Yasuyuki Watano

PHYLOGENETIC RELATIONSHIPS WITHIN CLADOPHORALES (ULVOPHYCEAE, CHLOROPHYTA) INFERRED FROM 18S rRNA GENE SEQUENCES, WITH SPECIAL REFERENCE TO AEGAGROPILA LINNAEI 1

The phylogenetic position of a freshwater green alga, Aegagropila linnaei (Cladophorales, Ulvophyceae), was investigated using nuclear 18S rRNA gene sequences. This alga has usually been called Cladophora aegagropila (L.) Rabenhorst or Cladophora sauteri (Nees ex Kütz.) Kütz. Based on morphology, it was formerly classified into the section Aegagropila or into the subgenus Aegagropila, together with several marine species of the genus Cladophora. This classification is not supported by the present phylogenetic analyses in which two very distinct Cladophorales clades are recognized. Aegagropila linnaei groups together in a well‐supported clade with Cladophora sp., Pithophora sp., Chaetomorpha okamurae, Arnoldiella conchophila, Wittrockiella lyallii, and Cladophora conchopheria. Aegagropila linnaei and its closely related species share some ultrastructural and biochemical characteristics, like pyrenoid structure, carotenoid composition, and cell wall composition. Freshwater species, included in the analysis, were located in two distantly related lineages, indicating that adaptation from a marine to a freshwater habitat has happened at least twice independently in the Cladophorales.

Genetic structure of hybrid zones between Pinus pumila and P. parviflora var. pentaphylla (Pinaceae) revealed by molecular hybrid index analysis

Pinus species have three differently inherited genomes: paternal chloroplast, maternal mitochondrial, and biparental nuclear. Our previous study on the hybrid zones between alpine Pinus pumila and montane to subalpine P. parviflora var. pentaphylla demonstrated contrasting patterns of introgression of two cytoplasmic genomes, i.e., the paternal cpDNA flowed from P. parviflora var. pentaphylla to P. pumila, and the maternal mtDNA flowed in the reverse direction. In the present study, we developed codominant nuclear DNA markers diagnostic or mostly diagnostic for each parental species by single-strand conformation polymorphism (SSCP) of polymerase chain reaction (PCR) products, using expressed sequence tag (EST) primers of Pinus taeda. To describe the introgressive patterns of the nuclear genes, the molecular hybrid index (MHI) showing the overall proportion of alleles inferred to be derived from P. pumila was determined for each plant collected in hybrid zones on Mt. Asahidake and Mt. Higashiazuma, Japan. At Mt. Asahidake, the MHI values changed clinally according to the altitudes at which the plants were collected. However, at Mt. Higashiazuma, there was a gap in the MHI values between the plants above and below the Abies and Tsuga forest zone (alt. 1800-1900 m). This suggested that the zone plays a role in creating an effective barrier to gene flow in the hybrid zone.

Incongruence among mitochondrial, chloroplast and nuclear gene trees in Pinus subgenus Strobus (Pinaceae)

Introgression has been considered to be one of main factors leading to phylogenetic incongruence among different datasets at lower taxonomic levels. In the plants of Pinaceae, the mtDNA, cpDNA, and nuclear DNA (nrDNA) may have different evolutionary histories through introgression because they are inherited maternally, paternally and biparentally, respectively. We compared mtDNA, cpDNA, and two low-copy nrDNA phylogenetic trees in the genus Pinus subgenus Strobus, in order to detect unknown past introgression events in this group. nrDNA trees were mostly congruent with the cpDNA tree, and supported the recent sectional and subsectional classification system. In contrast, mtDNA trees split the members of sect. Quinquefoliae into two groups that were not observed in the other gene trees. The factors constituting incongruence may be divided into the following two categories: the different splits within subsect. Strobus, and the non-monophyly of subsect. Gerardianae. The former was hypothesized to have been caused by the past introgression of cpDNA, mtDNA or both between Eurasian and North American species through Beringia. The latter was likely caused by the chimeric structure of the mtDNA sequence of P. bungeana, which might have originated through past hybridization, or through a horizontal transfer event and subsequent recombination.

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.

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.

Genetic differentiation and phylogeography of partially sympatric species complex Rhizophora mucronata Lam. and R. stylosa Griff. using SSR markers

BackgroundMangrove forests are ecologically important but globally threatened intertidal plant communities. Effective mangrove conservation requires the determination of species identity, management units, and genetic structure. Here, we investigate the genetic distinctiveness and genetic structure of an iconic but yet taxonomically confusing species complex Rhizophora mucronata and R. stylosa across their distributional range, by employing a suite of 20 informative nuclear SSR markers.ResultsOur results demonstrated the general genetic distinctiveness of R. mucronata and R. stylosa, and potential hybridization or introgression between them. We investigated the population genetics of each species without the putative hybrids, and found strong genetic structure between oceanic regions in both R. mucronata and R. stylosa. In R. mucronata, a strong divergence was detected between populations from the Indian Ocean region (Indian Ocean and Andaman Sea) and the Pacific Ocean region (Malacca Strait, South China Sea and Northwest Pacific Ocean). In R. stylosa, the genetic break was located more eastward, between populations from South and East China Sea and populations from the Southwest Pacific Ocean. The location of these genetic breaks coincided with the boundaries of oceanic currents, thus suggesting that oceanic circulation patterns might have acted as a cryptic barrier to gene flow.ConclusionsOur findings have important implications on the conservation of mangroves, especially relating to replanting efforts and the definition of evolutionary significant units in Rhizophora species. We outlined the genetic structure and identified geographical areas that require further investigations for both R. mucronata and R. stylosa. These results serve as the foundation for the conservation genetics of R. mucronata and R. stylosa and highlighted the need to recognize the genetic distinctiveness of closely-related species, determine their respective genetic structure, and avoid artificially promoting hybridization in mangrove restoration programmes.

Predominant inbreeding and its genetic consequences in a homosporous fern genus, Sceptridium (Ophioglossaceae)

Analysis of isozyme variation revealed the common occurrence of inbreeding in the homosporous fern genus Sceptridium. To examine the genetic consequences of frequent intraga- metophytic selfing, the population genetic structure of S. ternatum was analyzed with special ref- erence to multilocus organization within and among populations. The presence of multiple clones in populations was suggested by high levels of gametic phase linkage disequilibrium within pop- ulations. Each population contained 13-14 multilocus gamete genotypes, and 30 genotypes were counted in three populations examined. The universal genotype was rare and 73% of the genotypes were restricted to a single population. This pattern of genotype variation is concordant with the general pattern in agamospermous or vegetatively reproducing plants. This study also indicated that the local endemic S. triangularifolium has no genetic variation and suggested that this species is a monoclonal lineage derived from hybridization between S. nipponicum and S. ternatum. Large-scale surveys of isozyme variation in higher plants have provided evidence of the important role of the mating system in influ- encing the amounts and organization of genetic diversity within and among populations (Ham- rick and Godt 1990; Loveless and Hamrick 1984). Among the great diversity of mating systems in higher plants, the inbreeding of homospo- rous pteridophytes has a unique position, and may give important insights into the genetic consequences of inbreeding. Homosporous pte- ridophytes produce free-living, bisexual ga- metophytes capable of producing completely homozygous sporophytes through intragame-

Airborne‐pollen pool and mating pattern in a hybrid zone between Pinus pumila and P. parviflora var. pentaphylla

The reproductive isolation barriers and the mating patterns among Pinus pumila, P. parviflora var. pentaphylla and their hybrids were examined by flowering phenology and genetic assays of three life stages: airborne‐pollen grains, adults and seeds, in a hybrid zone on Mount Apoi, Hokkaido, Japan. Chloroplast DNA composition of the airborne‐pollen was determined by single‐pollen polymerase chain reaction. Mating patterns were analysed by estimating the molecular hybrid index of the seed parent, their seed embryos and pollen parents. The observation of flowering phenology showed that the flowering of P. pumila precedes that of P. parviflora var. pentaphylla by about 6 to 10 days within the same altitudinal ranges. Although this prezygotic isolation barrier is effective, the genetic assay of airborne‐pollen showed that the two pine species, particularly P. pumila, still have chances to form F1 hybrid seeds. Both parental species showed a strong assortative mating pattern; F1 seeds were found in only 1.4% of seeds from P. pumila mother trees and not at all in P. parviflora var. pentaphylla. The assortative mating was concluded as the combined result of flowering time differentiation and cross‐incompatibility. In contrast to the parental species, hybrids were fertilized evenly by the two parental species and themselves. The breakdown of prezygotic barriers (intermediate flowering phenology) and cross‐incompatibility may account for the unselective mating. It is suggested that introgression is ongoing on Mount Apoi through backcrossing between hybrids and parental species, despite strong isolation barriers between the parental species.

Pollen movement in a natural population of Arisaema serratum (Araceae), a plant with a pitfall-trap flower pollination system

Arisaema serratum possesses a pitfall-trap flower pollination system. However, little is known about the efficiency and pattern of pollen movement in A. serratum. Thus, the aims of this study are to (1) determine the paternal parents of the seeds and (2) elucidate pollen movement in a natural population. Paternity analysis using microsatellite markers was performed. Seeds were collected from a natural population of A. serratum in 2001 at Horigane, Japan. Small midges became trapped in female spathe tubes during the flowering period. We found that (1) seeds in a fruit were fertilized by multiple sires; (2) seeds sired by a paternal parent were either clumped, exclusively, or randomly distributed on the spadix, depending on the parent; (3) to a great extent, a few males contributed as sires; (4) distance from a female was not a factor in the inequality of reproductive success among males; (5) male reproductive success was not correlated with its size. We conclude that pollen carryover and the trap-flower pollination system are likely to result in multiple paternity and inequality in male success.