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.

ABSTRACT:Controlling combining chromosomes in meiosis is necessary to maintain genome constitutions. The Dryopteirs varia complex is one of the apogamous fern groups which perform recurrent hybridization with unequal meiosis, and produce spores after meiosis of the genome doubling in spore mother cells. We analyzed five low-copy nuclear markers (AK1, EST, GapCp, G6PD, and PgiC) and three regions of plastid DNA (rbL, ndhF and trnL-F). The genetic constitution of the five nuclear markers was the same in the D. varia complex. Therefore, recurrent hybridization seemed to be occurred without homoeologous chromosome paring. KEYWORDS: Apogamous ferns, Homoeologous, Hybridization, Unequal meiosisControlling combining chromosomes in meiosis is necessary to maintain genome constitutions (Riley and Chapman 1958; Wall et al. 1971;Griffiths et al. 2006). Especially, meiosis of hybrids does not succeed because appropriate partners to pair are absent (Hirabayashi 1969(Hirabayashi , 1970. To solve this problem, allopolyploid has doubled set of chromosomes originated from progenitors to pair (Kihara 1924(Kihara , 1930 Wagner 1954; Riley and Chapman 1958;Ebihara et al. 2005;Griffiths et al. 2006; Sessa et al. 2012). Furthermore, allopolyploids can get more genome from other progenitors through recurrent hybridizations. In this case, the set of chromosomes originated from a parental allopolyploid and another parental diploid are also saved (Kihara 1924(Kihara , 1930 Riley and Chapman 1958;Griffiths et al. 2006; Sessa et al. 2012).Many apogamous ferns also have to control behaviors of their chromosomes because they produce spores through delicate mechanism called 'Döpp-Manton scheme' (Döpp 1939;Manton 1950). In this mechanism, disomic chromosome pairing and meiosis is occurred after genome doubling of spore mother cells. However, if homoeologous chromosomes pair randomly, offspring may have various morphological characteristics because genome constitutions of them are edited, but it does not seem to be occurred in regular shaped spores (Manton 1950). In fact, if homoeologous chromosome pairing is occurred in spore mother cells, the offspring get difficulty to survive (Otsuki et al. 2012).Otherwise, apogamous ferns get genetic diversity through hybridization between sexual species (Grusz et al. 2009;Ebihara et al. 2012;Dyer et al. 2012;Hori et al. 2014). Apogamous ferns get genetic variation through hybridization maintaining maturity of their spores in three patterns: (1) tetraploid apogamous hybrid between triploid apogamous species and diploid sexual species (Walker 1962; Watano and Iwatsuki 1988;Grusz et al. 2009;Dyer et al. 2012); (2) tetraploid apogamous hybrid between diploid apogamous species and tetraploid sexual species (Walker 1962); (3) triploid apogamous hybrid between diploid apogamous species and diploid sexual species (Walker 1962; Suzuki and Iwatsuki 1990;Chao et al. 2012;Jaruwattanaphan et al. 2013); (4) triploid apogamous hybrid between triploid apogamous species and diploid sexual species (Hori et al. 2014); (...

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

The name Hymenasplenium laterepens was first proposed by Cheng and Murakami in 1998 but was not validly published. We recently collected and studied this taxon at its locus classicus in Xishuangbanna, Yunnan Province in China. Molecular phylogenetic analyses and morphological observation of H. laterepens and related species clearly indicate that this is a distinct taxonomic entity worthy of species rank. Phylogenetically, H. laterepens is closely related to H. apogamum and may be involved in the reticulate evolution of the H. apogamum complex, which is comprised of several taxa with different levels of ploidy. Morphologically, H. laterepens is distinguished by the combination of its triangular laminae, large pinnae with acute apices and deeply serrated margins, and ca. 117 chromosomes at meiosis I and 32 spores in each sporangium. This work provides a complete species description and comparison to related species within the genus Hymenasplenium, particularly to other relatives of the “H. unilaterale” group. Hymenasplenium laterepens is described here with diagnosis and type designation.

We analyzed molecular phylogenetically using two low-copy nuclear markers (AK1 and Esterase) and plastid ndhF DNA sequence in the Dryopteris atrata complex. According to nuclear allele constitution and plastid ndhF sequence, apogamous species of the D. atrata complex seemed to be of hybrid origin. The genome constitution of the D. atrata complex showed that apogamous fern species which produce regularly shaped spores can maintain genome constitution if it is of hybrid origin between apogamous species and sexual species. Otherwise, sterile hybrids will be going to hybrid collapse because they cannot prevent genetic segregation.

The Japanese apogamous fern Dryopteris yakusilvicola was previously thought to be a hybrid of D. sabaei and D. sparsa var. sparsa based on morphological characterization and enzyme electrophoresis. However, the phylogenetic relationships with D. hayatae, D. melanocarpa var. melanocarpa, D. melanocarpa var. elegans, and D. sparsa var. ryukyuensis have been unclear. We performed a molecular phylogenetic analysis of the DNA sequences of two plastid (trnL-F, rbcL) and one low-copy nuclear marker (AK1) of Dryopteris yakusilvicola and related species. The origin of D. yakusilvicola is not a hybrid between D. sparsa var. sparsa and D. sabaei, but the parents appear to have been undetected species closely related to D. melanocarpa var. elegans and D. sabaei. Analysis of additional species of the D. sparsa complex around Southeast Asia and additional nuclear markers are needed to clarify the relationships of this complex.

Athyrium christensenianum has been considered a fern hybrid of diploid sexual A. crenulatoserrulatum and tetraploid sexual A. decurrentialatum. Based on plastid (rbcL) and nuclear (AK1) DNA phylogeny, this study solved relationships between A. crenulatoserrulatum (allele A), A. decurrentialatum (B) and A. opacum (C). Relationships of the complex suggested A. christensenianum had at least five allele constitution: α, AABB (tetraploid sexual); β, AAB (triploid sterile); γ, ABB (triploid sterile); δ, ABBB (tetraploid sterile); ε, ABC (triploid sterile). In addition, this study expected the existence of undetected tetraploid sexual species which is originated from hybrid between ancestral diploid sexual A. decurrentialatum and diploid sexual A. opacum.

The genus Thylacopteris is a small, phylogenetically isolated genus belonging to the fern family Polypodiaceae. This study describes a new species, Thylacopteris minuta, based on collections obtained during field surveys of Shan State, Myanmar. This new species is distinct from other species of Thylacopteris in its small size and presence of sclerenchyma strands in the rhizome. This species is also distinct from the only other species of Thylacopteris with molecular data available, T. papillosa, in a plastid rbcL phylogeny of Polypodiaceae. This new discovery of Thylacopteris from Myanmar suggests that this genus is still overlooked in Southeast Asia.