Morphological and functional evolution of gametophytes in epilithic Hymenasplenium murakami-hatanakae (Aspleniaceae): The fifth family capable of producing the independent gametophytes.

The fern independent gametophytes that can maintain populations by vegetative reproduction without conspecific sporophytes have been considered an unusual phenomenon found in some epiphytic or epilithic species of Hymenophyllaceae, Pteridaceae, Lomariopsidaceae, and Polypodiaceae. By chance, the discovery of mysterious strap-like gametophytes on Izu-Oshima Island, Japan, has led to the hypothesis that Hymenasplenium murakami-hatanakae, a fern species belonging to Aspleniaceae, can also form independent gametophytes. Our investigation revealed gametophyte populations of H. murakami-hatanakae on three islands in the Izu Islands. Based on chloroplast DNA analysis of the gametophyte and sporophyte populations, the gametophytes were found to be maintained by vegetative reproduction without a new supply of spores from sporophytes. A comparison of the surrounding vegetation at the collection sites showed that environmental factors such as light and humidity may influence the maintenance of gametophyte populations. These results clearly show that H. murakami-hatanakae is one of the ferns capable of forming independent gametophytes. This is the first report of independent gametophytes from the suborder Aspleniineae (eupolypod II). The discovery of the independent gametophyte within a phylogenetic lineage previously thought not to form independent gametophytes will provide important insights into the morphological and functional evolution of gametophytes in ferns.

Lepisorusmedioximus (Polypodiales, Polypodiaceae), a new species from Shan State of Myanmar.

A new species of the species-rich fern genus Lepisorus (Polypodiales, Polypodiaceae) has been found to occur in Shan state, Myanmar. Lepisorusmedioximus is described based on morphological characters and phylogenetic evidence. Phylogenetic analyses showed that the specimens of L.medioximus formed a distinct clade nested in the Pseudovittaria clade. The morphological comparison demonstrated that the species is distinct from phylogenetically related species, namely L.elegans, L.contortus, and L.tosaensis, in the morphology of the rhizome scales, size, and shape of the lamina, position of sori, and paraphyses.

Molecular prospecting for cryptic species of the Hypholoma fasciculare complex: toward the effective and practical delimitation of cryptic macrofungal species.

Many macrofungal cryptic species remain unidentified. A possible solution is to increase the number of loci analyzed and use rigorous statistics for macrofungal species delimitation. To validate this assumption, cryptic species of the Hypholoma fasciculare complex, a group of common wood-decomposing fungi, were attempted to be delineated. Massively parallel sequencing of mitochondrial ribosomal RNA (mt_rRNA), nuclear ribosomal internal transcribed spacer (ITS) region, and 24 single-copy genes were performed for 96 specimens collected in Japan. Then, the species boundaries were inferred using comparative gene genealogies (mt_rRNA vs. ITS), Bayesian Poisson tree process (bPTP) model for the phylogeny of concatenated nuclear sequences, and analysis of molecular variance (AMOVA) for single nucleotide polymorphisms. In both the mt_rRNA and ITS phylogenies, the H. fasciculare complex was not divided into well-supported clades. Nevertheless, based on the bPTP, two mitochondrial haplotypes were inferred to represent distinct species (H. fasciculare and H. subviride). The results of AMOVA also indicated that the differentiation of nuclear loci can be explained mostly by differences between haplotype. These results suggest that it is necessary to increase the number of target loci to 20 or more and use both phylogeny-based and population genetics-based statistics for the accurate delimitation of macrofungal species.

Strong genetic structure revealed by microsatellite variation in Callicarpa species endemic to the Bonin (Ogasawara) Islands.

Adaptive radiation is the diversification of a founding population into multiple taxa that are differentially adapted to diverse ecological niches. The three Callicarpa (Lamiaceae) species endemic to the Bonin Islands are considered to represent an example of adaptive radiation on oceanic islands. All three species are distributed in the Chichijima Island Group and grow in different habitats, while only one species, C. subpubescens, is distributed among other island groups. Particularly, in the Hahajima and Mukojima Island Groups, C. subpubescens grows in various habitats and shows relatively high morphological variation. We investigated genotypes of the three Callicarpa species at 14 microsatellite markers to elucidate genetic differentiation within and between species or island groups and between different habitats or morphologies. We found that genetic differentiation within C. subpubescens in the Hahajima and Mukojima Island Groups was equally as high as that between the three species in the Chichijima Island Group, while differentiation within C. subpubescens in the Chichijima Island Group was much lower. Analyses such as a Bayesian clustering analysis showed that genetically distinct groups were associated with the three species in the Chichijima Island Group, whereas they showed strong genetic structure within C. subpubescens in the Hahajima and Mukojima Island Groups among different habitats and morphologies. These results indicated that ecological diversification occurred in the Hahajima and Mukojima Island Groups. Meanwhile, high genetic differentiation among different island groups was also observed, reflecting isolation by distance. It implies that non-ecological factors such as geographic isolation also played important roles in genetic differentiation in Callicarpa species in the Bonin Islands. These findings suggest that the Callicarpa species in the Bonin Islands are differentiated into multiple genetic groups by both ecological and non-ecological factors.

Origin of the Diplaziumhachijoense complex (Athyriaceae).

We analyzed the phylogeny of the Diplaziumhachijoense complex using plastid trnL-F and low-copy nuclear marker AK1 DNA sequences. Based on allele constitution, triploid apogamous species of the D.hachijoense complex appeared to have originated from the hybridization of triploid apogamous species and diploid sexual species by recurrent hybridization events. These results suggested that triploid apogamous ferns can achieve hybridization with diploid sexual species by producing diploid spores with irregular meiosis in sporogenesis. Furthermore, the present study predicted the involvement of several unknown species associated with hybridization. More sampling of Callipteris species from China and adjacent areas is required to determine the relationships among unknown species and the D.hachijoense complex.

Flower color changes in three Japanese hibiscus species: further quantitative variation of anthocyanin and flavonols.

One anthocyanin and four flavonols were detected from the petals of Hibiscus hamabo, H. tiliaceus and H. glaber. They were identified as cyanidin 3-0- sambubioside, gossypetin 3-O-glucuronide-8-O-glucoside, quercetin 7-O-rutinoside, gossypetin 3-O-glucoside and gossypetin 8-O-glucuronide by UV spectra, LC-MS, acid hydrolysis and HPLC. The flavonoid composition was essentially the same among the petals ofH. hamabo, H. tiliaceus and H. glaber, and there was little quantitative variation, except for cyanidin 3-O-sambubioside, the content of which in the petals ofH. tiliaceus and H. glaber was much higher than in that of H. hamabo. Flower colors of H. tiliaceus and H. glaber change from yellow to red, and that of H. hamabo changes from yellow to orange. These changes were caused by contents of anthocyanin and flavonols, which increased after flowering of H. hamabo, H. tiliaceus and H. glaber.

Environmental factors contribute to the formation and maintenance of the contact zone observed in deciduous broad-leaved tree species in Japan.

Contact zones are defined as areas where populations from different refugia meet during a postglacial expansion and distinct DNA lineages are mixedly distributed. In Japan, contact zones of various plants and animals were reported from the Kinki-Chugoku region. These contact zones appear to be maintained without any drastic topographic barriers such as those observed in the Alps and Pyrenees Mountains. In this study, the mechanisms underlying the formation and/or maintenance of these contact zones were investigated using six deciduous broad-leaved tree species (Carpinus laxiflora, C. tschonoskii, C. japonica, Magnolia obovata, Padus grayana, and Euonymus oxyphyllus). First, the precise location of the contact zones was examined by intensive genetic analysis of the six species. Second, the relationships between the geographic location of the contact zone and various environmental factors, including climate and topography, were investigated by generalized additive models to reveal the mechanisms of the formation and maintenance of the contact zones. As a result, four of the six examined plant species clearly showed a geographically common contact zone in Hyogo Prefecture and its adjacent areas. The results of the generalized additive models indicate that the pattern of low habitat suitability estimated by ecological niche modeling was the most important factor for determining the location of the common contact zone. These results suggest that areas with low habitat suitability in Hyogo Prefecture restrict the migration and gene flow of the four species in this region, and thus, they maintain the pattern of the contact zones. This study suggests that there are major effects of habitat suitability on the formation and maintenance of the contact zones.

Low-temperature reduction of brownmillerite CaFeO2.5 in LaAlO3/CaFeO2.5 heterostructures made on SrTiO3.

When LaAlO3/CaFeO2.5 thin-film heterostructures made on SrTiO3 were annealed with CaH2 at low temperatures below 300 °C, the brownmillerite CaFeO2.5 layer was reduced to CaFeO2 with an infinite-layer structure while both the LaAlO3 capping layer and the SrTiO3 substrate remained intact. The reduction behaviour strongly depends on the lattice matching of LaAlO3 to CaFeO2.5, suggesting that oxygen ions migrate through the coherently grown LaAlO3 layer of the heterostructure predominantly in the out-of-plane direction. The structural defects near the interface in the relaxed-structure LaAlO3 capping layer prevent the oxygen ions from migrating.

Reticulate evolution in the apogamous Dryopteris varia complex (Dryopteridaceae, subg. Erythrovariae, sect. Variae) and its related sexual species in Japan.

Apogamous fern species are often difficult to distinguish from related species because of their continuous morphological variations. To clarify the genetic relationships among the members of the Dryopteris varia complex, we analyzed the nucleotide sequences of the plastid gene rbcL and the nuclear gene PgiC. We also analyzed the diploid sexual species D. caudipinna and D. chinensis, which have not been included in the complex, but were recently shown to be closely related to the complex in a molecular phylogenetic study. The PgiC sequences of the diploid sexual species, D. varia, D. saxifraga, D. sp. 'protobissetiana' (undescribed diploid sexual species), D. caudipinna, and D. chinensis, were well differentiated and hence designated A, B, C, D, and E, respectively. Thus, the PgiC constitution of apogamous species in the complex was as follows: D. bissetiana, B + C; D. kobayashii, B + C + E); D. pacifica, A + C, A + B + C, or A + C + D; D. sacrosancta, A + C + E; and D. saxifragivaria, B + C. These results suggest that these apogamous species are formed by hybridizations of species including not only the three diploid sexual species of the D. varia complex (A, B, and C) but also the two diploid sexual species D. caudipinna (D) and D. chinensis (E), which do not belong to the complex.

Genetic differentiation and genetic diversity of Castanopsis (Fagaceae), the dominant tree species in Japanese broadleaved evergreen forests, revealed by analysis of EST-associated microsatellites.

The broadleaved evergreen forests of the East Asian warm temperate zone are characterised by their high biodiversity and endemism, and there is therefore a need to extend our understanding of its genetic diversity and phylogeographic patterns. Castanopsis (Fagaceae) is one of the dominant tree species in the broadleaved evergreen forests of Japan. In this study we investigate the genetic diversity, genetic structure and leaf epidermal morphology of 63 natural populations of C. sieboldii and C. cuspidata, using 32 Expressed Sequence Tag associated microsatellites. The overall genetic differentiation between populations was low (GST = 0.069 in C. sieboldii and GST = 0.057 in C. cuspidata). Neighbor-joining tree and Bayesian clustering analyses revealed that the populations of C. sieboldii and C. cuspidata were genetically clearly differentiated, a result which is consistent with the morphology of their epidermal cell layers. This suggests that C. sieboldii and C. cuspidata should be treated as independent species, although intermediate morphologies are often observed, especially at sites where the two species coexist. The higher level of genetic diversity observed in the Kyushu region (for both species) and the Ryukyu Islands (for C. sieboldii) is consistent with the available fossil pollen data for Castanopsis-type broadleaved evergreen trees during the Last Glacial Maximum and suggests the existence of refugia for Castanopsis forests in southern Japan. Within the C. sieboldii populations, Bayesian clustering analyses detected three clusters, in the western and eastern parts of the main islands and in the Ryukyu Islands. The west-east genetic differentiation observed for this species in the main islands, a pattern which is also found in several plant and animal species inhabiting Castanopsis forests in Japan, suggests that they have been isolated from each other in the western and eastern populations for an extended period of time, and may imply the existence of eastern refugia.

Genetic differentiation in Elaeocarpus photiniifolia (Elaeocarpaceae) associated with geographic distribution and habitat variation in the Bonin (Ogasawara) Islands.

Gene flow between populations in different environmental conditions can be limited due to divergent natural selection, thus promoting genetic differentiation. Elaeocarpus photiniifolia, an endemic tree species in the Bonin Islands, is distributed in two types of habitats, dry scrubs and mesic forests. We aim to elucidate the genetic differentiation in E. photiniifolia within and between islands and between the habitat types. We investigated genotypes of 639 individuals from 19 populations of E. photiniifolia and its closely-related E. sylvestris at 24 microsatellite loci derived from expressed sequence tags. The data revealed genetic differentiation (1) between E. photiniifolia and E. sylvestris (0.307 ≤ F ST ≤ 0.470), (2) between the E. photiniifolia populations of the Chichijima and Hahajima Island Groups in the Bonin Islands (0.033 ≤ F ST ≤ 0.121) and (3) between E. photiniifolia populations associated with dry scrubs and mesic forests in the Chichijima Island Group (0.005 ≤ F ST ≤ 0.071). Principal coordinate analysis and Bayesian clustering analysis also showed that genetically distinct groups were associated with the habitat types, and isolation by distance was not responsible for the genetic differentiation. These findings suggest that E. photiniifolia is divided into genetically differentiated groups associated with different environmental conditions in the Bonin Islands.

Geographical origin and sexual-system evolution of the androdioecious plant Gynochthodes boninensis (Rubiaceae), endemic to the Bonin Islands, Japan.

Gynochthodes boninensis is a woody climber endemic to the Bonin Islands, Japan. It is characterized by an androdioecious sexual system, which is rare in angiosperms. We conducted a molecular phylogenetic analysis of 29 taxa including 61 samples from the tribe Morindeae to elucidate the geographical origin of G. boninensis by determining its progenitor species. We also investigated evolutionary transitions among different sexual systems within this plant group. The combined ETS, ITS, and trnT-F sequence data showed that G. boninensis formed a monophyletic group, but it did not form a clade with G. umbellata, which was treated as the same species, whereas it formed a clade with G. parvifolia, which is distributed in southeastern Asia. This suggests that G. boninensis evolved independently from G. umbellata, and probably originated from a progenitor native to southeastern Asia. In the clade composed of the three species of G. boninensis, G. parvifolia, and G. umbellata, only G. boninensis is androdioecious, whereas the others are dioecious. Thus, the androdioecious sexual system of G. boninensis may have evolved from dioecy.

Geographical origin of Leucobryum boninense Sull. & Lesq. (Leucobryaceae, Musci) endemic to the Bonin Islands, Japan.

Leucobryum boninense is endemic to the Bonin Islands, Japan, and its related species are widely distributed in Asia and the Pacific. We aimed to clarify the phylogenetic relationships among Leucobryum species and infer the origin of L. boninense. We also describe the utility of the chloroplast trnK intron including matK for resolving the phylogenetic relationships among Leucobryum species, as phylogenetic analyses using trnK intron and/or matK have not been performed well in bryophytes to date. Fifty samples containing 15 species of Leucobryum from Asia and the Pacific were examined for six chloroplast DNA regions including rbcL, rps4, partial 5' trnK intron, matK, partial 3' trnK intron, and trnL-F intergenic spacer plus one nuclear DNA region including ITS. A molecular phylogenetic tree showed that L. boninense made a clade with L. scabrum from Japan, Taiwan and, Hong Kong; L. javense which is widely distributed in East and Southeast Asia, and L. pachyphyllum and L. seemannii restricted to the Hawaii Islands, as well as with L. scaberulum from the Ryukyus, Japan, Taiwan, and southeastern China. Leucobryum boninense from various islands of the Bonin Islands made a monophylic group that was closely related to L. scabrum and L. javense from Japan. Therefore, L. boninense may have evolved from L. scabrum from Japan, Taiwan, or Hong Kong, or L. javense from Japan. We also described the utility of trnK intron including matK. A percentage of the parsimony-informative characters in trnK intron sequence data (5.8%) was significantly higher than that from other chloroplast regions, rbcL (2.4%) and rps4 (3.2%) sequence data. Nucleotide sequence data of the trnK intron including matK are more informative than other chloroplast DNA regions for identifying the phylogenetic relationships among Leucobryum species.

Transmission ratio distortion of molecular markers in a doubled haploid population originated from a natural hybrid between Osmunda japonica and O. lancea.

In ferns, intra-gametophytic selfing occurs as a mode of reproduction where two gametes from the same gametophyte form a completely homozygous sporophyte. Intra-gametophytic selfing is considered to be prevented by lethal or deleterious recessive genes in several diploid species. In order to investigate the modes and tempo of selection acting different developmental stages, doubled haploids obtained from intra-gametophytic selfing within isolated gametophytes of a putative F1 hybrid between Osmunda japonica and O. lancea were analyzed with EST_derived molecular markers, and the distribution pattern of transmission ratio distortion (TRD) along linkage map was clarified. As the results, the markers with skewness were clustered in two linkage groups. For the two highly distorted regions, gametophytes and F2 population were also examined. The markers skewed towards O. japonica on a linkage group (LG_2) showed skewness also in gametophytes, and the TRD was generated in the process of spore formation or growth of gametophytes. Also, selection appeared to be operating in the gametophytic stage. The markers on other linkage group (LG_11) showed highest skewness towards O. lancea in doubled haploids, and it was suggested that the segregation of LG_11 were influenced by zygotic lethality or genotypic evaluation and that some deleterious recessive genes exist in LG_11 and reduce the viability of homozygotes with O. japonica alleles. It is very likely that a region of LG_11were responsible for the low frequencies of intra-gametophytic selfing in O. japonica.

Microsatellite markers for Leucobryum boninense (Leucobryaceae), endemic to the Bonin Islands, Japan.

PREMISE OF THE STUDY: Microsatellite primers were developed for Leucobryum boninense, endemic to the Bonin Islands, Japan, to investigate its level of genetic diversity and population genetic structure. • METHODS AND RESULTS: Using next-generation sequencing, 21 primer sets were developed, among which nine loci were polymorphic in the populations of the Bonin Islands. Among these polymorphic loci, the number of alleles per locus ranged from two to 10 (mean = 3.444) and the expected heterozygosity ranged from 0.066 to 0.801 (mean = 0.338). • CONCLUSIONS: These results indicate the utility of the nine microsatellite markers that we developed for population genetic studies of L. boninense.

Evidence of genetic segregation in the apogamous fern species Cyrtomium fortunei (Dryopteridaceae).

In apogamous ferns, all offspring from a parent are expected to be clonal. However, apogamous 'species' frequently demonstrate a large amount of morphological and genetic variations. Cyrtomium fortunei composed of four varieties (C. fortunei var. fortunei, var. clivicola, var. intermedium, and var. atropunctatum), is all reported to be apogamous triploids, but demonstrates large and continuous morphological variation. In previous studies, we showed that considerable genetic diversity was observed in many local populations of the apogamous fern 'species'. We hypothesized that genetic segregation has occurred, because neither sexual type nor intraspecific polyploidy have been observed in C. fortunei in Japan. Of 732 progeny examined (250 gametophytes and 482 sporophytes), obtained from a parental sporophyte whose pgiC genotype was estimated as aab, 11 (4.4%) gametophytes and 8 (1.7%) sporophytes showed a different genotype (aaa) from that of the parent sporophyte. We showed that genetic segregation occurs in apogamous C. fortunei in relatively high frequency. Moreover, we could first show that the segregation frequency in gametophytes is significantly higher than that in sporophytes of the next generation (χ² = 4.90, P = 0.027). It may suggest the existence of deleterious genes, which are expressed during the morphogenesis and growth of sporophytes.

Development of EST-SSR markers for Elaeocarpus photiniifolia (Elaeocarpaceae), an endemic taxon of the Bonin Islands.

PREMISE OF THE STUDY: Expressed sequence tag (EST)-derived microsatellite markers were developed for Elaeocarpus photiniifolia, an endemic taxon of the Bonin Islands. METHODS AND RESULTS: Initially, a complementary DNA (cDNA) library was constructed by de novo pyrosequencing of total RNA extracted from a seedling. A total of 267 primer pairs were designed from the library. Of the 48 tested loci, 25 loci were polymorphic among 41 individuals representing the entire geographical range of the species, with the number of alleles per locus and expected heterozygosity ranging from two to 14 and 0.09 to 0.86, respectively. Most loci were transferable to a related species, E. sylvestris. CONCLUSIONS: The developed markers will be useful for evaluating the genetic structure of E. photiniifolia.

Comparative phylogeography of four component species of deciduous broad-leaved forests in Japan based on chloroplast DNA variation.

A phylogeographic study of four tree species (Padus grayana, Euonymus oxyphyllus, Magnolia hypoleuca, and Carpinus laxiflora) growing in Japanese deciduous broad-leaved forests was conducted based on chloroplast DNA (cpDNA) variations. Using nucleotide sequences of 702-1,059 bp of intergenic spacers of cpDNA, 20, 27, eight, and eight haplotypes were detected among 251, 251, 226, and 262 individuals sampled from 67, 79, 75, and 71 populations of the above species, respectively. The geographical pattern of the cpDNA variations was highly structured in each species, and the following three regional populations were genetically highly differentiated among all four species: (1) the Sea of Japan-side area, (2) the Kanto region, and (3) southwestern Japan. Based on some interspecific similarities among the phylogeographic patterns, the following migration scenario of Japanese deciduous broad-leaved forests was postulated. During the last glacial maximum (LGM), the forests were separately distributed in six regions. After LGM, as the climate warmed, the forests in eastern Japan separately expanded from each of the refugia along the Sea of Japan-side or along the Pacific Ocean-side. In contrast, those in southwestern Japan retreated and moved to high altitudes from each of the continuous forests.

Two types of partial fertility in a diploid population of the fern Thelypteris decursive-pinnata (Thelypteridaceae).

Two types of abnormal sporophytes were observed in a population of diploid Thelypteris decursive-pinnata. Most sporophytes in this population exhibited regular chromosome pairing, resulting in the formation of 30 bivalents in meiosis I; however, they produced abortive spores to various degrees. Some formed large globose spores at low frequencies, most likely to be unreduced diplospores. The other type of abnormal sporophyte underwent synaptic failure to form 60 univalents at meiosis I, but produced fertile spores, mostly large globose ones at low frequencies. The globose spores were considered unreduced diplospores because the gametophytes arising from them produced tetraploid sporophytes by gametophytic selfing. One tetraploid formed only univalents at meiosis I. Allozyme variation was not detected in this population, although neighboring ordinary diploid populations exhibited it to a certain degree. The sympatric occurrence and allozyme uniformity of the two groups suggest that both are offspring of a founder sporophyte, which may have possessed two types of mutated recessive genes responsible for the spore sterility and the synaptic failure in meiosis. Unreduced spores formed by these two types may play an important role in the polyploid speciation of this species.

Fertility and precocity of Osmunda x intermedia offspring in culture.

The feasibility of later-generation hybrid production in ferns has not been previously studied, although it is a significant factor in relation to reproductive isolation. Osmunda x intermedia, a hybrid between O. japonica and O. lancea, is semifertile and has moderate spore germination rates. Under the artificial conditions of this study, F2 and F3 offspring were formed. Some of the F2 offspring showed precocity, and some of the F3 offspring also showed precocity. This fertility suggests that introgressive hybridization might be ongoing in nature. This also indicates a currently unknown genetic control over the timing of fertile frond production in Osmunda.