close relationship of members of the same syngen is that its representatives all share a common chromosome number, time of division, and zygote distribution pattern, characters which vary between syngens. The known range of 18 of the syngens is less than 500 km. The other two syngens may have a world-wide distribution; they both have a haploid chromosome number of 5. Crosses between geographically isolated clones in these two syngens and one other give evidence of both pre- and postzygotic partial isolation within a syngen. Analysis of collections from two revisited sites demonstrated that representatives of a syngen can be recovered after 19 years have elapsed, and that a single pond may contain as many as four syngens. The results obtained for P. morum are compared with the information available on other microalgae with a similar breeding system, and the possible causes and consequences of the species structure are discussed.     

A SIMPLIFIED,RAPID METHOD FOR IDENTIFYING MATING TYPE IN ALGAE: RESULTS WITH THE PANDORINA MORUM (CHLOROPHYCEAE) SPECIES COMPLEX1

As a collection expands, increasing numbers of test crosses are required to identify new isolates in algal species containing numerous pairs of mating types. A short-cut is described that utilizes a reduced number of preliminary test crosses. The method was used successfully with the colonial green flagellate, Pandorina morum Bory de St. Vincent, to identify a new pair of milling types (syngen) from Japan and five new isolates from China of a previously known syngen. When tested on the 20 previously described syngens, it revealed one unexpected multi-clone effect on mating. The method should be valuable for identifying newly collected clones, examining potential, interactions among incompatible mating types, and for screening other highly specific inter-organism interactions such as host-parasite infections.     

BIOGEOGRAPHY AND SPECIATION IN THE PANDORINA/VOLVULINA (CHLOROPHYTA) SUPERCLADE

Mating affinity, nuclear rDNA internal transcribed spacer (ITS) sequence, and geographic distribution of more than 100 isolates of Pandorina , Volvulina , and Yamagishiella were determined. Comparative analysis of ITS sequences reaffirmed the unity of Pandorina morum and its separation from all other species of Volvocaceae except those of Volvulina. This latter genus, represented by four species, appears to represent only morphological variants of several P. morum subclades. The P. morum clade (including also additional species such as P. smithii and P. colemaniae ) encompasses an evolutionary span, as determined by comparison of ITS, greater than either of the multispecies genera Gonium or Eudorina. There are at least 30 Pandorina / Volvulina syngens, sexually isolated groups, so far as can be determined, among the current collection of strains. In addition, as in other volvocacaean genera, two clones are homothallic, capable of forming zygotes within a genetic clone. The existence of so many syngens suggests that considerable evolutionary diversification of the genes controlling gamete compatibility and intercross survival has occurred, unaccompanied by significant morphological change. Within each syngen, genetic distance increases with geographic distance between collection sites. At least half of the isolates studied must have been introduced northward since the Pleistocene. Although we probably know more about characters in this group of algae than any other algal group, in part because they are so easy to culture, we are still largely ignorant of what circumscribes their niche in nature. The study of all these organisms, distributed throughout the world presumably by the activities of shorebirds, emphasizes the evolutionary role of mating genes in the inevitable formation of new genetically isolated subclades in these eukaryotes and provides initial data on their rate of appearance versus their rate of distribution over the earth.     

Intraspecies Analysis: Comparison of ITS Sequence Data and Gene Intron Sequence Data with Breeding Data for a Worldwide Collection of Gonium pectorale

The morphologically uniform species Gonium pectorale is a colonial green flagellate of worldwide distribution. The affinities of 25 isolates from 18 sites on five continents were assessed by both DNA sequence comparisons and sexual compatibility. Complete sequences were obtained (i) for the internal transcribed spacer ITS-1 and ITS-2 regions of ribosomal DNA and (ii) for each of three single-copy spliceosomal introns, two in a small G protein and one in the actin gene. ITS sequences appeared to homogenize sufficiently rapidly to behave as a single copy gene. Intron sequence differences between isolates in this species reached nucleotide substitution saturation, while ITS sequences did not. Parsimony and evolutionary distance analysis of the two types of DNA data gave essentially the same tree conformation. By all these criteria, the group of G. pectorale isolates fell into two main clades, A and B. Clade A, with isolates from four continents, was comprised of four subclades of quite closely related isolates, plus one strain of ambiguous affinity. Clade B was comprised of two subclades represented by South African and South American isolates, respectively; thus, only subclades of clade B showed geographical localization. With respect to mating, all isolates except one homothallic strain and one apparently sterile strain fell into either one or the other of two mating types. Pairings in all possible combinations revealed that isolates from the same site formed abundant zygotes, which germinated to produce new, sexually active organisms. Zygotes were also formed in many pairings of other combinations, including crosses of clade A with clade B organisms, but none of the latter produced viable germlings. The ability to mate and produce viable progeny that were themselves capable of sexual reproduction was restricted to members of subclades established on the basis of DNA sequence similarities. Thus, the grades of difference in both nuclear intron sequences and rDNA ITS sequences paralleled those observed in the sexual analysis. Received: 9 March 1998 / Accepted: 1 June 1998     

Comparison of the evolutionary distances among syngens and sibling species of Paramecium

The morphospecies of the genus Paramecium have several mating type groups, so-called syngens, composed of cells of complementary mating types. The Paramecium aurelia complex is composed of 15 sibling species assigned to the species from the syngen. To increase our understanding of the evolutionary relationships among syngen and sibling species of the genus Paramecium, we investigated the gene sequences of cytosol-type hsp70 from 7 syngens of Paramecium caudatum and 15 sibling species of P. aurelia. Molecular phylogenetic trees indicated that the P. aurelia complex could be divided into four lineages and separated into each sibling species. However, we did not find any obvious genetic distance among syngens of P. caudatum, and they could only be separated into two closely related groups. These results indicated that the concept of syngens in P. caudatum differs quite markedly from that of the P. aurelia complex. In addition, we also discuss the relationships among these species and other species, Paramecium jenningsi and Paramecium multimicronucleatum, which were once classified as varieties of P. aurelia.     

Mating Type Inheritance in Glaucoma*

SYNOPSIS. Mating was observed in collections of Glaucoma from 2 localities in Illinois. The collections could be divided into 2 syngens on the basis of mating type reactions. Corticotype analysis showed that syngen 1 was intermediate in meridian number between Glaucoma chattoni and Glaucoma scintillans, while syngen 2 had the same range as Glaucoma scintillans (Lee, unpublished). The major features of mating type inheritance in syngen 1 are that exconjugant clones usually have identical mating types and that mating types I and II appear in an approximately 1:1 ratio in successive generations. This is the result expected in genic mating type inheritance if one of the original parents was a homozygote and the other a heterozygote at the mating type locus. No significant immaturity period was found in these strains. Only 2 viable pairs were obtained from syngen 2 crosses, but results from these suggest that mating type inheritance may be epigenetic and a longer immaturity period may characterize this syngen.     

Comparison of the Esterases and Acid Phosphatases in Paramecium multimicronucleatum,Syngens 1–5, P. jenningsi,P. caudatum,and the P. aurelia Complex1

ABSTRACT. Forty-eight stocks in Paramecium jenningsi, syngens 1–5 of P. multimicronucleatum, P. caudatum, P. primaurelia, P. biaurelia, and P. tetraurelia were grown axenically and tested for their esterases and acid phosphatases using starch gel electrophoresis. The five esterases and the acid phosphatases previously characterized in species of the P. aurelia complex were also found in P. jenningsi, and three to four of the esterases and the acid phosphatases were found in the P. multimicronucleatum species complex and in P. caudatum. Additional subtypes were observed for each of the enzyme phenotypes in these new (though here unnamed) species of Paramecium. Two of the new acid phosphatase subtypes, which depart radically in mobility and in pattern, were found in syngen 3 of P. multimicronucleatum and in P. caudatum. Except for syngens 1 and 5 in P. multimicronucleatum, the degree of similarity between syngens 1, 5 and 2, 3, and 4 appears to be very low—perhaps even lower than that seen for species in the aurelia complex. More realistically, the syngens of P. multimicronucleatum should be considered as separate species although they are not here given separate taxonomic names. Limited sharing of subtypes occurred between species in different species complexes. This observation suggests that the molecular distances between species complexes may be even greater than between species within a complex.     

Three-locus analysis in conjunction with strain crosses confirms the existence of reproductively isolated populations in Paramecium jenningsi

Paramecium jenningsi (Diller & Earl, 1958) was formerly considered to be a species with only one syngen (genetic species) based on an inter-strain cross of two strains, cytological analysis, and an investigation of esterases and acid phosphatases. However, the existence of syngens within the species was later suggested by genetic studies, i.e. classical strain crosses of new strains and molecular PCR-based analyses (RAPD, RFLP), as well as by sequencing the H4 gene fragment. This issue still needs to be clarified by the application of molecular markers, genetic tests and cytological preparations. In the present study, we tested 12 strains of P. jenningsi originating from Asia, North America and Africa. Trees reconstructed on the basis of three genome fragments (ITS1-5.8S-ITS2-5’LSU, COI and CytB) show that P. jenningsi is divided into two distinct clusters (PJ1, PJ3) and one branch (PJ2) which correspond to reproductively isolated groups revealed by strain crosses. A study based both on strain crosses and a three-locus comparison gives the opportunity for a more complete identification of the reproductively isolated populations of P. jenningsi and other ciliate species.     

Enzyme Variation in Paramecium caudatum

Stocks of four “syngens” (syngens 1, 3, 12, 13) of Paramecium caudatum could not be distinguished on the basis of isozymic variations of six enzymes. Using the most common enzyme form as the standard for the syngen, we found a higher coefficient of identity between syngens (>90%) than within syngens (73%). These observations, combined with evidence for fertile matings among the groups, suggest that the groups do not warrant the status of syngens. True syngenic variation in P. caudatum is, however, clearly established on the basis of isozymic and breeding studies of wild stocks collected from various places. Some stocks from England have a close affinity with those from Japan, but stocks from Scotland and California must be placed in separate syngenic sets. Thus, P. caudatum is indeed a species complex, within which evolutionarily distinct groups (species = syngens) are identifiable. The definition of syngens on the basis of initial agglutination response is, however, unjustified.     

The Syngens of Paramecium bursaria: New Mating Types and Intersyngenic Mating Reactions

SYNOPSIS. Three syngens of Paramecium bursaria have been identified amongst stocks collected in Scotland. These syngens probably correspond to the previously-described syngens 4, 5 and 6; they have been so named. In all 3 syngens 8 mating types have been found. An extensive series of intersyngenic mating reactions has been discovered between stocks of syngens 4 and 5, and between stocks of syngen 2 and syngens 4 and 5.     

Cortical Patterns in Two Syngens of Glaucoma*

SYNOPSIS. Strains of 2 syngens of Glaucoma (16) were found to have different cortical characteristics. All clones of a particular strain had approximately the same range of corticotypes, approximately the same means and high and very similar variances. The chief differences between the syngens was the corticotypic range. The patterns of variation of cortical elements of syngen 2 appeared to be primarily extensions of the patterns of syngen 1. The range of meridian numbers of different species of Glaucoma overlapped with each other and could not be distinguished by this criterion alone.     

Genetic control of lactate dehydrogenase isozymes in Paramecium caudatum

The LDH isozymes were surveyed in bacterized cultures of syngens 1, 3, 12, and 13 of Paramecium caudatum by polyacrylamide gel electrophoresis. All the examined stocks of different syngens except one stock in syngen 3 had a single common LDH isozyme, and intra- and intersyngenic variation was not observed except for the one stock in syngen 3. Breeding data using the exceptional stock indicated that the LDH isozymes of P. caudatum are controlled by two codominant alleles at a single locus whose products aggregate randomly, forming a dimer.     

The Axenic Culture of Strains of the Various Syngens of Paramecium aurelia*

SYNOPSIS Strains of the various syngens of Paramecium aurelia respond differently to the culture media developed for Strain 299 of syngen 8. Not only is there a variety of response between the syngens, but strains belonging to the same syngen respond differently to the 3 growth media.     

Intraspecies Variability in the Esterases and Acid Phosphatases of Paramecium jenningsi and Paramecium multimicronucleatum: Assignment of Unidentified Paramecia; Comparison with the P. aurelia Complex1

ABSTRACT. Enzyme electrophoresis was exploited to identify stocks of paramecia previously not identified to particular species. Stocks collected in India and one from Panama belong to Paramecium jenningsi, while others collected in Panama or in Brazil are assignable to syngen 2 of P. multimicronucleatum on the basis of similarity of their esterase and acid phosphatase phenotypes. Inclusion of these doubled the numbers of stocks available in the two species, thereby facilitating examination of intraspecies variation and comparison of particular features of intraspecies variation found for the P. aurelia complex. Variant stocks were observed in P. jenningsi and in syngens 2, 3, and 4 of P. multimicronucleatum. In some cases the variant lacked the enzyme; in others, a change in mobility of the enzyme occurred that resulted in an electrophoretic form similar to one common in another species. Unique phenotypes were displayed by the variants of syngen 2 in P. multimicronucleatum. Hypervariability for Esterase B was observed in this syngen, where, in addition, several subtypes were seen for three other esterases. Unique phenotypes and hypervariability were also noted in P. biaurelia. Clustered variations were observed in these species and in the P. aurelia species. Unlike the situation for members of the aurelia complex, where lack of geographical differentiation between stocks in the same species is a unique feature, some such differentiation does occur in P. multimicronucleatum-2. The frequency of variant stocks in P. jenningsi was similar to that observed in the aurelia sibling species. In contrast, a significantly higher frequency of variant stocks was found in syngens 2, 3, and 4 of P. multimicronucleatum.     

Intersyngenic variations in the esterases of bacterized Paramecium aurelia

The esterase isozymes were surveyed in bacterized stocks representative of all 14 syngens of Paramecium aurelia by starch gel electrophoresis. The properties of substrate specificity and independent variation of particular isozymes permit the ordering of the differences observed among stocks. Differences can arise from several sources: bacterial variation, intrasyngenic variation, and intersyngenic variation. Bacterial esterases tend to be found in certain zonal areas (see Rowe et al., 1971) and produce minor stock differences, which are erratic in their distribution. Unlike the situation found in Tetrahymena pyriformis, major intrasyngenic variations are rare in P. aurelia except in syngen 2. This lack of intrasyngenic variation is significant in view of the wide differences in geographic origin and micronuclear chromosome numbers among stocks within a syngen. It suggests that certain esterase genotypes must be under stringent selection within a syngen. The lack of intrasyngenic variation permits assessment of intersyngenic relationships. Syngens differ in a complex way from each other, suggesting that several gene differences may be involved. The syngens can be classified on the basis of their esterases. Syngens which have been shown to be more closely related in terms of cross-mating, breeding systems, and other criteria tend to be more similar in their esterase isozymes. The isozyme technique confirms relationships previously suggested among syngens and offers the promise of eventual assessment of evolutionary distances among syngens. However, establishment of these relationships will be clearer in the absence of bacteria.Supported by a Research Grant, GM-15879, from the National Institute of General Medical Sciences, U.S. Public Health Service     

Isozyme band variation within the algal species Pandorina morum

Ten clones from five collections of the freshwater green alga Pandorina morum, taken from four sites and representing three syngens, were analyzed for electrophoretic variants of five enzyme. Isozyme bands of different mobility were found in isolates from different syngens, different sites, and even the same site and syngen; the pattern of variation differed with the enzyme studied. The results suggest care is needed in choosing isozyme mobilities as syngen or species characters.     

The linear 20 kb mitochondrial genome of Pandorina morum (Volvocaceae,Chlorophyta)

A physical restriction map of the mitochondrial genome from one clone (TCC 854) of the sexually isolated populations (syngens) of the morphologically uniform species Pandorina morum Bory has been constructed using restriction endonucleases Ava I, Bam HI, Bgl II, Eco RI, Kpn I, and Pst I. The 20 kb linear genome can easily be separated from plastid DNA, nuclear satellite rDNA, and main band (nuclear) DNA on a Hoechst/CsCl buoyant density gradient. The Pandorina mitochondrial DNA shows sufficient similarity to the 16 kb mitochondrial genome of Chlamydomonas reinhardtii to cross-hybridize, and also hybridizes with a probe containing maize mitochondrial 18S rRNA genes. Double digests, self-probing, and Bal31 exonuclease experiments suggest that 1.8 to 3.3 kb of sequence is repeated at each end of the genome as an inverted repeat. Mitochondrial genome sizes of other P. morum syngens were found to range from ca. 20 to ca. 38 kb. The mitochondrial genome should be valuable for taxonomic studies; it can be used for comparative organellar studies; and it should be of interest to compare with that of other plant and animal mitochondrial genomes.     

Interspecies Crosses in Paramecium aurelia (Syngen 4 by Syngen 8)*

Two syngens (biologic species) of Paramecium aurelia that appear to be closely related were crossed. Parental stocks carrying different homozygous recessive marker genes were utilized to identify true hybrids (those that had undergone cross-fertilization followed by normal nuclear processes). From these crosses of syngens 4 and 8, 32% of the conjugants survived but only 9% (27% of the survivors) were true hybrids. All 19 viable hybrids recovered were cytoplasmically descended from the syngen 8 parent; but the viable nonhybrids were cytoplasmically descended from either of the 2 parents and with equal frequency from each. Surprisingly, the hybrids could not be infected with the symbiont kappa, even though they should have carried a K gene donated by their syngen 4 parent which is necessary and sufficient to allow infection of the syngen 4 parent stocks. The hybrids required special care to cultivate and were sterile, thus indicating that they are at an evolutionary dead end. However, one type of nonhybrid clone produced by the intersyngenic conjugants was able to produce viable progeny. It is speculated that genetic elements of the cortex and in the cytoplasm (e.g. mitochondria) could be transferred intersyngenically via this type of nonhybrid clone.     

Analysis of natural diversity of symbiotic relationships in the Paramecium bursaria--Holospora curviuscula system

Bacteria of the genus Holospora belong to obligatory endonucleobionts of ciliates of the genus Paramecium. The bacteria show specificity towards the particular host species and the types of nuclei they infect: macro- or micronuclei. During a long-term screening of P. bursaria clones, belonging to three different syngens, Holospora inhibited cells of two syngens only. Using the number of host clones and symbiont isolates, it was shown that H. curviuscula was unable to pass successfully through the syngen barrier even under experimental infection. Considering the species level of specificity in Holospora associations of P. caudatum we suggest the existence of a greater evolutionary divergence in P. bursaria syngens than in syngens of P. caudatum. We have revealed that in incompatible combinations "host clone--symbionts isolate" the complicated bacterial life cycle may be blocked at definite stages depending on genetic features of both partners. Thus, the recognition of the full block spectrum could break the continuous infection process down to independently controlled steps. The block spectrum revealed in the system of P. bursaria--H. curviuscula demonstrates its significant similarity to block spectra of other systems within the Holospora--Paramecium complex. A block of transverse binding formation has been first revealed in Holospora dividing in the nucleus.     

Phylogenetic relationships of Paramecium jenningsi strains (classical analysis and RAPD studies)

The present studies with application of classical strain crosses and RAPD-PCR analyses showed the existence of different genetic species (syngens) within Paramecium jenningsi. So far the existence of only one syngen has been accepted. It was found that strains from Saudi Arabia, India, and China compose one genetic species (syngen 1) and six strains from Japan compose second genetic species (syngen 2).