綿野 泰行

Abstract Cycads represent an example of the success of evolutionary stasis. Despite their early origin, they survived multiple events of mass extinction and diversified in modern tropical ecosystems during the Cenozoic without major changes in their morphology. What factors have allowed their persistence and diversification despite their conservative nature? We reviewed documentation on the micro‐ and macro‐evolutionary processes involved in the diversification of the Neotropical genus Dioon. Dioon comprises 18 species from varied habitats in Mexico and Honduras, and serves as a model to understand the patterns of cycad diversification. Here, we synthesize evidence reached from different fields, especially biogeography, phylogenetics, population ecology, and speciation, to propose a mechanism that can explain the current patterns of biodiversity in Dioon. At the macroevolutionary scale, a Paleogene origin for Dioon is more likely than an alternative hypothesis of a Neogene origin. Dioon lineages have dispersed along with the expansion of tropical forests throughout main mountain chains. Subsequently, climate change, and particularly aridification, promoted the fragmentation of the tropical forests, allowing the main Dioon clades to evolve in isolation at distinct biogeographic regimes. At the microevolutionary scale, low seed dispersal capability, stochastic demographic processes, and niche conservatism restrict the lineages to isolate themselves at narrow habitats, promoting local adaptation in populations. Local adaptation seems to be a process achieved through many generations under stabilizing selection. Altogether, these processes shaped the diversification in Dioon. This review attempts to stimulate further research on cycads and other biological groups that have diversified despite their apparent evolutionary stasis.

Allopolyploidization often leads to disruptive conflicts among more than two sets of subgenomes, leading to genomic modifications and changes in gene expression. Although the evolutionary trajectories of subgenomes in allopolyploids have been studied intensely in angiosperms, the dynamics of subgenome evolution remain poorly understood in ferns, despite the prevalence of allopolyploidization. In this study, we have focused on an allotetraploid fern—Phegopteris decursivepinnata—and its diploid parental species, P. koreana (K) and P. taiwaniana (T). Using RNA-seq analyses, we have compared the gene expression profiles for 9,540 genes among parental species, synthetic F₁ hybrids, and natural allotetraploids. The changes in gene expression patterns were traced from the F₁ hybrids to the natural allopolyploids. This study has revealed that the expression patterns observed in most genes in the F₁ hybrids are largely conserved in the allopolyploids; however, there were substantial differences in certain genes between these groups. In the allopolyploids compared with the F₁ hybrids, the number of genes showing a transgressive pattern in total expression levels was increased. There was a slight reduction in T-dominance and a slight increase in K-dominance, in terms of expression level dominance. Interestingly, there is no obvious bias toward the T- or K-subgenomes in the number and expression levels overall, showing the absence of subgenome dominance. These findings demonstrated the impacts of the substantial transcriptome change after hybridization and the moderate modification during allopolyploid establishment on gene expression in ferns and provided important insights into subgenome evolution in polyploid ferns.

Abstract Genome sizes are known to vary within and among closely related species, but the knowledge about genomic factors contributing to the variation and their impacts on gene functions is limited to only a small number of species. This study identified a more than twofold heritable genome size variation among the unicellular Zygnematophycean alga, Closterium peracerosum-strigosum-littorale (C. psl.) complex, based on short-read sequencing analysis of 22 natural strains and F1 segregation analysis. Six de novo assembled genomes revealed that genome size variation is largely attributable to genome-wide copy number variation (CNV) among strains rather than mating type-linked genomic regions or specific repeat sequences such as rDNA. Notably, about 30% of genes showed CNV even between strains that can mate with each other. Transcriptome and gene ontology analysis demonstrated that CNV is distributed nonrandomly in terms of gene functions, such that CNV was more often observed in the gene set with stage-specific expression. Furthermore, in about 30% of these genes with CNV, the expression level does not increase proportionally with the gene copy number, suggesting presence of dosage compensation, which was overrepresented in genes involved in basic biological functions, such as translation. Nonrandom patterns in gene duplications and corresponding expression changes in terms of gene functions may contribute to maintaining the high level of CNV associated with extensive genome size variation in the C. psl. complex, despite its possible detrimental effects.

Lepisorus rufofuscus T. Fujiw. (Polypodiaceae, Polypodiales), a Japanese species previously treated as L. angustus Ching, is described as new based on integrated evidence from phylogenetics, cytology, and morphology. It is morphologically segregated from L. angustus by the broadly lanceolate or ovate-lanceolate rhizome scales and the ovate leaf scales in the former, but narrowly lanceolate and lanceolate leaf scales in the latter. Although it shows close phylogenetic affinity with L. contortus (Christ) Ching, it is distinct from the latter in basic chromosome number and can be morphologically distinguished from L. contortus in the rhizome scales; opaque center band broader and longer in the former but narrower and shorter in the latter.

Latitude is correlated with environmental components that determine the distribution of biodiversity. In combination with geographic factors, latitude-associated environmental variables are expected to influence speciation, but empirical evidence on how those factors interplay is scarce. We evaluated the genetic and environmental variation among populations in the pair of sister species Dioon sonorense-D. vovidesii, two cycads distributed along a latitudinal environmental gradient in northwestern Mexico, to reveal their demographic histories and the environmental factors involved in their divergence. Using genome-wide loci data, we determined the species delimitation, estimated the gene flow, and compared multiple demographic scenarios of divergence. Also, we estimated the variation of climatic variables among populations and used ecological niche models to test niche overlap between species. The effect of geographic and environmental variables on the genetic variation among populations was evaluated using linear models. Our results suggest the existence of a widespread ancestral population that split into the two species ~829 ky ago. The geographic delimitation along the environmental gradient occurs in the absence of major geographic barriers, near the 28th parallel north, where a zonation of environmental seasonality exists. The northern species, D. vovidesii, occurs in more seasonal environments but retains the same niche of the southern species, D. sonorense. The genetic variation throughout populations cannot be solely explained by stochastic processes; the latitudinal-associated seasonality has been an additive factor that strengthened the species divergence. This study represents an example of how speciation can be achieved by the effect of the latitude-associated factors on the genetic divergence among populations.

<title>Abstract</title>Evolution of mating systems has become one of the most important research areas in evolutionary biology. <italic>Cyrtomium falcatum</italic> is a homosporous fern species native to eastern Asia. Two subspecies belonging to a sexual diploid race of <italic>C. falcatum</italic> are recognized: subsp. <italic>littorale</italic> and subsp. <italic>australe</italic>. Subspecies <italic>littorale</italic> shows intermediate selfing rates, while subsp. <italic>australe</italic> is an obligate outcrosser. We aimed to evaluate the process of mating system evolution and divergence for the two subspecies using restriction site associated DNA sequencing (RAD-seq). The results showed that subsp. <italic>littorale</italic> had lower genetic diversity and stronger genetic drift than subsp. <italic>australe</italic>. Fluctuations in the effective population size over time were evaluated by extended Bayesian skyline plot and Stairway plot analyses, both of which revealed a severe population bottleneck about 20,000 years ago in subsp. <italic>littorale</italic>. This bottleneck and the subsequent range expansion after the LGM appear to have played an important role in the divergence of the two subspecies and the evolution of selfing in subsp. <italic>littorale</italic>. These results shed new light on the relationship between mating system evolution and past demographic change in fern species.

<title>ABSTRACT</title> Variation in partner quality is commonly observed in diverse cooperative relationships, despite the theoretical prediction that selection favoring high-quality partners should eliminate such variation. Here, we investigated how genetic variation in partner quality could be maintained in the nitrogen-fixing mutualism between Lotus japonicus and Mesorhizobium bacteria. We reconstructed de novo assembled full-genome sequences from nine rhizobial symbionts, finding massive variation in the core genome and the similar symbiotic islands, indicating recent horizontal gene transfer (HGT) of the symbiosis islands into diverse Mesorhizobium lineages. A cross-inoculation experiment using 9 sequenced rhizobial symbionts and 15 L. japonicus accessions revealed extensive quality variation represented by plant growth phenotypes, including genotype-by-genotype interactions. Variation in quality was not associated with the presence/absence variation in known symbiosis-related genes in the symbiosis island; rather, it showed significant correlation with the core genome variation. Given the recurrent HGT of the symbiosis islands into diverse Mesorhizobium strains, local Mesorhizobium communities could serve as a major source of variation for core genomes, which might prevent variation in partner quality from fixing, even in the presence of selection favoring high-quality partners. These findings highlight the novel role of HGT of symbiosis islands in maintaining partner quality variation in the legume–rhizobia symbiosis.

Niche conservatism is the tendency of lineages to retain the same niche as their ancestors. It constrains biological groups and prevents ecological divergence. However, theory predicts that niche conservatism can hinder gene flow, strengthen drift and increase local adaptation: does it mean that it also can facilitate speciation? Why does this happen? We aim to answer these questions.We examined the variation of chloroplast DNA, genome-wide single nucleotide polymorphisms, morphological traits and environmental variables across the Dioon merolae cycad populations. We tested geographical structure, scenarios of demographic history, and niche conservatism between population groups.Lineage divergence is associated with the presence of a geographical barrier consisting of unsuitable habitats for cycads. There is a clear genetic and morphological distinction between the geographical groups, suggesting allopatric divergence. However, even in contrasting available environmental conditions, groups retain their ancestral niche, supporting niche conservatism.Niche conservatism is a process that can promote speciation. In D. merolae, lineage divergence occurred because unsuitable habitats represented a barrier against gene flow, incurring populations to experience isolated demographic histories and disparate environmental conditions. This study explains why cycads, despite their ancient lineage origin and biological stasis, have been able to diversify into modern ecosystems worldwide.

Although polyploidy is pervasive and its evolutionary significance has been recognized, it remains unclear how newly formed polyploid species become established. In particular, the impact of multiple origins on genetic differentiation among populations of a polyploid species and whether lineages of independent origins have different evolutionary potentials remain open questions. We used population genetic and phylogenetic approaches to identify genetic differentiation between lineages with independent origins within an allotetraploid fern, Lepisorus nigripes. A total of 352 individuals from 51 populations were collected throughout the distribution range. To examine the genetic structure, multilocus genotyping, Bayesian population structure analysis, and neighbor-net analysis were carried out using single-copy nuclear genes. Phylogenetic trees were constructed to detect recurrent polyploid origins. Proportions of abortive spores were analysed as the measure of postzygotic reproductive isolation. Two genetically distinct lineages, the East-type and the West-type, were distributed mainly in the eastern and western parts, respectively, of the Japanese archipelago. Phylogenetic analyses indicated independent origins of these types and detected additional independent origins within each type. We also revealed limited genetic recombination between both types, even in their sympatric regions. F1 hybrids between the East- and West-types showed a reduction in fertility. It is likely that the East- and West-types formed independently in the eastern and western parts of Japan, respectively. The limited genetic recombination and reduced fertility of hybrids suggest that the two types are at an incipient stage of speciation. Two polyploid lineages with independent geographic origins could develop reproductive isolation barrier(s).

Asplenium serratipinnae (Aspleniaceae: Polypodiales), an allotetraploid species between the diploid race of A. normale and A. oligophlebium is described as new. It is endemic to Japan and morphologically most similar to A. normale, but differs in having narrower pinnae with an auriculate to hastate acroscopic base and deeply serrated margins.

To investigate the genetic diversity and understand the process of horizontal gene transfer (HGT) in nodule bacteria associated with Lotus japonicus, we analyzed sequences of three housekeeping and five symbiotic genes using samples from a geographically wide range in Japan. A phylogenetic analysis of the housekeeping genes indicated that L. japonicus in natural environments was associated with diverse lineages of Mesorhizobium spp., whereas the sequences of symbiotic genes were highly similar between strains, resulting in remarkably low nucleotide diversity at both synonymous and nonsynonymous sites. Guanine-cytosine content values were lower in symbiotic genes, and relative frequencies of recombination between symbiotic genes were also lower than those between housekeeping genes. An analysis of molecular variance showed significant genetic differentiation among populations in both symbiotic and housekeeping genes. These results confirm that the Mesorhizobium genes required for symbiosis with L. japonicus behave as a genomic island (i.e., a symbiosis island) and suggest that this island has spread into diverse genomic backgrounds of Mesorhizobium via HGT events in natural environments. Furthermore, our data compilation revealed that the genetic diversity of symbiotic genes in L. japonicus-associated symbionts was among the lowest compared with reports of other species, which may be related to the recent population expansion proposed in Japanese populations of L. japonicus.

The Japanese Lepisorus thunbergianus complex contains diploid and tetraploid races of L. thunbergianus and a hexaploid species, L. mikawanus. Here, we performed molecular phylogenetic analysis on this complex to delimit species and to elucidate the evolutionary origins of tetraploid and hexaploid species. Chloroplast DNA (cpDNA) phylogeny supported the monophyly of the complex. Based on a single-copy nuclear gene (PgiC) tree, the tetraploid L. thunbergianus samples could be classified into two variants: an allotetraploid of hybrid origin between diploid L. thunbergianus and Japanese L. angustus and another allotetraploid of hybrid origin between diploid L. thunbergianus and an unknown diploid race of L. tosaensis. These variants can be recognized morphologically and distinguished from their parent species. Hence, here we described these allopolyploids as new species, L. nigripes and L. kuratae, respectively. The hexaploid species L. mikawanus has three types of PgiC alleles, each of which was derived from diploid L. thunbergianus, L. tosaensis, and Japanese L. angustus, while cpDNA shows that it is included in Japanese L. thunbergianus clade. Based on the cpDNA phylogeny and PgiC nucleotide sequences, we therefore concluded that L. mikawanus is an allohexaploid that originated through hybridization between tetraploid species, L. nigripes and an unknown ancestral diploid race of L. tosaensis.

Cycads are considered to be the most threatened plant group on Earth. Thus, the identification of important biological units for conservation is crucial for their management and protection. Taxonomic studies have enormously contributed to cycad conservation, since species descriptions formally recognize biodiversity components that can be considered in conservation. However, because multidisciplinary studies that evaluate the biodiversity within cycad species are almost non-existent intraspecific evolutionarily significant units (ESUs) have not been considered when making conservation plans. The cycad Dioon sonorense (Zamiaceae) has a relatively wide distribution range along a vegetation gradient in Northwest Mexico: the southern populations of D. sonorense occur in tropical forests, whereas the northern populations occur in more xeric environments in the Sonoran Desert. Although both habitats might have promoted important evolutionary trends within D. sonorense, only the southern populations are being conserved. Here, we evaluated whether intrinsic differentiation among populations of D. sonorense exists, and whether northern populations may merit attention in conservation programs. We analyzed the variation of genome-wide loci to evaluate the genetic structure among six populations. We found two clusters, northern and southern, that can be recognized using 361 neutral loci. Such genetic structure is consistent with the differentiation observed from the variation of 16 macromorphological and 9 epidermal traits. Southern and northern regions are clearly differentiated in genetic, morphological and anatomical analyses. These multidisciplinary results suggest that the northernmost populations of D. sonorense represent an ESU that should be included in conservation programs. This study highlights that the evaluation of intraspecific variation may contribute effective strategies for cycad conservation, and that evolutionary processes should be considered in the delimitation of conservation units.

• Background and Aims Biogeographic transition zones are promising areas to study processes of biogeographic evolution and its influence on biological groups. The Mexican transition zone originated due to the overlap of Nearctic and Neotropical biota, which promoted great biological diversification. However, since most previous studies in this area were focused on revealing the phylogeography of Nearctic plants, how historical biogeographic configuration influenced the expansion and diversification of the Neotropical flora remains almost unknown. Using the cycad genus Dioon (Zamiaceae), this study aimed to test whether the biogeographic provinciality of the Mexican transition zone reflects the history of diversification of Neotropical plants. • Methods Two chloroplast DNA (cpDNA) regions were analysed from 101 specimens of 15 Dioon species to reveal the distribution of haplogroups. In addition, genome-wide single nucleotide polymorphisms (SNPs) from 84 specimens were used to test the concordance between phylogenetic clusters and the biogeographic provinces. An ultrametric tree was constructed from the sequences containing SNPs to reconstruct the biogeographic events of vicariance and dispersal of Dioon across the Neotropical biogeographic provinces. • Key Results Four Dioon lineages with strong phylogeographic structures were recognized using both cpDNA and SNP data. The lineages correspond to two clades that originated from a common ancestor in Eastern Mexico. One clade expanded and diversified in South-east Mexico and Central America. Another clade diversified into three lineages that dispersed to North-east, South and North-west Mexico. Each lineage was biogeographically delimitated. Biogeographic provinces might have provided disparate ecological conditions that facilitated speciation in Dioon since the Miocene. • Conclusions The current genetic structure and species diversity of Dioon depict the history of expansion and diversification of the northernmost Neotropical provinces. Past biogeographic connectivities were favoured by elevated topographies, since mountain systems served as corridors for the migration of Dioon and as refugia of tropical communities that diversified during the formation of modern Neotropical forests.

Background and Aims Aridification is considered a selective pressure that might have influenced plant diversification. It is suggested that plants adapted to aridity diversified during the Miocene, an epoch of global aridification (15 million years ago). However, evidence supporting diversification being a direct response to aridity is scarce, and multidisciplinary evidence, besides just phylogenetic estimations, is necessary to support the idea that aridification has driven diversification. The cycad genus Dioon (Zamiaceae), a tropical group including species occurring from humid forests to arid zones, was investigated as a promising study system to understand the associations among habitat shifts, diversification times, the evolution of leaf epidermal adaptations, and aridification of Mexico. Methods A phylogenetic tree was constructed from seven chloroplast DNA sequences and the ITS2 spacer to reveal the relationships among 14 Dioon species from habitats ranging from humid forests to deserts. Divergence times were estimated and the habitat shifts throughout Dioon phylogeny were detected. The epidermal anatomy among Dioon species was compared and correlation tests were performed to associate the epidermal variations with habitat parameters. Key Results Events of habitat shifts towards arid zones happened exclusively in one of the two main clades of Dioon. Such habitat shifts happened during the species diversification of Dioon, mainly during the Miocene. Comparative anatomy showed epidermal differences between species from arid and mesic habitats. The variation of epidermal structures was found to be correlated with habitat parameters. Also, most of the analysed epidermal traits showed significant phylogenetic signals. Conclusions The diversification of Dioon has been driven by the aridification of Mexico. The Miocene timing corresponds to the expansion of arid zones that embedded the ancestral Dioon populations. As response, species in arid zones evolved epidermal traits to counteract aridity stress. This case study provides a robust body of evidence supporting the idea that aridification is an important driver of biodiversity.

Patterns of genetic structure are essential for a comprehensive understanding of the evolution and biogeography of a species. Here, we investigated the genetic patterns of one of the most widespread and abundant mangrove species in the Indo-West Pacific, Sonneratia alba J. Sm., in order to gain insights into the ecological and evolutionary drivers of genetic structure in mangroves. We employed 11 nuclear microsatellite loci and two chloroplast regions to genotyped 25 S. alba populations. Our objectives were to (1) assess the level of genetic diversity and its geographic distribution and (2) determine the genetic structure of the populations. Our results revealed significant genetic differentiation among populations. We detected a major genetic break between Indo-Malesia and Australasia, and further population subdivision within each oceanic region in these two major clusters. The phylogeographic patterns indicated a strong influence of vicariance, oceanic barriers and geographic distance on genetic structure. In addition, we found low genetic diversity and high genetic drift at range edge. This study advances the scope of mangrove biogeography by demonstrating a unique scenario whereby a widespread species has limited dispersal and high genetic divergence among populations.

Xylocarpus granatum (Meliaceae) is one of the most widespread core component species of mangrove forests in the Indo-West Pacific (IWP) region, and as such is suitable for examining how genetic structure is generated across spatiotemporal scales. We evaluated the genetic structure of this species using maternally inherited chloroplast (cp) and bi-parentally inherited nuclear DNA markers, with samples collected across the species range. Both cp and nuclear DNA showed generally similar patterns, revealing three genetic groups in the Indian Ocean, South China Sea (with Palau), and Oceania, respectively. The genetic diversity of the Oceania group was significantly lower, and the level of population differentiation within the Oceania group was significantly higher, than in the South China Sea group. These results revealed that in addition to the Malay Peninsula-a common land barrier for mangroves-there is a genetic barrier in an oceanic region of the West Pacific that prevents gene flow among populations. Moreover, demographic inference suggested that these patterns were generated in relation to sea level changes during the last glacial period and the emergence of Sahul Shelf which lied northwest of Australia. We propose that the three genetic groups should be considered independent conservation units, and that the Oceania group has a higher conservation priority.

PREMISE OF THE STUDY: Delimitation of cryptic species provides an understanding of biodiversity and opportunities to elucidate speciation processes. Extensive flavonoid variation has been reported in the tetraploid cytotype of the fern, Asplenium normale, although related species have no intraspecific variations in flavonoid composition. We hypothesized that Japanese A. normale still harbors multiple cryptic species with different flavonoid compositions, and tested this hypothesis using chemotaxonomic and multilocus genotyping approaches. METHODS: We determined the multilocus genotypes (MLGs) of 230 samples from 37 populations for one chloroplast DNA region and three nuclear genes. MLGs were used to delimit reproductively isolated lineages by population-genetic approaches. We also tested the correspondence between genetically recognized groups and flavonoid compositions. To identify the origins of putative cryptic species, we conducted phylogenetic analysis of the DNA markers used in genotyping. KEY RESULTS: The genetic clusters and flavonoid compositions showed clear correspondence. We recognized three putative cryptic species in tetraploid Asplenium normale in Japan. Phylogenetic analyses revealed that cryptic species I and III originated from allopolyploidization between a diploid A. normale and an unknown diploid of A. boreale, and cryptic species II originated from allopolyploidization between a diploid A. normale and A. oligophlebium. CONCLUSIONS: Our study demonstrated that intraspecific variation of secondary metabolites can be a good indicator of cryptic species in ferns. The presence of the two cryptic species having the same progenitor diploid pair suggests that speciation between allopolyploid lineages of independent origin may be more common than previously considered.

The impact of variation in mating system on genetic diversity is a well-debated topic in evolutionary biology. The diploid sexual race of Cyrtomium falcatum (Japanese holly fern) shows mating system variation, i.e., it displays two different types of sexual expression (gametangia formation) in gametophytes: mixed (M) type and separate (S) type. We examined whether there is variation in the selfing rate among populations of this species, and evaluated the relationship between mating system, genetic diversity and effective population size using microsatellites. In this study, we developed eight new microsatellite markers and evaluated genetic diversity and structure of seven populations (four M-type and three S-type). Past effective population sizes (Ne) were inferred using Approximate Bayesian computation (ABC). The values of fixation index (F-IS), allelic richness (A(R)) and gene diversity (h) differed significantly between the M-type (F-IS: 0.626, A(R): 1.999, h: 0.152) and the S-type (F-IS: 0.208, A(R): 2.718, h: 0.367) populations (when admixed individuals were removed from two populations). Although evidence of past bottleneck events was detected in all populations by ABC, the current N-e of the M-type populations was about a third of that of the S-type populations. These results suggest that the M-type populations have experienced more frequent bottlenecks, which could be related to their higher colonization ability via gametophytic selfing. Although high population differentiation among populations was detected (F-ST = 0.581, F-ST' = 0.739), there was no clear genetic differentiation between the M- and S-types. Instead, significant isolation by distance was detected among all populations. These results suggest that mating system variation in this species is generated by the selection for single spore colonization during local extinction and recolonization events and there is no genetic structure due to mating system.

Bruguiera hainesii (Rhizophoraceae) is one of the two Critically Endangered mangrove species listed in the IUCN Red List of Threatened Species. Although the species is vulnerable to extinction, its genetic diversity and the evolutionary relationships with other Bruguiera species are not well understood. Also, intermediate morphological characters imply that the species might be of hybrid origin. To clarify the genetic relationship between B. hainesii and other Bruguiera species, we conducted molecular analyses including all six Bruguiera species using DNA sequences of two nuclear genes (CesA and UNK) and three chloroplast regions (intergenic spacer regions of trnL-trnF, trnS-trnG and atpB-rbcL). For nuclear DNA markers, all nine B. hainesii samples from five populations were heterozygous at both loci, with one allele was shared with B. cylindrica, and the other with B. gymnorhiza. For chloroplast DNA markers, the two haplotypes found in B. hainesii were shared only by B. cylindrica. These results suggested that B. hainesii is a hybrid between B. cylindrica as the maternal parent and B. gymnorhiza as the paternal one. Furthermore, chloroplast DNA haplotypes found in B. hainesii suggest that hybridization has occurred independently in regions where the distribution ranges of the parental species meet. As the IUCN Red List of Threatened Species currently excludes hybrids (except for apomictic plant hybrids), the conservation status of B. hainesii should be reconsidered.

Premise of the study: Twenty-seven nuclear microsatellite markers were developed for the mangrove fern, Acrostichum aureum (Pteridaceae), to investigate the genetic structure and demographic history of the only pantropical mangrove plant. Methods and Results: Fifty-six A. aureum individuals from three populations were sampled and genotyped to characterize the 27 loci. The number of alleles and expected heterozygosity ranged from one to 15 and 0.000 to 0.893, respectively. Across the 26 polymorphic loci, the Malaysian population showed much higher levels of polymorphism compared to the other two populations in Guam and Brazil. Cross-amplification tests in the other two species from the genus determined that seven and six loci were amplifiable in A. danaeifolium and A. speciosum, respectively. Conclusions: The 26 polymorphic microsatellite markers will be useful for future studies investigating the genetic structure and demographic history of of A. aureum, which has the widest distributional range of all mangrove plants.

Background: Mangrove 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. Results: Our 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. Conclusions: Our 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.

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.

Aim Mangroves are intertidal plants with sea-dispersed propagules, hence their population structure can offer valuable insights into the biogeographical processes driving population subdivision in coastal species. In this study, we used molecular markers and ocean circulation simulations to examine the effects of ocean currents and land masses on the genetic structure of the major mangrove species Rhizophora mucronata. Location Southeast Asia. Methods We assessed the genetic structure of 13 R. mucronata populations from continental Southeast Asia and Sumatra using 10 microsatellite loci. We first examined the relative effects of geographical distance and land mass (the Malay Peninsula) in shaping the genetic structure of R. mucronata in Southeast Asia. We then characterized the genetic structure of R. mucronata and compared it to the simulated ocean circulation patterns within our study region. Results Despite the low genetic diversity, significant genetic structuring was detected across R. mucronata populations. Contrary to observations on other mangrove species, genetic differentiation in R. mucronata was not found across the coasts of the Malay Peninsula, nor was it correlated with geographical distance. Instead, the most distinct genetic discontinuity was found at the boundary between the Andaman Sea and the Malacca Strait, and this can be explained by the prevailing ocean currents in this region. Main conclusions Our study presents novel evidence that the genetic structure of R. mucronata is maintained by ocean current-facilitated propagule dispersal.

© 2014, Natural Product Incorporation. All rights reserved. New flavone glycoside, genkwanin 4′-O-β-glucopyranosyl-(1→2)-O-α-rhamnopyranoside was isolated from the fronds of new chemotype of Asplenium normale D.Don, together with two known C-glycosylflavones, vicenin-2 and lucenin-2. The chemical structure of the isolated glycoside was established by UV, LC-MS, characterization of acid hydrolysates, and 1H and 13C NMR spectroscopy.

Human impacts have seriously damaged mangroves, and conservation of mangroves will require information on local and regional population genetic structures. Here, we report the development and polymorphism of eleven novel microsatellite markers, developed using next-generation sequencing on 56 samples of widespread mangrove species Xylocarpus granatum (Meliaceae) from nine populations across the Indo-West Pacific region. All loci were found to be polymorphic, with the number of alleles per locus ranging from four to 19. In a population from Sabah (Malaysia), the mean observed and expected heterozygosity per locus was 0.59 and 0.58, respectively. No null allele, significant linkage disequilibrium or deviation from Hardy-Weinberg equilibrium was detected among all loci. The eleven markers developed can be valuable tools to conservation genetics of this species across its distributional range.

Wind-pollinated trees such as conifers have been considered relatively resistant to the deleterious effects of fragmentation and low density by virtue of their abundant production of airborne pollen. However, some studies indicated that population density affects the mating system even in wind-pollinated trees. In the present study, we examined the mating system and genetic structure of an endangered population (Boso) of Pinus parviflora var. parviflora, whose size has fallen by 80 % in only the last quarter century. Genetic analyses using four nuclear microsatellite markers showed that the population was in Hardy-Weinberg equilibrium and harbored a high level of genetic variation comparable to that of a larger population. Additionally, only weak genetic structure was detected among local patches. On the other hand, the outcrossing rate of seeds in the Boso population was much lower (0.277 from MLTR and 0.297 from paternity analysis) than that found in a larger population of the species (0.778 from MLTR). The outcrossing rate for each mother tree was found to correlate positively with local density around the tree in the Boso population. This suggests that the extensive selfing would be a direct result of pollen limitation caused by the extremely low density of the population: an average of 0.201 trees per hectare for local patches. Because the adult population was in HWE, it is likely that the selfed seeds produced at present cannot contribute to population regeneration. The present study suggests that low population density would increase extinction risk even for wind-pollinated conifer populations.

Polyploidy, hybridization, and agamospory have been considered important mechanisms in fern speciation. By integrating the methods of cytology, molecular phylogeny, and morphology, we examined the origins of polyploid species in the Pteris cretica group, which comprises five agamosporous taxa and six sexual species. Phylogenetic analysis was conducted using both cpDNA (rbcL and trnV-trnM) and a low-copy nuclear gene (gapCp). The combined results of cytology and the phylogenetic trees suggested that the sexual diploid P. kidoi had played a central role in the diversification of polyploid species in the P. cretica group. Some triploid clones of agamosporous P. cretica var. cretica originated through hybridization between the diploid cytotype of P. cretica var. cretica and the sexual diploid P. kidoi. The sexual polyploid species, P. henryi, P. multifida, P. ryukyuensis, and P. yamatensis, and the hexaploid race of P. deltodon have arisen through allopolyploidization between their respective ancestral parent species and P. kidoi. Additionally, the agamosporous triploid P. nipponica and P. cretica var. albolineata might have originated through hybridization between P. cretica var. cretica and an unknown ancestral diploid parent of P. ryukyuensis. Agamosporous P. cretica var. cretica harbored considerable genetic variation within both the diploid and triploid cytotypes. Because we could not find clear genetic differences between the diploid and triploid P. cretica, both cytotypes might be autopolyploids, or alternatively have originated through hybridization among the same members of ancestral sexual species. © 2013 by the American Society of Plant Taxonomists.

Species of the genus Elysia Risso, 1818 have relatively few external characters useful for species identification, and features such as papillae and/or color markings on the body surface are often used to classify and identify species. Elysia atroviridis Baba, 1955 and E. setoensis Hamatani, 1968, for example, have been described as distinct based mainly on such characters. Close examinations of 46 specimens identifiable as either species collected from nine localities in Japan revealed that many specimens exhibited mixed features of the two species. Detailed comparisons of radular morphology could also not differentiate the two species. With phylogenetic analyses based on COI and 16S rRNA (1072 bp in total), all of the examined specimens formed a single clade with very little variation among specimens. The AMOVA of the specimen haplotypes showed there was no significant genetic differentiation in relation to differences in external morphology, geographic region, or algal host. Our results indicate that E. atroviridis and E. setoensis are conspecific. In contrast, cryptic species were found in two other clades of congeners, indicating that further taxonomic scrutiny is needed within the genus.

A set of 14 new microsatellite markers was developed for mangrove species Rhizophora mucronata (Rhizophoraceae) by using pyrosequencing. Fifty-six samples from 9 populations of R. mucronata in the Indo-West Pacific region were genotyped; all loci were polymorphic, with the number of alleles ranging from 2 to 9. The mean expected heterozygosity per locus was 0.16 in a population from Sabah, no significant linkage disequilibrium was found among loci, and significant deviation from Hardy-Weinberg equilibrium was found in 3 loci. The polymorphic microsatellite markers with samples covering most of the species' distribution range can be applied in genetic diversity studies covering a broad geographical range of the species.

A set of 15 new microsatellite loci was developed and characterized for the widespread mangrove tree species Sonneratia alba (Lythraceae) by using next-generation sequencing. Forty-eight S. alba samples from seven populations in the Indo-West Pacific region were genotyped; all loci were polymorphic, with the number of alleles ranging from three to eight. The mean observed heterozygosity per locus was 0.21 for a population from Sabah, Malaysia. No null allele or significant linkage disequilibrium was detected, indicating the robustness of the markers. Only one locus (SA103) showed deviation from Hardy-Weinberg equilibrium. As characterization of these microsatellite loci was done with samples covering most of the species' distribution range, the markers can be applied to genetic diversity studies over the broad geographical range of the species.

Premise of the study: We developed novel microsatellite markers in Arisaema serratum, a perennial herb that possesses pitfall flowers and exhibits labile sex expression, to facilitate research on parentage and pollination biology in this species. Methods and Results: By using procedures for enrichment of desired microsatellite-containing fragments and PCR-based isolation of microsatellite arrays, we detected 18 novel microsatellite loci. Thirteen were highly polymorphic: the number of alleles per locus ranged from six to 46, the observed heterozygosities ranged from 0.320 to 0.940, and the expected heterozygosities ranged from 0.440 to 0.976. Nine of the 13 markers successfully amplified regions in congeneric species. Conclusions: These highly polymorphic markers will facilitate further studies on the mode of pollination and other aspects of reproductive biology in A. serratum

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.

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 F(1) hybrid seeds. Both parental species showed a strong assortative mating pattern; F(1) 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.

A late Pliocene (1.8-1.9 Ma) wetland fossil forest community that was dominated by Metasequoia and Glyptostrobus was reconstructed based on the species composition of the stumps and other plant macrofossil assemblages. The plant fossils were recovered from a fossil forest preserved in deposits of the Kobiwako Group that are exposed in the Echi River, Shiga Prefecture, central Japan. Fossil wood of Metasequoia and Glyptostrobus was distinguished based on anatomical characteristics. Apportionment of the wood from different horizons in the fossil forest indicates Metasequoia grew over a long period of time in a stable environment, while Glyptostrobus and Alnus grew in unstable environments characterized by short-interval floods. The fossil forest as a whole represents a fluvial back-marsh environment. The upland forest was composed of mixed evergreen conifers and deciduous broad-leaved trees including Chamaecyparis pisifera, Tsuga, Magnolia, and Acer. Aquatic and wetland herbs such as Cyperus, Carex, Scirpus, Polygonum, and Menyanthes grew in and around the wetland forest. The assemblage of fossil plants recovered from the fossil forest consists of plants that are currently distributed in the cool temperate climate zone, such as Picea, Thuja, Betula maximowicziana, Pterocarya rhoifolia, and Menyanthes trifoliata. Glyptostrobus is distributed only in subtropical areas at present, but it also grew under a cool temperate climate zone in Japan during the latest Pliocene.

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.

Species complexes consisting of ill-defined "species" are widely known among ferns, and their involvement with reticulate evolution is expected. Nevertheless approaches to reticulation history with DNA markers are not yet commonly adopted. We have successfully elucidated the biological status of the Vandenboschia radicans complex in East Asian islands by combining analyses of ploidy level, a cpDNA marker (rbcL), and a nuclear DNA marker (GapC). The results based on 266 individuals collected from 174 localities throughout Japan and Taiwan suggest that complicated hybridizations have occurred involving at least three parental diploid species from within the V. radicans complex and Vandenboschia liukiuensis, which was formerly considered to be distinct from this complex. Triploids are the most common cytotype, but they show no evidence of apogamous reproduction, while all nonhybrid diploids are rare and have very limited distribution. Possible accounts of this phenomenon will be briefly discussed including the possibility of relict distribution and occasional apogamous reproduction.