The phylogenetic reconstruction of the Neotropical cycad genus Ceratozamia (Zamiaceae) reveals disparate patterns of niche evolution.

The cycad genus Ceratozamia comprises 40 species from Mexico, Guatemala, Belize, and Honduras, where cycads occur throughout climatically varied montane habitats. Ceratozamia has the potential to reveal the history and processes of species diversification across diverse Neotropical habitats in this region. However, the species relationships within Ceratozamia and the ecological trends during its evolution remain unclear. Here, we aimed to clarify the phylogenetic relationships, the timing of clade and species divergences, and the niche evolution throughout the phylogenetic history of Ceratozamia. Genome-wide DNA sequences were obtained with MIG-seq, and multiple data-filtering steps were used to optimize the dataset used to construct an ultrametric species tree. Divergence times among branches and ancestral niches were estimated. The niche variation among species was evaluated, summarized into two principal components, and their ancestral states were reconstructed to test whether niche shifts among branches can be explained by random processes, under a Brownian Motion model. Ceratozamia comprises three main clades, and most species relationships within the clades were resolved. Ceratozamia has diversified since the Oligocene, with major branching events occurring during the Miocene. This timing is consistent with fossil evidence, the timing estimated for other Neotropical plant groups, and the major geological events that shaped the topographic and climatic variation in Mexico. Patterns of niche evolution in the genus do not accord with the Brownian Motion model. Rather, non-random evolution with shifts towards more seasonal environments at high latitudes, or shifts towards humid or dry environments at low latitudes explain the diversification of Ceratozamia. We present a comprehensive phylogenetic reconstruction for Ceratozamia and identify for the first time the environmental factors involved in clade and species diversification within the genus. This study alleviates the controversies regarding the species relationships in the genus and provides the first evidence that latitude-associated environmental factors may influence processes of niche evolution in cycads.

Extensive Copy Number Variation Explains Genome Size Variation in the Unicellular Zygnematophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

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 2-fold 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.

Subgenome evolutionary dynamics in allotetraploid ferns: insights from the gene expression patterns in the allotetraploid species Phegopteris decursivepinnata (Thelypteridacea, Polypodiales).

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.

Establishment of an allotetraploid fern species, Lepisorus yamaokae Seriz., between two highly niche-differentiated parental species.

PREMISE: The successful establishment of polyploid species is hypothesized to be promoted by niche differentiation from the parental species or by range shifts during climate oscillations. However, few studies have considered both of these factors simultaneously. We resolved the origin of a tetraploid fern, Lepisorus yamaokae, and explored a pattern of niche differentiation among the allotetraploid and parental species in past and current climates. METHODS: We reconstructed phylogenetic trees based on plastid marker and single-copy nuclear genes to resolve the allopolyploid origin of L. yamaokae. We also evaluated climatic niche differentiation among L. yamaokae and its two parental species using species distribution models in geographic space and principal component analysis. RESULTS: We infer that L. yamaokae had a single allotetraploid origin from L. annuifrons and L. uchiyamae. Climatic niche analyses show that the parental species currently occupy different niche spaces. The predicted distribution of the parental species at the Last Glacial Maximum (LGM) suggests more opportunities for hybridization during the LGM or during other recent temporary range shifts. Lepisorus yamaokae has a narrower niche than the additive niche of the parental species. We also observed niche conservatism in L. yamaokae. CONCLUSIONS: Range shifts of the parental species during climatic oscillations in the Quaternary likely facilitated the formation and establishment of L. yamaokae. Further, the genetic intermediacy of L. yamaokae may have enabled a niche shift in its microenvironment, resulting in its successful establishment without a macroclimatic niche shift in L. yamaokae.

Speciation along a latitudinal gradient: The origin of the Neotropical cycad sister pair Dioon sonorense-D. vovidesii (Zamiaceae).

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.

Mating system evolution and genetic structure of diploid sexual populations of Cyrtomium falcatum in Japan.

Evolution of mating systems has become one of the most important research areas in evolutionary biology. Cyrtomium falcatum is a homosporous fern species native to eastern Asia. Two subspecies belonging to a sexual diploid race of C. falcatum are recognized: subsp. littorale and subsp. australe. Subspecies littorale shows intermediate selfing rates, while subsp. australe 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. littorale had lower genetic diversity and stronger genetic drift than subsp. australe. 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. littorale. 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. littorale. These results shed new light on the relationship between mating system evolution and past demographic change in fern species.

Massive rhizobial genomic variation associated with partner quality in Lotus-Mesorhizobium symbiosis.

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.

Independent allopatric polyploidizations shaped the geographical structure and initial stage of reproductive isolation in an allotetraploid fern, Lepisorus nigripes (Polypodiaceae).

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).

Niche conservatism promotes speciation in cycads: the case of Dioon merolae (Zamiaceae) in Mexico.

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.

Exploring Genetic Diversity and Signatures of Horizontal Gene Transfer in Nodule Bacteria Associated with Lotus japonicus in Natural Environments.

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.

Phylogenetic analysis reveals the origins of tetraploid and hexaploid species in the Japanese Lepisorus thunbergianus (Polypodiaceae) complex.

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.

The phylogeography of the cycad genus Dioon (Zamiaceae) clarifies its Cenozoic expansion and diversification in the Mexican transition zone.

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.

Aridification as a driver of biodiversity: a case study for the cycad genus Dioon (Zamiaceae).

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.

Allotetraploid cryptic species in Asplenium normale in the Japanese Archipelago, detected by chemotaxonomic and multi-locus genotype approaches.

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 Relationship between Mating System and Genetic Diversity in Diploid Sexual Populations of Cyrtomium falcatum in Japan.

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 (FIS), allelic richness (AR) and gene diversity (h) differed significantly between the M-type (FIS: 0.626, AR: 1.999, h: 0.152) and the S-type (FIS: 0.208, AR: 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 Ne 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 (FST = 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.

Development and characterization of 27 microsatellite markers for the mangrove fern, Acrostichum aureum (Pteridaceae).

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.

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

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.

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.