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 fl agellate of worldwide distribution. The affinities of 25 isolates from 18 s ites 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 e ach of three single-copy spliceosomal introns, two in a small G protein and one in the actin gene. ITS sequences appeared to homogenize sufficiently r apidly 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 clade s, A and B. Clade A, with isolates from four continents, was comprised of f our subclades of quite closely related isolates, plus one strain of ambiguo us affinity. Clade B was comprised of two subclades represented by South Af rican and South American isolates, respectively; thus, only subclades of cl ade B showed geographical localization. With respect to mating, all isolate s except one homothallic strain and one apparently sterile strain fell into either one or the other of two mating types. Pairings in all possible comb inations revealed that isolates from the same site formed abundant zygotes, which germinated to produce new, sexually active organisms. Zygotes were a lso formed in many pairings of other combinations, including crosses of cla de A with clade B organisms, but none of the latter produced viable germlin gs. The ability to mate and produce viable progeny that were themselves cap able of sexual reproduction was restricted to members of subclades establis hed on the basis of DNA sequence similarities. Thus, the grades of differen ce in both nuclear intron sequences and rDNA ITS sequences paralleled those observed in the sexual analysis.