Shigeo Masuyama

Molecular systematics of the Asplenium nidus complex from Mt. Halimun National Park, Indonesia: evidence for reproductive isolation among three sympatric rbcL sequence types.

Asplenium nidus is an epiphytic fern with large simple leaves. Because A. nidus lacks the good taxonomic characters available for species recognition, multiple cryptic species may exist within A. nidus. In Mt. Halimun National Park, West Java, Indonesia, three rbcL sequence types of A. nidus were recorded. All plants regardless of rbcL sequence were 2n = 144. Crossing experiments among these rbcL types were conducted, and it was observed that the molecularly distinct types were reproductively isolated because hybrids failed to form between at least two pairs of rbcL types. These results suggest that these rbcL types are cryptic species because they are reproductively isolated but they are difficult to distinguish morphologically. Thus, the characters of DNA sequences information are useful in the discovery of cryptic species in ferns.

Reproductive biology of the fernPhegopteris decursive-pinnata

Four diploid plants and four tetraploid plants ofPhegopteris decursive-pinnata were investigated for determination of the reproductive characteristics of their gametophytes and two major features were recognized. First, gametophytes of the diploids showed an ontogenetic sequence of gametangium formation which is unfavorable for intragametophytic selfing, whereas those of the tetraploids showed that favorable for intragametophytic selfing. Second, 41 to 72% of the isolated gametophytes of the diploids produced sporophytes in the intragmetophytic selfing tests, whereas all of the isolated gametrophytes of the tetraploids produced sporophytes in the tests. Based on these developmental and genetic features of gametophytes, the dissimilar mating systems of the diploids and the tetraploids of this species are discussed.

Independent origins of tetraploid cryptic species in the fern Ceratopteris thalictroides.

Ceratopteris thalictroides (L.) Brongn is a tetraploid fern species that contains at least three cryptic species, the south, the north and the third type. In this study we combined data from both chloroplast DNA (cpDNA) and nuclear DNA sequences of three diploid species and three cryptic species of C. thalictroides to unravel the origin of the cryptic species, particularly of the reticulate relationships among the diploid and tetraploid taxa in the genus Ceratopteris. Of the three diploid species examined, C. cornuta had cpDNA identical to that of the tetraploid third type plants, and this diploid species is a possible maternal ancestor of the tetraploid third type. Analysis of the homologue of the Arabidopsis thaliana LEAFY gene (CLFY1) identified ten alleles in the genus Ceratopteris, with six alleles found in C. thalictroides. The unrooted tree of the CLFY1 gene revealed four clusters. Each cryptic species showed fixed heterozygosity at the CLFY1 locus and had two alleles from different clusters of the CLFY1 tree. Consideration of the cpDNA sequences, CLFY1 genotypes of the cryptic species and CLFY1 gene tree in concert suggested that the cryptic species of C. thalictroides had originated through independent allopolyploidization events involving C. cornuta and two unknown hypothetical diploid species.

Cryptic species in the fern Ceratopteris thalictroides (L.) Brongn. (Parkeriaceae). I. molecular analyses and crossing tests

Abstract For the taxonomic revision of the problematic species Ceratopteris thalictroides, molecular analyses and crossing tests were conducted for 16 sources in the world. An analysis of allozyme composition of five enzymes revealed the presence of three intraspecific entities, which were called the south type, the north type, and the third type. An analysis of the nucleotide sequences of chloroplast DNA also distinguished the same entities. Crossing tests showed that the south type was completely cross-sterile with the other two types, and that the other two were considerably cross-sterile with each other. These results suggest that the three entities should be regarded as different biological species. Although the south type and the other two meet in several regions, complete cross-sterility between them seems to sustain their genetic distinctiveness in spite of occasional crossing. The results from the present study suggest that widely distributed fern species are apt to comprise several cryptic species.

Genetic differentiation in populations of the polymorphic fernCeratopteris thalictroides in Japan

Analysis of isozyme variation was carried out for 27 natural populations ofCeratopteris thalictroides in Japan. Of fifteen enzyme loci examined, eight loci were genetically polymorphic. At six loci,Lap, Pgi-2, Pgm-3, Pgm-4, Idh-2, and Skd-2, a marked genetic differentiation was observed between populations to the south of Okinawa Island and those to the north of the island. Okinawa Island contained a mixture of both southern and northern variants. Thus, two genetically distinct types (the south type and the north type) ofC. thalictroides occur allopatrically in Japan. Neis genetic identity (I) between the two was 0.64, which was within the range of the I values between congeneric pteridophyte species. Regional fixation of a null allele was detected for one duplicated PGI locus in the north type ofC. thalictroides. This finding supports the recent hypothesis of genetic diploidization of polyploids through gene silencing.

Inbreeding in Natural Populations of the Annual Polyploid Fern Ceratopteris thalictroides (Parkeriaceae)

The degree of gametophytic selfing in natural populations was examined for the annual polyploid fern Ceratopteris thalictroides in several ways. Examination of sexual ontogeny of the gametophytes showed that two types of gametophytes were usually observed: large, cordate hermaphrodites with mature antheridia and archegonia and small, spatulate or amorphous males with numerous antheridia. The occurrence of the former may be correlated with gametophytic selfing, while that of the latter is indicative of the presence of antheridiogen, which can promote gametophytic crossing. Selfing potential was tested by growing isolated gametophytes. In this test, only 3 of 26 spore sources exhibited the presence of genetic load, and even the highest frequency of self-sterile gametophytes in these three was only 3.8%. These gametophytic results predicted a predominantly selfing mating system for this species. This prediction was tested using electropho- retic analysis of allozymes. Polymorphic band patterns were detected for IDH and PGI, and the genetic constitution of these variations was verified through progeny tests. Based on the frequencies of these allelically segregating variants, the rates of gametophytic selfing and F statistics were estimated for a population composed of five subpopulations. The estimated rates of gametophytic selfing were remarkably high, ranging from 0.600 to 1.000 with the mean FIS of 0.886. The estimated FST was 0.109 in IDH and 0.203 in PGI, values suggesting the occurrence of intrapopulational substructuring. The high level of gametophytic selfing in C. thalictroides is probably related to its polyploid nature and annually colonizing habit. An antheridiogen system may operate to compen- sate for the loss of genetic variability through predominant inbreeding.

Hybrid sterility between two isozymic types of the fernCeratopteris thalictroides in Japan

JapaneseCeratopteris thalictroides are composed of two allopatric entities called the south type and the north type which are isozymically distinct. Both types are tetraploid at 2n=156. To examine the crossability of these isozymic types, artificial hybridization was conducted among ten representative populations. Allozymes of PGI were used as markers to identify the hybrids among populations. Hybrids within the north type produced normal-shaped spores whose germination rate reached 60.7 to 96.0%. In contrast, hybrids between the north type and the south type formed irregular-shaped spores which were aborted with a low germination rate of 0.0 to 7.0%. It is thus evident that hybrid sterility is present between these types. Morphological analyses revealed that the two types were part of a continuous cline of variability and hence morphologically inseparable. The two types can thus be regarded so far as cryptic species that are morphologically indistinguishable but reproductively isolated.

Reproductive biology of the fern Phegopteris decursive-pinnata. II: Genetic analyses of self-sterility in diploids

It has been revealed that gametophytes of diploid plants ofPhegopteris decursive-pinnata have a low capability for intragametophytic selfing (Masuyama, 1979). In the present study, intergametophytic mating tests were conducted for the self-sterile gametophytes of four diploids to demonstrate the genetic factors responsible for such a low capability for selfing. The results of the tests indicated that the gametophytes carried two or more kinds of recessive embryonic lethal factors which were non-allelic with each other and that the occurrence frequency of the gametophytes with an identical recessive lethal factor was 13% to 27% in the gametophyte families of these four diploids The karyological study of a diploid sporophyte suggested not the tetraploid but the diploid constitution of somatic chromosomes. Based on these data, the diploid inheritance of two or three special deleterious genes with a synergistic interaction responsible for the embryonic lethality was hypothesized to elucidate the self-sterility in the diploids of this species.

Cryptic species in the fern Ceratopteris thalictroides (Parkeriaceae) : III. Referential diagnostic characters of three cryptic species

Three cryptic species of Ceratopteris thalictroides, named the south type, the north type and the third type, were examined for their morphological characteristics, using sporophytes cultivated under common conditions. The discriminant analysis for leaf characters followed by one-way layout ANOVA or Kruskal–Wallis test for selected combinations of characters revealed that the following characters may be effective for identifying the three types: the relative lengths of stipe to blade and to pinna, the degree of dissection, the segment densities on rachis and pinna rachis, and the elongation degree of ultimate segments. The number of annulus cells on sporangia also proved to be a possible distinguishing character. As morphological data were obtained from a limited number of cultivated sporophytes, they are regarded as not definitive, but only referential diagnostic characters of the types and should be utilized not solely, but collectively, to avoid identification errors of the types. An identification trial using herbarium specimens proved these diagnostic characters to be useful to a considerable degree.