Abstract
The Pteris fauriei group (Pteridaceae) has a wide distribution in Eastern Asia and includes 18 species with similar but varied morphology. We collected more than 300 specimens of the P. fauriei group and determined ploidy by flow cytometry and inferred phylogenies by molecular analyses of chloroplast and nuclear DNA markers. Our results reveal a complicated reticulate evolution, consisting of seven parental taxa and 58 hybrids. The large number of hybrid taxa have added significant morphological complexity to the group leading to difficult taxonomic issues. The hybrids generally had broader ranges and more populations than their parental taxa. Genetic combination of different pairs of parental species created divergent phenotypes of hybrids, exhibited by both morphological characteristics and ecological fidelities. Niche novelty could facilitate hybrid speciation. Apogamy is common in this group and potentially contributes to the sustainability of the whole group. We propose that frequent hybridizations among members of the P. fauriei group generate and maintain genetic diversity, via novel genetic combinations, niche differentiation, and apogamy.
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Introduction
Hybridization instantly creates novel combinations of genes and genomes1 and, therefore, can lead to rapid species differentiation2,3,4,5. Reticulate evolution of ferns was first reported in Asplenium species6. Subsequently, a growing number of studies have documented reticulate evolution in ferns, such as in Dryopteris, Pteris, and Vandenboschia7,8,9,10. However, the role of reticulate evolution in promoting diversification remains unclear. For example, with various phenotypes arising quickly, hybridization may promote adaptive radiation3,11,12,13. In angiosperms, rapid diversification is driven by various morphological, physiological, and genetic characters, as well as the origin of physical barriers that may combine to promote different ecological fidelities between parental and hybrid species14,15,16,17,18.
Hybrids may spread to new ecological habitats, differing from those of the parental species, thus reducing competition with parental species and potentially facilitating hybrid speciation19,20. A long-term field experiment of Helianthus provided direct evidence of hybridization driving adaptive radiation 21. In other words, ecological divergence of hybrids is likely to be related to the success of hybrid speciation 22,23,24,25, but the role of niche differentiation in the evolution of hybrid species in ferns has been little studied26,27,28. Ecological differentiation in hybrid zones was first proposed in the Pteris quadriaurita complex7,29, and then reported in Polystichum imbricans (D.C.Eaton) D.H.Wagner and Polystichum munitum (Kaulf.) C.Presl (Dryopteridaceae)30.
In ferns, hybridization is often accompanied by apogamy31,32. Apogamy is found in 3% to 10% of fern species33,34 and results from the deregulation of reproductive pathways35. Apogamous hybrid ferns are reported in many cryptic complexes, especially in Aspleniaceae36,37, Dryopteridaceae38,39,40, and Pteridaceae9,28,41. Because apogamous species often have limited genetic variation compared to close sexual relatives 42, apogamy has been considered a dead end for fern evolution43; however, it is common and could play a special role in Pteris29,44,45, where 21% of species reproduce only by apogamy and 8% of species have both sexual and apogamous individuals. Apogamous gametophytes could produce functional male gametes and serve as paternal parents when crossed with gametophytes of allied sexual species29,46,47. Similar scenarios have been proposed in the Pteris cretica, Dryopteris varia, and Diplazium hachijoense complexes48,49,50.
Interestingly, apogamous Pteris species are most prevalent in East Asia and South Asia and mostly found in sect. Campteria45. The Pteris fauriei group (Pteridaceae), belonging to sect. Campteria, includes more than 20 taxa (18 species) with bipinnatifid laminae51. Apogamy of many taxa in this group is probably associated with hybridization51,52. In addition, similar morphology among species in this group is thought to be due to hybridization. For example, Kuo53 suggested that Pteris wulaiensis C. M. Kuo was a hybrid between P. faurei Hieron. and P. bella Tagawa. The Pteris fauriei group is mainly distributed in Eastern Asia and taxa have diverse geographic distributions and niche fidelities. Pteris fauriei, P. arisanensis Tagawa, P. biaurita L., and P. latipinna Y.S.Chao & W.L.Chiou are common and distributed widely51,54, but most taxa in the P. fauriei group are endemic to small regions. For example, P. boninensis H.Ohba, P. laurisilvicola Sa.Kurata, P. natiensis Tagawa, P. satsumana Sa.Kurata, and P. yakuinsularis Sa. Kurata are limited to Japan55, P. confusa T.G.Walker is found in India and Sri Lanka56, P. austrotaiwanensis Y.S.Chao is only reported in south Taiwan50, and P. kawabatae Sa.Kurata, P. minor (Hieron.) Y.S.Chao, and P. wulaiensis are recorded in Japan and Taiwan51,57. In regards to niche diversity, most taxa occur in shady environments, like P. latipinna, or semi-open habitats, like P. fauriei, although P. minor occurs in open habitats51,58.
We addressed the following questions of the P. fauriei group: (1) Are any species of the P. fauriei group of hybrid origin? Chloroplast and nuclear DNA data were analyzed phylogenetically and integrated with ploidy level data to infer a possible network of hybridization. (2) Is morphological diversity related to hybridization? Morphological characteristics and genetic composition of each taxon were compared. (3) Is niche diversity related to hybridization? General attributes of ecological niches were compared among taxa. (4) What is the role of apogamy in the evolution of the P. fauriei group? Contributions and/or disadvantages of apogamy in hybridization were also discussed.
Results
Morphological and habitat characters
Samples of the Pteris fauriei group were collected from Eastern Asia, including Japan, Taiwan, China, Vietnam, and Malaysia, and included 20 taxa (18 species; Supplementary Table 1). No material of P. kiuschiuensis var. centrochinensis was collected for molecular analysis, and P. confusa and P. oshimensis var. paraemeiensis were amplified only for cpDNA markers. Some sampled plants morphologically matched the type specimens (Table 1); other plants were attributed to the most morphologically similar species. We found that the taxa had different niche attributes, such as light intensity and elevation (Table 1). All species occurred in shady or semi-shady environments, except P. minor, which was found in open and sunny areas. Most taxa grew at low elevations (≤ 1000 m), but P. arisanensis, P. biaurita, and P. setulosocostulata were located at both low and higher elevations (> 1000 m).
Ploidy levels and reproductive modes
There were diploids and triploids in the P. fauriei group based on flow cytometry data; some named taxa had both diploids and triploids. Previous studies on ploidy levels in the P. fauriei group are cited in Table 1. There were seven taxa with homozygous nDNA genotypes that could be potential parental taxa (inferred by nDNA described in the section below): P. arisanensis, P. biaurita, P. boninensis, P. latipinna, P. minor, P. oshimensis var. oshimensis, and P. wulaiensis. These are all diploids except for P. arisanensis, which has some homozygous triploid individuals. Only three of the diploid taxa—P. boninensis, P. minor, and P. oshimensis var. oshimensis—had 64 spores per sporangium, which indicated that they reproduce sexually. The other plants had 32 spores per sporangium, which we inferred to be apogamous (Table 1; Supplementary Table 1). It has been reported that sexual Pteris species produce 64 spores per sporangium whereas apogamous species produce 32 spores per sporangium, especial in P. fauriei and P. minor59. Each plant produced only one type of sporangium.
The phylogeny of chloroplast DNA and nuclear DNA
The sequences of rbcL, matK, IBR3, and Knox3 of the P. fauriei group in this study (Supplementary Table 1) were clustered and named as haplotypes and allele types. Phylogenetic statistics are shown in Table 2. There were 20 cpDNA haplotypes; some were specific to one species and some were shared by several species. We included different taxa, different species and/or one species with different cpDNA haplotypes, of the P. fauriei group in the cpDNA phylogenetic analyses. For each species, samples with different haplotypes were included, and the samples with morphology identical to type specimens were marked (Fig. 1). The cpDNA topology was similar to the previously published Pteris phylogeny 60, which resolved the P. fauriei group as part of sect. Campteria, although the supporting value was not very high.
Chloroplast DNA phylogeny of haplotypes of Pteris fauriei group. ML bootstrap support values are indicated on each branch. * indicates the samples with the greatest morphological similarity to corresponded type specimens.
Taxa belonging to the P. fauriei group are coded based on their haplotypes, such as ca, cc, and cf. Most species clustered within the main fauriei clade, but P. confusa (cc), P. aff. confusa (cu), P. kiuschiuensis (ck), and P. setulosocostulata (ch), as well as some plants of P. arisanensis (cx, cxx) and P. biaurita (ci, cxx) had distant phylogenetic positions outside of the main clade. Only P. austrotaiwanensis (ct), P. boninensis (cb), P. confusa (cc), P. kiuschiuensis (ck), P. natiensis (cn), P. pseudowulaiensis (cw), and P. setulosocostulata (ch) had their own unique cpDNA haplotypes (Table 1, Fig. 1); most taxa had more than one cpDNA haplotype. Some taxa shared haplotypes, indicating shared maternal lineages, such as P. arisanensis and P. biaurita (cxx), P. arisanensis and P. latipinna (ca); P. fauriei, P. minor, P. laurisilvicola, and P. oshimensis var. oshimensis (cf); P. oshimensis var. paraemeiensis and P. satsumana (cs); and P. wulaiensis and P. fauriei (cy). The two varieties of P. oshimensis have different cpDNA haplotypes. Based on this fact, together with their distinctly different morphologies, we suggest that P. oshimensis var. paraemeiensis is a new species awaiting taxonomic revision.
The phylogenetic analysis of the Knox3 alleles supported six clades, which we labeled B, C, D, E, F + G, and H (Fig. 2a.). Group A is related to P. minor, and groups F and G correspond to P. arisanensis and P. biaurita, respectively, and are interdigitated. The IBR3 topology (Fig. 2b) approximately corresponded to that based on Knox3. Together with ploidy data, the genotypes of IBR3 and Knox3 were determined (Supplementary Table 1). Most of the genotypes were found in two or more samples from each taxon, except P. kawabatae, in which each individual sampled had a unique genotype (Table 1). The IBR3 gene exhibited fewer alleles than the Knox3 gene (Table 1) and had fewer genotypes, labeled as S, T, V, W, X, and Y. For example, samples with Knox3 genotype A1A1 and A4A4 were both IBR3 genotype T1T1 (Table 1). In some taxa, however, IBR3 exhibited more variation than Knox3, such as samples with Knox3 genotype F7G1* showing five IBR3 genotypes (Table 1). We also phased the alleles of the two nDNA genes, to infer subgenome evolution of this group (Fig. 3). There is a mean posterior probability of − 3437.35, and the effective sample size (ESS) was 292. The results support that the two nDNA genes present similar evolutionary histories, and the inferred phylogeny is generally consistent with the pattern of two DNA phylogenies in Fig. 2. Potential parental taxa with homozygous nDNA genotypes were shown empty in the nDNA columns. Most of the posterior probabilities of the phase assignments were high (heatmap in blue), and potential missing data (not be amplified alleles) are shown as having low posterior probability (heatmap in red).
Nuclear DNA phylogeny of the Knox3 gene (a) and the IBR3 gene (b) of the Pteris fauriei group. ML bootstrap support values are indicated on each branch.
The phasing inference of the Knox 3 gene and the IBR3 gene of the Pteris fauriei group by homologizer75. Bayesian posterior probabilities of branches are indicated, and the values < 0.5 are not shown. The two heatmap columns show the corresponding alleles of the two nDNA genes and are colored by the marginal posterior probability of the phase assignment. Another column presents the mean marginal probability across the two loci of the phasing assignment per tip. Taxa in red are putative parents. All the samples of the Pteris fauriei group are the same as those listed in Table 1.
The following analyses were based on the Knox3 gene results mainly because of their higher resolution than the IBR3 gene and show clearer lineages than the phasing patterns to infer hybridization relationships between potential paternal taxa and hybrids. Subsequently, a phylogenetic network was inferred based on Knox3 alleles and genotypes (Fig. 4). The crossing patterns (taxa in Table 1) were inferred. Lineages of A to G groups were colored separately, and then the lineages of putative hybrid taxa were indicated. The topology presents an evolutionary radiation involving hybridization; many hybrid taxa arose from only a few parental lineages.
Phylogenetic network of the Pteris fauriei group, based on Knox3 gene. Color of line indicates lineage of Knox3 grouping. A group in red; B group in light purple; C group in orange; D group in yellow green; E group, in blue; F group in green; G group in limon. The dash black lines mean hybrid taxa. The taxa correspond to samples in Table 1.
Parent and hybrid taxa assignment
By comparing cpDNA haplotypes (Fig. 1) and the phasing of nDNA genotypes (Fig. 3), the maternal lineages of the nDNA genotypes were inferred (Table 1). For example, the cf, cf’, and cb cpDNA haplotypes were always present along with the group A allele of nDNA, and ca of cpDNA was found with allele D7 of Knox3 nDNA. A reticulogram based on the Knox3 gene was constructed, onto which we mapped ploidy levels, habitats, and reproductive modes (Fig. 5); the taxa correspond to the taxa in Table 1. Sexual plants were few, so we assumed that the probabilities of backcrossing with parents and introgression were low, especially with the apogamous diploid. Furthermore, the two nDNA markers presented similar topologies.
A reticulogram of the Pteris fauriei group, based on the Knox3 sequences. The taxa correspond to the genotypes in Table 1. Maternal contributors of hybrids are shown as red arrows and paternal contributors as blue arrows. Ploidy levels are indicated by symbols: ellipse for diploids, triangle for triploids, and rectangle for unknown ploidy. Different kind of habitats are separated by colors inside symbols. Red or green symbol outlines indicate reproductive mode (sex and apogamy, respectively). Undiscovered taxa have dashed-lined outlines. Alleles of Knox3 and cpDNA haplotypes of each taxon are also presented.
Based on the Knox3 gene, 72 genotypes were inferred, including 14 putative parental genotypes and 58 genotypes of hybrid origin (Table 1). All species were identified based on morphology. Plants with homozygous genotypes or some sexual diploid genotypes possessing alleles from the same groups, are proposed as possible parental taxa. Seven putative parental taxa were discovered. Strictly speaking, only samples morphologically identical with type specimens of P. arisanensis (F group, F7F7F7), P. boninensis (A7A7), P. minor (A group except A7 & A4, A1A1, A1A13, A1A6, A6A6), and P. latipinna (D7D7) were homozygous, which suggests that they were not hybrid taxa. Most taxa in the P. fauriei group, however, appeared to be of hybrid origin. Although the other three putative species had homozygous samples, such as P. biaurita belonging to group G (samples with G1G1), P. oshimensis var. oshimensis belonging to A4 (Kuo 3445, A4A4), and P. wulaiensis belonging to D4 (Ebihara et al. 3234, D4D4), they also had heterozygous taxa. It was difficult to separate the homozygous samples and heterozygous samples based on their morphology.
Pteris latipinna (D7D7) was the putative parental lineage of 18 hybrid taxa (Fig. 4), far more than any other species. Pteris wulaiensis (D4D4) was the second most important parental lineage, contributing to eight taxa. Homozygous P. oshimensis var. oshimensis (A4A4) was inferred to be the maternal parent of hybrid P. oshimensis var. oshimensis (A4D1) and P. cf. fauriei (A4D7).
The Knox3 alleles of F and G groups, corresponding to P. arisanensis and P. biaurita, respectively, were clustered together within one clade (Fig. 2). The two species can be identified by morphology (especially the costal veins, described in the section below) and were phylogenetically close based on Knox3. Some taxa were inferred to arise from hybridization of the two species, with each species serving as either paternal or maternal parent, such as F16G1 (P. arisanensis (♀) × P. biaurita (♂)) and F7G1 (P. biaurita (♀) × P. arisanensis (♂)). However, the possible parental individuals (F7F7F7 in P. arisanensis; G1G1, G1G4, G1G3 in P. biaurita) were all apogamous. We have not found sexual diploid individuals of the two species. Pteris latipinna was also involved in hybrid formation with each of those two species, such as D7F4F5 in P. arisanensis and D7G1 in P. biaurita.
Unique alleles only appeared in a few rare taxa. The C3 allele was found in P. kawabatae (C3D1, C3D7E2, C3E2), P. kiuschiuensis var. kiuschiuensis (C3H1, C3D4H1), P. natiensis (C3D7, C3D4D7), and P. satsumana (B1C3). Allele E2 was only found in P. kawabatae (C3D7E2, C3E2). Alleles H3, H5, and H10 were unique to Pteris setulosocostulata (D4H4H10, B2H3H5). Allele H1 was found in P. kawabatae (B4D5H1) and P. kiuschiuensis var. kiuschiuensis (C3H1, C3D4H1, D4D7H1). The relationships of P. setulosocostulata to P. kawabatae and P. kiuschiuensis var. kiuschiuensis were unclear because the sample sizes were small for the latter two taxa.
Genotypes resolving puzzles of morphology and habitats
The diverse morphology of the P. fauriei group appeared to be an outcome of a large number of hybridizations. By comparing morphological characters51 and molecular data (Table 1), the association of specific morphological character states with particular genotypes was explored. Some character states were only found in individuals with particular alleles (Table 3). We present apparent associations between morphological and ecological variation and genotypic markers as hypotheses that could be tested with more detailed analyses. We found that hybrid taxa exhibited the morphology associated with their parents (Table 1).
Plants with homozygous genotypes provided more apparent evidence of character state/genotype associations, than did heterogeneous taxa (Tables 1 and 3). Number of pairs of basiscopic secondary pinnae were useful key characters in this group. In P. minor individuals with two or more pairs of basiscopic secondary pinnae or with tripartite laminae possessed alleles A1, A6, and A13. Pteris latipinna (D7D7) had the largest pinnae and the fewest lateral pinnae of all taxa; other taxa with wide pinnae had allele D7, including P. fauriei, P. kawabatae, P. laurisilvicola, and P. natiensis. It appears that the P. latipinna genome is associated with broad pinnae.
The triangular costal veins of P. arisanensis are more-or-less intermediate between areolate costal veins, such as in P. biaurita, and the free venation of some other species, suggesting a possible hybrid origin of P. arisanensis. Homozygous P. arisanensis (F7F7F7, Chao2135), however, had triangular costal veins, and homozygous P. biaurita (G1G1, Chao3056) had areolate costal veins, thus hybridization does not appear to account for the areolate veins of the former species. By contrast, hybrids (F7G1*, F16G1) between two species exhibit areolate costal veins and were identified as P. biaurita.
It is thought that putative hybrids had intermediate phenotypes between two parental taxa but others were more similar to one or the other parent67. Hybrids with different genotypes could be morphologically similar if they had the same allele, from the same parental taxon. For example, P. kawabatae, P. kiuschiuensis, P. natiensis, and P. satsumana have basal pinna segments connecting to midribs and they share allele C3. Although no sample had a homozygous genotype of C3, the C3 allele was associated with this character. The segments’ angle against midribs in P. kiuschiuensis var. kiuschiuensis and P. setulosocostulata was 80°–85°, wider than all the other taxa, and both taxa had H type alleles. Thus the H allele group appeared to be associated with the larger angle of the segments against midribs.
Some putative hybrids showed ecological intermediacy between parental taxa. For example, Pteris fauriei (A1A6D7, A6D7*) occurs in semi-shady environments, whereas the parental taxon P. minor (A1A1, A1A13, A1A6, A6A6) grows in open habitats and P. latipinna (D7D7) in shady habitats.
Most plants in the P. fauriei group are found below 1000 m elevation, except some plants of P. arisanensis (D7F4F5, F7F7F7) and P. biaurita (F7G1*, F16G1) can occur up to 2000 m. The Knox3 alleles of the F and G groups appeared to be associated with elevational range. Homozygous samples (i.e., putative parents) of P. biaurita (G1G1) were located in Southeast Asia at low elevation, whereas F group alleles were associated with occurrence at high elevation, above 1000 m. Hybrids (with both F and G group alleles) had broad elevational ranges (P. biaurita F7G1*, F16G1, low & high; Table 1, Supplementary Table 1).
Discussion
In recent evolutionary radiations, genetic and morphological divergences are low because of the short evolutionary time scales involved68,69,70. According to Chao, et al.60, the main fauriei clade arose around 5 Myr, and most taxa diversified around 2 Myr. Our cpDNA tree topology is based on the same markers as that study and supported a recent radiation of the P. fauriei group because most taxa were separated by distinctly shorter branches, corresponding to short time periods inferred in the study of Chao, et al.60. We revealed a complex hybridization network in the P. fauriei group, composed of morphologically similar taxa but also putative hybrid taxa exhibiting morphological intermediacy between probable parental species. This large number of hybrid taxa adds significant morphological complexity to the group leading to difficult taxonomic issues51. Furthermore, the evolution of the P. fauriei group accounts for a significant amount of the diversity of sect. Campteria in Pteris, i.e., 18 species, and especially the apogamous elements in Asia45. Sect. Campteria consisting of more than 60 species, could be the biggest section in Pteris71 and has more hybridizations, such as in the P. quadriaurita complex41. Hybridization is a key mechanism facilitating the diversification of sect. Campteria.
Among the 18 species of the P. fauriei group, some species are phylogenetically distinct from the main clade. We have clarified the hybridization patterns of P. arisanensis and/or P. biaurita. Although P. confusa was thought to be a synonym of P. arisanensis56, our data suggest that they are different species51. We found that P. confusa and P. praetermissa have the same material lineage. However, because of limited materials, the hybridization involving P. confusa7 is still unclear.
The assignment of homoeologs is complicated in taxa involved in hybridization and polyploidy72,73,74. Recently, more applicable methods for phasing gene copies into polyploid subgenomes are now in development, focusing on several loci75 or target capture data76. Even though we found no evidence of possible introgression in the P. fauriei group, we revealed that multiple hybridizations contributed to offspring exhibiting diverse phenotypes. Frequent hybridization with low introgression is found in hybrid zones of cacti, apparently due to postzygotic isolation77.
Diversification of the P. fauriei group appears to be related to different environments. For example, light intensity of P. fauriei (apogamous; semi-shady), is an intermediate habitat between its parental taxa P. minor (sexual; open) and P. latipinna (apogamous; shady). By contrast, homozygous plants of P. arisanensis and P. biaurita were located at high and low elevation, respectively, and putative hybrids of the two species showed a wider elevational range than the parents, essentially representing the addition of ranges of both parental taxa.
Cases of apparently extinct diploid parental taxa are common in many fern complexes9,48,49,78 and in the P. fauriei group as well. The distributions of extant parental taxa are much narrower than those of the hybrids, which might imply the decline of parental taxa. For example, P. latipinna is an important parent, while the known populations are fewer than ten in China and Taiwan and P. minor is limited to Iriomote Isl. (Japan) and Taiwan51. Their hybrid P. fauriei occurs across a broader area from Japan, Taiwan, and eastern China. Pteris oshimensis var. oshimensis is distributed across Japan, but the paternal plants are only found in Amami Is, Japan51,52. Thus, the ranges of hybrids often exceed those of their parents. The wide distribution of the P. fauriei group also suggests that hybridization has enhanced range expansion of the entire group79.
After long-distance dispersal, apogamy and gametophytic selfing could be adaptive because either is more likely to produce a new population than would sexual reproduction between gametophytes80. Apomictic angiosperms (i.e., asexually reproducing via seeds) tend to predominate in environments unfavorable for sexual reproduction, such as at higher latitudes and elevations, and have a wider distribution than their sexual relatives81,82. The apogamous triploid fern Myriopteris gracilis has a wide distribution which might be related to its ability to reproduce asexually83. Niche differentiation of apogamous and sexual fern taxa has also been documented previously38,84,85 similar to what we have observed in the P. fauriei group in the current study58. However, whether those differences result from reproductive modes and/or higher fitness of certain genotypes needs to be explored further.
Within the P. fauriei complex, apogamy is the main reproductive mode with only three sexual taxa. In general, asexual taxa, and lineages with low genetic variation, are thought to be less “adaptable” than are sexual taxa42. Furthermore, without recombination, deleterious mutations could accumulate and decrease the fixation of beneficial mutations (Muller’s ratchet)86,87. However, new genetic combinations from hybridization could induce phenotypic differentiation of a hybrid from its progenitors21,88,89. In the P. fauriei group, the extant hybrid taxa, even being apogamous, are apparently the survivors of natural selection and have putatively retained adaptive genotypes that arose from hybridizations, even though some of their parental taxa may be extinct.
Furthermore, apogamous taxa have evolutionary advantages that might overcome the potential deleterious effects of low genetic variation. Epigenetic modification has been proposed to explain high fitness of apogamous taxa34. Moreover, Klekowski90 proposed that genetic segregation is possible via homoeologous chromosome pairing during sporogenesis leading to genetic variation in apogamous ferns as was found in the study of Cyrtomium fortunei (Dryopteridaceae)91,92.
Of the seven diploid putative parental taxa, five are apogamous. We hypothesize that some parental taxa with apogamy in the P. fauriei group were primarily sexual and then apogamy developed subsequent to the production of hybrids. Apogamous homozygous diploids are more likely derived from sexual diploids rather than from hybrid taxa. The exact mechanisms of sexual taxa giving rise to apogamous taxa could be related to environmental factors such as light, water, and sugar93,94,95. Some critical genes expressed during reproduction could control the process of apogamy96,97,98,99,100. Apogamous gametophytes could contribute functional male gametes, which could account for the origin of some triploid taxa in the P. fauriei group.
The putative reticulate evolution of the P. fauriei group demonstrates a pattern of hybridization in evolutionary radiation, with limited or no introgression. Parental taxa have close phylogenetic relationships and novel genetic combinations in hybrids led to a rapid increase of phenotypic variation, such as diversification of niche fidelities, compared to parental taxa. Apogamy leads to the genetic fixation of some successful hybrid lineages under natural selection. While some parental species could be extinct, some of their genetic diversity will be preserved within the hybrids. Hybridization could lead to abundant taxa, more phenotypic variation, and broader distributions (as discussed above). Our ongoing and future research will explore the relationship of niche differentiation to hybridization, especially by exploring ecophysiological characters and environmental conditions. We hope to know if hybridization plays a role to maintain genetic variation and increase adaptation of ferns.
Methods
Sampling
Voucher specimens of samples were deposited at the herbarium of the Taiwan Forestry Research Institute (TAIF; Supplementary Table 1). All sampled plants were identified based on the morphology of type specimens and original protologues; the plants that were morphologically the most similar to the types were marked as representatives of specific species. To identify the plants, morphological characteristics were examined following Chao, et al.51. Use of plant material in the study complied with relevant institutional, national, and international guidelines and legislation.
Ploidy analysis and reproductive systems
Ploidy levels of samples were determined by flow cytometry, if fresh leaves were available, following the methods Chao, et al.9. Chromosome numbers of some samples had been counted in previous studies46,52. Previously published accounts of ploidy levels and reproductive modes of the P. fauriei group were also used (Table 1).
The fact that the sexual Pteris plants produce 64 spores per sporangium and apogamous plants produce 32 or fewer spores per sporangium was used to infer the reproductive mode of each plant, corresponding to sexual or apogamous29,31,59,101. Five mature sporangia were picked randomly from each plant and spores were counted under a microscope.
DNA isolation, amplification
Materials for molecular analyses were preserved in silica gel. Total genomic DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method102. Two plastid gene markers, rbcL and matK, were amplified by the primers employed in previous Pteris studies57. The two nuclear DNA (nDNA) putative single-copy genes, Knox3 and IBR3, were analyzed by single-strand conformation polymorphism (SSCP; see description below) and sequenced on the Illumina Miseq platform. Five primers for Knox3 were designed based on transcriptome sequencing data103: Knox3PF2 (ACA TTC AAG GAG CAG CTT CAG C) and Knox3PR3 (CTC TTG CCT AAC GCG CTC CAT G) for the SSCP analysis, Knox3PF2NE (CTT CAG CAG CAT GTA CGG GTT CA) and Knox3PR3 NGS (CTC TTG CCT AAC GCG CTC CAT G) for the SSCP product amplification, and Knox3PF2NE and Knox3PR4 (CAT CCT CAT CGT CCG ACA TGG T) for Miseq analyses. We tested the IBR3 primers IBR3.1_4321F2 and IBR3.1_4321R2104 and modified them for Pteris species for the Miseq analyses: primers Pteris.IBR3.1_F (CGC ATA TTC ACA GAC CCT) and Pteris.IBR3.1_R (GCC AGA TAT TGT TTA GCC CAC C). All primers for Miseq were designed to target sequences shorter than 500 bp. Both forward and reverse primers for Knox3 and IBR3 were synthesized with an 8-base barcode to produce amplicons; the barcodes were designed as recommended by Roche105.
Single-strand conformation polymorphism
Some Knox3 data were analyzed by SSCP. The SSCP analyses were conducted by isolating the nDNA loci from PCR products for each individual through separation on SSCP gels, following the methods of Ebihara, et al.8. Individual bands were isolated from gels and purified by the Gel/PCR DNA Fragment Extraction Kit (Geneaid Biotech Ltd. Taipei, Taiwan) and amplified by primers Knox3PF2NE and Knox3PR3 for further Sanger sequencing.
Library preparation, sequencing and quality assessment for NGS
Each amplicon was extracted with the illustra GFX PCR DNA and Gel Band Purification Kit (GE, UK). The amplicon mixtures were pooled with equal quantities of DNA, then a total amount of 150 ng of amplicons was used as input material for the DNA library preparations. The sequencing library was generated using the Truseq Nano DNA HT Sample Prep Kit (Illumina, USA) following the manufacturer’s recommendations, and index codes were added to each sample. DNA fragments were ligated with the adapter for Illumina sequencing, followed by further PCR amplification. Then the PCR products were purified (SPRIselect reagent, Beckman) and DNA size spectra were determined using an Agilent 2100 Bioanalyzer and quantified with a Qubit fluorometer (Invitrogen, Carlsbad, California, USA.). Finally, the DNA libraries were sequenced using the Illumina Miseq platform, and 300 bp paired-end reads were generated. Sequencing output was deposited in GenBank (Supplementary Table 1).
Bioinformatic analysis for nDNA data
Raw data were cleaned by FastQC106 and Trimmomatic107 to remove the adapter and low quality bases and reads. The forward and reverse reads were merged using PEAR108. The cleaned data were demultiplexed and clustered (including chimeras removed), and chimeras were removed using PURC109. We kept the clusters comprising the three (for diploid samples) or four highest number of reads (for triploid or ploidy unknown samples). The barcode sequences were identified and removed, that is, each read was assigned to its source accession, then primers were trimmed. The sequences obtained from SSCP and Sanger sequencing were used as the reference sequences for the clustering of consensus sequences. The sequences representing clusters of less than 100 original reads were removed. The consensus sequences of each sample were the alleles of nDNA genes and were ready for downstream phylogenetic analyses. The sequences of the Knox3 gene, both from SSCP (and Sanger sequencing) and NGS, were pooled together.
Data analysis
Sequences were automatically aligned using MUSCLE110 and then manually edited with BioEdit 7.1.3111. Using DnaSp112, the sequences of the cpDNA and two nDNA markers were clustered by haplotypes and type of alleles, respectively. To exclude errors in amplicon data, we only kept the alleles found in two or more samples. We hypothesized that the taxa that had only one allele type were homozygous and classified them as parental taxa (non-hybrid taxa), regardless of whether their reproduction modes were sexual or apogamous. Allele numbers of genotypes were supported by ploidy data. When the allele number was smaller than the ploidy level, it was difficult to infer the exact allelic composition; in those cases, “*” stands for unassorted alleles8,9.
Maximum likelihood analyses were performed for cpDNA haplotypes and nDNA sequences using the program GARLI v.2.0.1019113. Pteris species in sect. Campteris were included, and species in other sections, including P. bella Tagawa, P. formosana Baker, P. kidoi Sa.Kurata, P. longipes D.Don, and P. wallichiana J.Agardh, were chosen as outgroups60,71. For the three sequence datasets—cpDNA, Knox 3, and IBR3—three phylogenetic analyses were conducted by ten independent runs, from different random sequence-order starting trees, based on automatic termination following 10,000 generations without a significant topological change. The ML bootstrap support for each clade was assessed by performing 1000 bootstrap replicates, each replicate with one single tree search with the same search parameters as above. A 50% majority rule consensus tree was then calculated using PAUP* v. 4.0b10114. Gaps were treated as missing data. Then the nDNA genotypes, Knox3 and IBR3, were compared visually to the cpDNA markers to infer possible hybridization patterns. A phylogenetic network was visualized with Dendroscope 3115.
We also used homologizer75 to phase the alleles of the two nDNA genes (linking the tree of the two genes) and infer a phylogeny. The potential parental taxa with homozygous nDNA genotypes were fixed firstly to infer the pattern of the subgenome (or allele) evolution of this group. The two genes were modelled by an independent GTR substitution model and exponential priors (mean = 0.1) on branch lengths. The analysis was based on a Bayesian Markov chain Monte Carlo (MCMC) approach for four runs; the run with highest posterior (mean) was selected. Trees were run for 15,000 generations and the first 0.1 trees were discarded as burn-in. We assessed convergence by Tracer v.1.7116 (ESS over 200) and summarized the trees into a single tree and rooted it by FigTree117. Then the phasing estimates were plotted by RevGadgets118.
References
Nolte, A. W. & Tautz, D. Understanding the onset of hybrid speciation. Trends Genet. 26, 54–58 (2010).
Martin, C. H. The cryptic origins of evolutionary novelty: 1000-fold faster trophic diversification rates without increased ecological opportunity or hybrid swarm. Evolution 70, 2504–2519 (2016).
Seehausen, O. Hybridization and adaptive radiation. Trends Ecol. Evol. 19, 198–207. https://doi.org/10.1016/j.tree.2004.01.003 (2004).
Haufler, C. H. Species and speciation. In The Biology and Evolution of Ferns and Lycophytes (eds T. A. Ranker & C. H. Haufler) Ch. 12, 303–331 (Cambridge University Press, 2008).
Haufler, C. H. Species concepts and speciation in pteridophytes. In Pteridology in Perspective (eds J. M. Camus, M. Gibby, & R. J. Johns) 291–305 (Royal Botanic Gardens, Kew, 1996).
Wagner, W. H. J. Reticulate evolution in the Appalachian Aspleniums. Evolution 8, 103–118 (1954).
Walker, T. G. Hybridization in some species of Pteris L. Evolution 12, 82–92 (1958).
Ebihara, A. et al. Nuclear DNA, chloroplast DNA, and ploidy analysis clarified biological complexity of the Vandenboschia radicans complex (Hymenophyllaceae) in Japan and adjacent areas. Am. J. Bot. 92, 1535–1547 (2005).
Chao, Y.-S., Dong, S.-Y., Chiang, Y.-C., Liu, H.-Y. & Chiou, W.-L. Extreme multiple reticulate origins of the Pteris cadieri complex. Int. J. Mol. Sci. 13, 4523–4544. https://doi.org/10.3390/ijms13044523 (2012).
Sessa, E. B., Zimmer, E. A. & Givnish, T. J. Reticulate evolution on a global scale: A nuclear phylogeny for New World Dryopteris (Dryopteridaceae). Mol. Phylogen. Evol. 64, 563–581 (2012).
Wan, D. et al. Multiple ITS copies reveal extensive hybridization within Rheum (Polygonaceae), a genus that has undergone rapid radiation. PLoS ONE 9, e89769 (2014).
Krak, K., Caklová, P., Chrtek, J. & Fehrer, J. Reconstruction of phylogenetic relationships in a highly reticulate group with deep coalescence and recent speciation (Hieracium, Asteraceae). Heredity 110, 138–151 (2013).
Rundell, R. J. & Price, T. D. Adaptive radiation, nonadaptive radiation, ecological speciation and nonecological speciation. Trends Ecol. Evol. 24, 394–399 (2009).
Folk, R. A. et al. Rates of niche and phenotype evolution lag behind diversification in a temperate radiation. Proc. Natl. Acad. Sci. 116, 10874–10882 (2019).
Guzmán, B., Lledó, M. D. & Vargas, P. Adaptive radiation in mediterranean Cistus (Cistaceae). PLoS ONE 4, e6362 (2009).
Fernández-Mazuecos, M. et al. Resolving recent plant radiations: Power and robustness of genotyping-by-sequencing. Syst. Biol. 67, 250–268 (2017).
Stroud, J. T. & Losos, J. B. Ecological opportunity and adaptive radiation. Annu. Rev. Ecol. Evol. Syst. 47, 507–532 (2016).
Simões, M. et al. The evolving theory of evolutionary radiations. Trends Ecol. Evol. 31, 27–34 (2016).
Rieseberg, L. H. et al. Major ecological transitions in wild sunflowers facilitated by hybridization. Science 301, 1211–1216 (2003).
Ortego, J., Gugger, P. F., Riordan, E. C. & Sork, V. L. Influence of climatic niche suitability and geographical overlap on hybridization patterns among southern Californian oaks. J. Biogeogr. 41, 1895–1908 (2014).
Mitchell, N., Owens, G. L., Hovick, S. M., Rieseberg, L. H. & Whitney, K. D. Hybridization speeds adaptive evolution in an eight-year field experiment. Sci. Rep. 9, 6746 (2019).
Harrison, R. G. Hybrids and hybrid zones: Historical perspective. In Hybrid zones and the evolutionary process (ed. Harrison, R. G.) 3–12 (Oxford University Press, 1993).
Taylor, S. A., Larson, E. L. & Harrison, R. G. Hybrid zones: Windows on climate change. Trends Ecol. Evol. 30, 398–406 (2015).
Harrison, R. G. & Rand, D. M. Mosaic hybrid zone and the nature of species boundaries. In Speciation and its Consequences (eds Otte, D. & Endler, J. A.) 11–133 (Sinauer Associates, 1989).
Abbott, R. J. Plant speciation across environmental gradients and the occurrence and nature of hybrid zones. J. Syst. Evol. 55, 238–258 (2017).
Wang, L. et al. Phylogeography of the Sino-Himalayan fern Lepisorus clathratus on “The Roof of the World”. PLoS ONE 6, e25896 (2011).
Sigel, E. M., Windham, M. D., Haufler, C. H. & Pryer, K. M. Phylogeny, divergence time estimates, and phylogeography of the diploid species of the Polypodium vulgare complex (Polypodiaceae). Syst. Bot. 39, 1042–1055 (2014).
Kao, T.-T., Pryer, K. M., Freund, F. D., Windham, M. D. & Rothfels, C. J. Low-copy nuclear sequence data confirm complex patterns of farina evolution in notholaenid ferns (Pteridaceae). Mol. Phylogen. Evol. 138, 139–155 (2019).
Walker, T. G. Cytology and evolution in the fern genus Pteris L. Evolution 16, 27–43 (1962).
Kentner, E. K. & Mesler, M. R. Evidence for natural selection in a fern hybrid zone. Am. J. Bot. 87, 1168–1174 (2000).
Manton, I. Problems of Cytology and Evolution in the Pteridophyta (Cambridge University Press, 1950).
Haufler, C. H. et al. Sex and the single gametophyte: Revising the homosporous vascular plant life cycle in light of contemporary research. Bioscience 66, 928–937 (2016).
Walker, T. G. Chromosomes and evolution in pteridophytes. In Chromosomes in Evolution of Eukaryotic Groups Vol. 2 (ed A. K. Sharma) 101–141 (CRC Press, Boca Raton, FL, 1984).
Grusz, A. L. A current perspective on apomixis in ferns. J. Syst. Evol. 54, 656–665 (2016).
Grimanelli, D., Leblanc, O., Perotti, E. & Grossniklaus, U. Developmental genetics of gametophytic apomixis. Trends Genet. 17, 597–604 (2001).
Braithwaite, A. F. The Asplenium aethiopicum complex in South Africa. Bot. J. Linn. Soc. 93, 343–378 (1986).
Dyer, R., Pellicer, J., Savolainen, V., Leitch, I. & Schneider, H. Genome size expansion and the relationship between nuclear DNA content and spore size in the Asplenium monanthes fern complex (Aspleniaceae). BMC Plant Biol. 13, 1–13 (2013).
Hori, K., Kuo, L.-Y., Chiou, W.-L., Ebihara, A. & Murakami, N. Geographical distribution of sexual and apogamous types of Dryopteris formosana and Dryopteris varia (Dryopteridaceae) in Taiwan. Acta Phytotaxon. Geobot. 68, 23–32 (2017).
Hori, K. et al. Hybridization of the Dryopteris erythrosora complex (Dryopteridaceae, Polypodiidae) in Japan and adjacent areas. Hikobia 17, 299–313 (2018).
Sessa, E. B., Zhang, L.-B., Väre, H. & Juslén, A. What we do (and don’t) know about ferns: Dryopteris (Dryopteridaceae) as a case study. Syst. Bot. 40, 387–399 (2015).
Manton, I. Chromosomes and fern phylogeny with special reference to “Pteridaceae”. J. Linn. Soc. Bot. 56, 73–92 (1958).
Bengtsson, B. O. Asex and evolution: a very large-scale overview. In Lost Sex (eds Isa Schön, Koen Martens, & Peter van Dijk) 1–19 (Springer, 2009).
Liu, H.-M. et al. The evolutionary dynamics of apomixis in ferns: a case study from polystichoid ferns. J. Botany 2012, Article ID 510478, doi:https://doi.org/10.1155/2012/510478 (2012).
Chao, Y.-S., Liu, H.-Y., Chiang, Y.-C. & Chiou, W.-L. Polyploidy and speciation in Pteris (Pteridaceae). J. Botany 2012, Article ID 817920, 817927 pages, doi:https://doi.org/10.1155/2012/817920 (2012).
Picard, K. T., Ranft, H., Grusz, A. L., Windham, M. D. & Schuettpelz, E. When it only takes one to tango: Assessing the impact of apomixis in the fern genus Pteris. Am. J. Bot. 108, 2220–2234. https://doi.org/10.1002/ajb2.1761 (2021).
Huang, Y.-M., Hsu, S.-Y., Hsieh, T.-H., Chou, H.-M. & Chiou, W.-L. Three Pteris species (Pteridaceae: Pteridophyta) reproduce by apogamy. Bot. Stud. 52, 79–87 (2011).
Chiou, W.-L., Huang, Y.-M., Hsieh, T.-H. & Hsu, S.-Y. Diplazium megaphyllum (Bak.) Christ, a rare fern in Taiwan, reproduces by apogamy. Taiwan J. For. Sci. 21, 39–47 (2006).
Jaruwattanaphan, T., Matsumoto, S. & Watano, Y. Reconstructing hybrid speciation events in the Pteris cretica Group (Pteridaceae) in Japan and adjacent regions. Syst. Bot. 38, 15–27. https://doi.org/10.1600/036364413X661980 (2013).
Hori, K. & Murakami, N. Origin of the Diplazium hachijoense complex (Athyriaceae). PhytoKeys 124, 57–76. https://doi.org/10.3897/phytokeys.124.35242 (2019).
Hori, K., Okuyama, Y., Watano, Y. & Murakami, N. Recurrent hybridization without homoeologous chromosome paring in the Dryopteris varia complex (Dryopteridaceae). Chromosome Botany 13, 9–24 (2018).
Chao, Y.-S. et al. Taxonomic and nomenclatural novelties in the Pteris fauriei group (Pteridaceae). Taiwania 66, 307–316. https://doi.org/10.6165/tai.2021.66.307 (2021).
Nakato, N. & Ebihara, A. Chromosome numbers of 18 ferns in Japan: Toward completion of chromosome information in Japanese ferns. Bull. Natl. Mus. Nat. Sci. Ser. B 42, 25–40 (2016).
Kuo, C.-M. Additional knowledge for the fern flora of Taiwan 7. Pteris wulaiensis Kuo, a new species. Bot. Bull. Acad. Sin. 30, 143–145 (1989).
Shieh, W.-C. Pteridaceae. In Flora of Taiwan Vol. 1 (eds T.-C. Huang et al.) 222–233 (Editorial Committee of Flora of Taiwan, second Edition, 1994).
Iwatsuki, K., Kato, M. & Yamazaki, T. Pteridaceae. In Flora of Japan: Pteridophyta and Gymnospermae Vol. 1 (eds K. Iwatsuki, T. Yamazaki, David E. Boufford, & Hideaki Ohba) 89–97 (Kodansha, Tokyo, 1995).
Fraser-Jenkins, C. R. Taxonomic Revision of Three Hundred Indian Subcontinental Pteridophytes with a Revised Consus-List. (Bishen Singh Mahendra Pal Singh, 2008).
Chao, Y.-S. et al. New distribution of Pteris kawabatae Sa. Kurata and re-circumscription of Pteris arisanensis Tagawa. Taiwania 58, 305–310. https://doi.org/10.6165/tai.2013.58.305 (2013).
Huang, Y.-M., Chou, H.-M., Wang, J.-C. & Chiou, W.-L. The distribution and habitats of the Pteris fauriei complex. Taiwania 52, 49–58. https://doi.org/10.6165/tai.2007.52(1).49 (2007).
Huang, Y.-M., Chou, H.-M., Hsieh, T.-H., Wang, J.-C. & Chiou, W.-L. Cryptic characteristics distinguish diploid and triploid varieties of Pteris fauriei (Pteridaceae). Can. J. Bot. 84, 261–268. https://doi.org/10.1139/B05-160 (2006).
Chao, Y.-S., Rouhan, G., Amoroso, V. B. & Chiou, W.-L. Molecular phylogeny and biogeography of the fern genus Pteris (Pteridaceae). Ann. Bot. 114, 109–124. https://doi.org/10.1093/aob/mcu086 (2014).
Tsai, J.-L. & Shieh, W.-C. A cytotaxonomic survey of the pteridophytes in Taiwan (2) chromosome and spore characteristics. J. Sci. Engin. 21, 57–70 (1984).
Mitui, K. A cytological survey on the pteridophytes of the Bonin Islands. J. Jap. Bot. 48, 247–254 (1973).
Fraser-Jenkins, C. R. & Matsumoto, S. New cytotaxonomic studies on some Indo-Himalayan ferns. Indian Fern J. 32, 36–79 (2015).
Kurita, S. Chromosome number of some Japanese ferns III. J. College Arts Sci. Chiba Univ., Natural Science Series 8, 463–468 (1962).
Mitui, K. Chromosomes and speciation in ferns. Sci. Rep. the Tokyo Kyoiku Daigaku, B 13, 285–333 (1968).
Mitui, K. Chromosome studies on Japanese ferns (2). J. Jap. Bot. 41, 60–64 (1966).
Bird, K. A., VanBuren, R., Puzey, J. R. & Edger, P. P. The causes and consequences of subgenome dominance in hybrids and recent polyploids. New Phytol. 220, 87–93. https://doi.org/10.1111/nph.15256 (2018).
Meudt, H. M., Prebble, J. M. & Lehnebach, C. A. Native New Zealand forget-me-nots (Myosotis, Boraginaceae) comprise a Pleistocene species radiation with very low genetic divergence. Plant Syst. Evol. 301, 1455–1471 (2015).
Gruenstaeudl, M., Santos-Guerra, A. & Jansen, R. K. Phylogenetic analyses of Tolpis Adans. (Asteraceae) reveal patterns of adaptive radiation, multiple colonization and interspecific hybridization. Cladistics 29, 416–434 (2013).
Wang, J., Ai, B., Kong, H. & Kang, M. Speciation history of a species complex of Primulina eburnea (Gesneriaceae) from limestone karsts of south China, a biodiversity hotspot. Evol. Appl. 10, 919–934 (2017).
Zhang, L. & Zhang, L.-B. Phylogeny and systematics of the brake fern genus Pteris (Pteridaceae) based on molecular (plastid and nuclear) and morphological evidence. Mol. Phylogen. Evol. 118, 265–285 (2018).
Bertrand, Y. J. et al. Assignment of homoeologs to parental genomes in allopolyploids for species tree inference, with an example from Fumaria (Papaveraceae). Syst. Biol. 64, 448–471 (2015).
Rothfels, C. J. Polyploid phylogenetics. New Phytol. 230, 66–72 (2021).
Oxelman, B. et al. Phylogenetics of allopolyploids. Annu. Rev. Ecol. Evol. Syst. 48, 543–557 (2017).
Freyman, W. A., Johnson, M. G. & Rothfels, C. J. homologizer: Phylogenetic phasing of gene copies into polyploid subgenomes. bioRxiv, https://doi.org/10.1101/2020.10.22.351486 (2020).
Nauheimer, L. et al. HybPhaser: A workflow for the detection and phasing of hybrids in target capture datasets. bioRxiv., https://doi.org/10.1101/2020.10.27.354589. (2021).
Khan, G. et al. Maintaining genetic integrity with high promiscuity: Frequent hybridization with low introgression in multiple hybrid zones of Melocactus (Cactaceae). Mol. Phylogen. Evol. 142, 106642 (2020).
Sigel, E. M. Genetic and genomic aspects of hybridization in ferns. J. Syst. Evol. 54, 638–655 (2016).
Pfennig, K. S., Kelly, A. L. & Pierce, A. A. Hybridization as a facilitator of species range expansion. Proc. R. Soc. B 283, 20161329 (2016).
Sessa, E. B., Testo, W. L. & Watkins, J. E. Jr. On the widespread capacity for, and functional significance of, extreme inbreeding in ferns. New Phytol. 211, 1108–1119 (2016).
Hörandl, E., Cosendai, A.-C. & Temsch, E. M. Understanding the geographic distributions of apomictic plants: A case for a pluralistic approach. Plant Ecol. Divers. 1, 309–320 (2008).
Hörandl, E. The complex causality of geographical parthenogenesis. New Phytol. 171, 525–538 (2006).
Wickell, D. A., Windham, M. D., Wang, X., Macdonald, S. J. & Beck, J. B. Can asexuality confer a short-term advantage? Investigating apparent biogeographic success in the apomictic triploid fern Myriopteris gracilis. Am. J. Bot. 104, 1254–1265 (2017).
Tanaka, T., Isaka, Y., Hattori, M. & Sato, T. Ecological and phylogenetic approaches for diversification of apogamous ferns in Japan. Plant Syst. Evol. 300, 2041–2050 (2014).
Ebihara, A. & Nitta, J. H. An update and reassessment of fern and lycophyte diversity data in the Japanese Archipelago. J. Plant Res. 132, 723–738. https://doi.org/10.1007/s10265-019-01137-3 (2019).
Muller, H. J. The relation of recombination to mutational advance. Mutat. Res. Fundam. Mol. Mech. Mutagen 1, 2–9 (1964).
Bachtrog, D. & Gordo, I. Adaptive evolution of asexual populations under Muller’s ratchet. Evolution 58, 1403–1413 (2004).
Mallet, J. Hybrid speciation. Nature 446, 279–283 (2007).
Alix, K., Gérard, P. R., Schwarzacher, T. & Heslop-Harrison, J. Polyploidy and interspecific hybridization: Partners for adaptation, speciation and evolution in plants. Ann. Bot. 120, 183–194 (2017).
Klekowski, E. J. Jr. Sexual and subsexual systems in homosporous pteridophytes: A new hypothesis. Am. J. Bot. 60, 535–544 (1973).
Ootsuki, R., Sato, H., Nakato, N. & Murakami, N. Evidence of genetic segregation in the apogamous fern species Cyrtomium fortunei (Dryopteridaceae). J. Plant Res. 125, 605–612 (2012).
Ootsuki, R., Shinohara, W., Suzuki, T. & Murakami, N. Genetic variation in the apogamous fern Cyrtomium fortunei (Dryopteridaceae). Acta Phytotaxon. Geobot. 62, 1–14. https://doi.org/10.18942/apg.KJ00007694690 (2011).
Cordle, A. R., Irish, E. E. & Cheng, C.-L. Apogamy induction in Ceratopteris richardii. Int. J. Plant Sci. 168, 361–369 (2007).
Steil, W. N. Apogamy, apospory and parthenogenesis in the pteridophytes II. Bot. Rev. 17, 90–104. https://doi.org/10.1007/bf02861787 (1951).
Steil, W. N. Apogamy, apospory, and parthenogenesis in the Pteridophytes. Bot. Rev. 5, 433–453. https://doi.org/10.1007/bf02878704 (1939).
Fu, Q. & Chen, L.-Q. Comparative transcriptome analysis of two reproductive modes in Adiantum reniforme var. sinense targeted to explore possible mechanism of apogamy. BMC Genet. 20, 1–14. https://doi.org/10.1186/s12863-019-0762-8 (2019).
Banks, J. A. Sex determination in the fern Ceratopteris. Trends Plant Sci. 2, 175–180 (1997).
Banks, J. A. Sex-determining genes in the homosporous fern Ceratopteris. Development 120, 1949–1958 (1994).
Eberle, J. R. & Banks, J. A. Genetic interactions among sex-determining genes in the fern Ceratopteris richardii. Genetics 142, 973–985 (1996).
Wen, C. K., Smith, R. & Banks, J. A. ANI1: A sex pheromone–induced gene in Ceratopteris gametophytes and its possible role in sex determination. Plant Cell 11, 1307–1317 (1999).
Chao, Y. S., Liu, H. Y., Huang, Y. M. & Chiou, W. L. Reproductive traits of Pteris cadieri and P. grevilleana in Taiwan: Implications for their hybrid origins. Bot. Stud. 51, 209–216 (2010).
Allen, G., Flores-Vergara, M., Krasynanski, S., Kumar, S. & Thompson, W. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat. Protoc. 1, 2320–2325 (2006).
Matasci, N. et al. Data access for the 1,000 Plants (1KP) project. GigaScience 3, 17. https://doi.org/10.1186/2047-217x-3-17 (2014).
Rothfels, C. J. et al. Transcriptome-mining for single-copy nuclear markers in ferns. PLoS ONE 8, e76957 (2013).
Roche, T. B. Amplicon fusion primer design guidelines for GS FLX Titanium series Lib‐A chemistry. 454 Sequenc. Tech. Bull. 13, 1–3 (2009).
Andrews, S. FastQC: a quality control tool for high throughput sequence data, <http://www.bioinformatics.babraham.ac.uk/projects/fastqc> (2010).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Zhang, J., Kobert, K., Flouri, T. & Stamatakis, A. PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics 30, 614–620 (2013).
Rothfels, C. J., Pryer, K. M. & Li, F. W. Next-generation polyploid phylogenetics: rapid resolution of hybrid polyploid complexes using PacBio single-molecule sequencing. New Phytol. 213, 413–429 (2017).
Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).
Hall, T. A. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Sym. Ser. 41, 95–98 (1999).
Rozas, J. et al. DnaSP 6: DNA sequence polymorphism analysis of large datasets. Mol. Biol. Evol. 34, 3299–3302 (2017).
Zwickl, D. J. Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion PhD thesis, The University of Texas (2006).
Swofford, D. L. PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods), version 4. Sinauer, Sunderland, MA, USA (2002).
Huson, D. H. & Scornavacca, C. Dendroscope 3: An interactive tool for rooted phylogenetic trees and networks. Syst. Biol. 61, 1061–1067 (2012).
Rambaut, A., Drummond, A. J., Xie, D., Baele, G. & Suchard, M. A. Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Syst. Biol. 67, 901–904 (2018).
FigTree v. 1.2.3. Available from http://tree.bio.ed.ac.uk/software/figtree/ v. 1.2.3 (2009).
Tribble, C. M. et al. RevGadgets: An R package for visualizing Bayesian phylogenetic analyses from RevBayes. Methods Ecol. Evol. 13, 314–323. https://doi.org/10.1111/2041-210X.13750 (2022).
Acknowledgements
The authors thank Yi-Han Chang, Cheng-Wei Chen, Chun-Ming Chen, Dr. Shi-Yong Dong, Dr. Ai-Qun Hu, Dr. Yao-Moan Huang, Tian-Chuan Hsu, Hsin-Chieh Hung, Raff Knapp, Jui-Hsuan Kuo, Dr. Li-Yaung Kuo, Dr. Wai-Chao Leong, Pi-Fong Lu and Koichi Ohora, for collecting specimens, to the curators of the herbaria TAIF and TNS for the loan of herbarium specimens and for permission extract DNA, and to anonymous reviewers for their valuable suggestions. This work was supported by the Ministry of Science and Technology of Taiwan (MOST 105-2311-B-037-003, MOST106-2311-B-037-005-MY3) and the Kaohsiung Medical University Research Foundation (KMU-M111017) to Y.S.C.
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Y.S.C conceived and designed the research. Y.S.C and Y.W.H. carried out the experiments and performed the analyses; J.M.T. managed the computer science; Y.S.C. and A.E. collected the samples and data. Y.S.C., A.E., W.L.C., and T.A.R. discussed the results and wrote the manuscript.
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Chao, YS., Ebihara, A., Chiou, WL. et al. Reticulate evolution in the Pteris fauriei group (Pteridaceae). Sci Rep 12, 9145 (2022). https://doi.org/10.1038/s41598-022-11390-7
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DOI: https://doi.org/10.1038/s41598-022-11390-7
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