On Hybrid Formation in the Rock Fern Asplenium x alternifolium (Aspleniaceae,Pteridophyta)

Length polymorphism in a non-coding spacer (trnL_UAA-trnF_GAA) in the chloroplast DNA was used in the investigation of the origin of the most common and conspicuous European fern hybrid, Asplenium x alternifolium (Aspleniaceae, Pteridophyta). The origins of A. x alternifolium, the hybrid between A. trichomanes s.l. and A. septentrionale s.l. was studied at three ploidy levels, diploid, triploid and tetraploid. The cpDNA technique allowed us to investigate the mode of hybrid formation between sexual species for the first time over a wide geographic range and with a large sample size. Morphological variation in this hybrid has previously been attributed to different reciprocal parental combinations, and to the influence of chloroplast genes on morphogenesis. Our results demonstrate that one parent, A. septentrionale s.l., acts predominantly as the female parent in these hybrids, with only one population of A. x alternifolium showing reciprocal hybridity. The discovery of predominantly unidirectional hybrid formation in this hybrid may be explained by the different breeding systems of the parental taxa. The role of gametophyte ecology is also assessed.     

江苏铁角蕨的系统位置和多倍体起源

运用分子系统发育分析的方法研究了江苏铁角蕨（Asplenium kiangsuense）的系统位置及其与庐山铁角蕨（Agulingense）的关系,并探讨了该类群可能的多倍体起源方式。结果显示：江苏铁角蕨与庐山铁角蕨可能为同源四倍体,是组成倒挂铁角蕨复合体（Anormale complex）的成员之一;二者在形态特征与基因序列方面均表现一致,接受英文版中国植物志的处理,即把庐山铁角蕨处理为江苏铁角蕨的异名。     

中国铁角蕨属(铁角蕨科)一新名称

提出新名称 Asplenium dulongjiangense Y.F.Deng以代替晚出同名 Asplenium qiujiangense Chingex S.H.Wu non Nakaike,1 986。     

Exploring the reticulate evolution in the Asplenium pekinense complex and the A. varians complex (Aspleniaceae)

The Asplenium pekinense complex mainly comprises one diploid, A. sarelii Hook. (rare), one autotetraploid, A. pekinense Hance (best known and very common), and shares two allotetraploids, A. anogrammoides Christ (common but often misidentified) and A. altajense (Komarov) Grubov (rare and endemic) with the A. varians complex. The latter is further constituted by two diploids, A. tenuicaule Hayata (widespread) and A. semivarians Viane & Reichstein (rare), as well as other three tetraploids, A. kansuense Ching (barely known), A. varians Wallich ex Hooker & Greville (well‐known, relatively common, and morphologically variable), and A. kukkonenii Viane & Reichstein (rare and often misidentified). These two species complexes are notorious for their taxonomic difficulty based on general morphology, which is mainly caused by their history of reticulate evolution. Here, we collected most species within the two complexes, and obtained ploidy information by spore size measurement and flow cytometry investigation. Phylogenetic analyses using DNA markers representing maternally inherited chloroplast and biparentally inherited nuclear genomes helped to reconstruct the reticulate evolution history. The present results support previous hypotheses that A. sarelii is the ancestor of both A. pekinense and A. anogrammoides, as well as that A. tenuicaule is the common progenitor of A. anogrammoides, A. varians, and A. kukkonenii. We also unraveled the autotetraploid origin of A. kansuense from A. tenuicaule for the first time, and found that A. altajense shares essentially identical genomes with A. anogrammoides.     

Leaf anatomy and the occurrence of false veins in Asplenium (Aspleniaceae, Pteridophyta)

False veins in African afro-alpine (2000–4700 m) Asplenium species with long creeping rhizomes and highly dissected leaves are morphologically and anatomically similar to true veins but differ in the absence of a vascular bundle. False veins in Aspleniaceae may have originated by the fusion of leaf lobes, and are more similar to those in Angiopteris and Thelypteris than to those in Davallia and Hymenophyllaceae. Because false veins are long and extend from the leaf margin to the junction of the neighbouring true veins in A. actiniopteroides , A. goetzei , A. majus , A. praegracile , A . sp. nov. and A. uhligii , but short, not reaching this junction in A. decompositum , A. demerkense , A. kassneri , A. linckii and A. mildbraedii , and even absent in A. aethiopicum , A. lademannianum , A. simii , A. stipicellatum and A. volkensii , they can be used for identification in this enigmatic group of ferns.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 145 , 187–194.     

Isozyme variation and genetic relationships among taxa in the Asplenium obovatum group (Aspleniaceae, Pteridophyta)

The Asplenium obovatum group consists of diploid and tetraploid taxa; the origin of the tetraploid A. obovatum subsp. lanceolatum was previously considered to have occurred via autopolyploidy, involving one of the diploids of the group, either A. obovatum subsp. obovatum var. obovatum or var. protobillotii. To test this hypothesis, electrophoretic analyses of eight enzyme systems encoded by fourteen putative loci and cytological studies of the artificial hybrid between both diploid varieties were conducted. Alleles of the loci Lap-1, Mdh-2, Mdh-3, Pgm-1, Pgm-1', and 6Pgd-1 emerged as genetic markers for the diploids and were present in an additive pattern in most of the analyzed individuals of the tetraploid. Cytological results indicated a high degree of genomic homology between the diploids. These results indicated that the tetraploid behaves as a segmental allopolyploid. Our results showed that both diploids were involved in the origin of the tetraploid. We propose the new combination Asplenium obovatum subsp. protobillotii for one of the diploids.     

Chloroplast DNA inheritance in theStellaria longipes complex (Caryophyllaceae)

Inheritance of chloroplast DNA (cpDNA) was examined in F₁ progenies derived from three crosses and three corresponding reciprocal crosses betweenStellaria porsildii andS. longifolia. Chloroplast DNA restriction fragments were analyzed using methods of nonradioactive digoxigenin-11-dUTP labeling and chemiluminescent detection with Lumi-Phos 530. Distinct interspecific restriction fragment polymorphisms were identified and used to demonstrate the mode of cpDNA inheritance. Mode of cpDNA inheritance differed among crosses. Two crosses in whichS. porsildii, SP2920-21, was the maternal parent exhibited three different types of plastids, maternal, paternal and biparental, among the F₁ hybrids, suggesting a biparental cpDNA inheritance and plastid sorting-out inStellaria.     

地黄属植物的DNA条形码研究

地黄属(Rehmannia)为玄参科(Scrophulariaceae)药用植物,广泛分布于中国中东部及北部地区。由于地黄属植物经历了快速成种,导致其属内物种间形态性状差异较小,运用传统的形态学分类方法已难以准确地鉴定物种,近年来迅速发展起来的DNA条形码技术为快速、准确地鉴别物种提供了新思路。本研究选用3个叶绿体DNA非编码区片段(trn L-trn F、trn M-trn V和trn S-trn G)及核基因ITS片段,运用PWG-distance和TreeBuilding两种方法对地黄属5个物种75个个体进行了DNA条形码分析。结果表明:单个叶绿体DNA片段或核基因ITS片段对地黄属物种的鉴别率较低(0%~20%),组合的叶绿体DNA片段分辨能力虽然高于单个DNA片段,但并不能将地黄属5个物种完全区分开;trn S-trn G+ITS片段组合的分辨率可达100%,能够将地黄属5个物种准确区分,与所有叶绿体DNA片段和核基因ITS片段组合(trn L-trn F+trn M-trn V+trn S-trn G+ITS)的辨别率相同,因此推荐trn S-trn G+ITS作为地黄属植物的标准条形码。此外,利用DNA条形码鉴别物种时,可采用叶绿体DNA片段和核DNA片段组合的方法来提高物种鉴定的成功率。     

Gametophyte morphology and gametangial ontogeny of Asplenium foreziense and related taxa (Aspleniaceae: Pteridophyta)

Gametophytes of Asplenium foreziense and related taxa have been studied by culture of spores on mineral agar and soil. Those of A. obovatum ssp. obovatum var. protobillotii and var. deltoideum , ssp. numidicum , and of A. macedonicum are described for the first time. Gametophyte development follows the Adiantum type in the A. obovatum group, and the Aspidium type in A. fontanum . Both types of development have been found in A. foreziense , depending on the sporophytic sample. The taxa with hairy gametophytes show significant differences in hair density. As in most of the homosporous ferns, antheridia are formed first and in a high proportion of gametophytes in the A. obovatum group and in A. fontanum , except for one sample; most of these male gametophytes become bisexual. In A. foreziense and A. macedonicum archegonia are formed first or at the same time as antheridia, but the proportion of female gametophytes is higher than in the other taxa; some of the gametophytes become bisexual, most of them differentiated from the female ones. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 139 , 87–98.     

Phylogenetic analysis of Asplenium subgenus Ceterach (Pteridophyta: Aspleniaceae) based on plastid and nuclear ribosomal ITS DNA sequences

Phylogenetic relationships among 20 taxa of the fern genus Asplenium subgenus Ceterach (Filicopsida, represented by 73 accessions) were investigated using DNA sequence data from the nuclear ribosomal internal transcribed spacers (ITS nDNA) and plastid trnL-F intergenic spacer. In addition, a single sample per taxon was used in an analysis of the plastid rbcL gene. Chromosome counts were determined for all the samples, and these demonstrated a range from diploid to octoploid. Analyses of the DNA sequence data indicated that Asplenium subgenus Ceterach is polyphyletic, implicating homoplasy in the characters previously used to circumscribe this taxon. Plastid trnL-F and rbcL analyses resulted in identical tree topologies. The trees produced from the separate plastid and nuclear matrices agree in (1) the recognition of identical groups of accessions corresponding to A. dalhousiae, A. ceterach, A. aureum, A. cordatum, A. phillipsianum, and A. haughtonii; (2) the division of A. subg. Ceterach into two subclades, a Eurasian-Macaronesian and a strictly African alliance; (3) the position of A. dalhousiae as a member of the former subclade; (4) the lack of genetic variation in A. cordatum despite its morphological variability; and (5) the clustering of each autopolyploid with their diploid ancestor. However, the plastid and nuclear trees differ in their placement of A. haughtonii and A. dalhousiae, which might be due to different evolutionary histories of nuclear and plastid genomes, and is possibly an indication of ancient hybridization. The analyses confirm the existence of several strictly African taxa. Asplenium phillipsianum and A. cordatum each form species complexes of diploid and autopolyploid taxa, from which a third, morphologically intermediate, allotetraploid species has originated. Asplenium haughtonii is a distinct endemic species from Saint Helena. The maternally inherited plastid sequences support the hypothesis that A. aureum is an ancestor of A. lolegnamense and of A. octoploideum. Because gene conversion did not eliminate divergent ITS alleles in the allopolyploids, their reticulate ancestry could be demonstrated. Biparentally inherited nrITS sequences support the allopolyploid status of A. aureum, A. lolegnamense, and A. punjabense, indicating they share the ancestral A. javorkeanum genome.     

Chloroplast DNA variation and the phylogeny ofAsplenium sect.Hymenasplenium (Aspleniaceae) in the New World tropics

Asplenium sect.Hymenasplenium is a well-defined group in Aspleniaceae, distinguished by several morphological and cytological characters. However, interspecific relationships in the section were not clear. In this paper, we report the phylogenetic relationships of 9 Neotropical species of sect.Hymenasplenium determined by chloroplast DNA restriction site variation analyses. From the obtained phylogenetic tree, two major clades: one withA. obtusifolium, A. riparium, A. volubile andA. repandulum and another withA. delitescens, A. ortegae, A. purpurascens, A. laetum andA. hoffmannii were recognized.Asplenium delitescens was shown to have a polyphyletic origin. It was also shown that the epiphytic habit evolved only once in the New World species of sect.Hymenasplenium.     

Chloroplast DNA inheritance in Populus

Summary The inheritance of chloroplast (cp) DNA was examined in F₁ hybrid progenies of two Populus deltoides intraspecific controlled crosses and three P. deltoides × P. nigra and two P. deltoides × P. maximowiczii interspecific controlled crosses by restriction fragment analysis. Southern blots of restriction digests of parental and progeny DNAs were hybridized to cloned cpDNA fragments of Petunia hybrida. Sixteen enzymes and five heterologous cpDNA probes were used to screen restriction fragment polymorphisms among the parents. The mode of cpDNA inheritance was demonstrated in progenies of P. deltoides × P. nigra crosses with 26 restriction fragment polymorphisms of cpDNA differentiating P. deltoides from P. nigra, as revealed by 12 enzyme-probe combinations, and in progenies of P. deltoides × P. maximowiczii crosses with 12 restriction fragment polymorphisms separating P. deltoides from P. maximowiczii, as revealed by 7 restriction enzyme-probe combinations. In all cases, F₁ offspring of P. deltoides × P. nigra and P. deltoides × P. maximowiczii crosses had cpDNA restriction fragments of only their maternal P. deltoides parent. The results clearly demonstrated uniparental-maternal inheritance of the chloroplast genome in interspecific hybrids of P. deltoides with P. nigra and P. maximowiczii. Intraspecific P. deltoides hybrids also had the same cpDNA restriction fragments as their maternal parent. Maternal inheritance of the chloroplast genome in Populus is in agreement with what has been observed for most other angiosperms.     

Chloroplast and mitochondrial DNA composition of triploid and tetraploid somatic hybrids between Lycopersicon esculentum and Solanum tuberosum

The chloroplast (cp) DNA type and mitochondrial (mt) DNA composition of 17 somatic hybrids between a cytoplasmic albino tomato and monoploid potato (A7-hybrids) and 18 somatic hybrids between a nitrate reductase-deficient tomato and monoploid potato (C7-hybrids) were analyzed. Thirteen A7-hybrids and 9 C7-hybrids were triploids (with one potato genome); the other hybrids were tetraploid. As expected, all A7-hybrids contained potato cpDNA. Of the C7-hybrids 7 had tomato cpDNA, 10 had potato cpDNA and 1 hybrid contained both tomato and potato cpDNA. The mtDNA composition of the hybrids was analyzed by hybridization of Southern blots with four mtDNA-specific probes. The mtDNAs in the hybrids had segregated independently from the cpDNAs. Nuclear DNA composition (i.e. one or two potato genomes) did not influence the chloroplast type in the C7-hybrids, nor the mtDNA composition of A7- or C7-hybrids. From the cosegregation of specific mtDNA fragments we inferred that both tomato and potato mtDNAs probably have a coxII gene closely linked to 18S+5S rRNA genes. In tomato, atpA, and in potato, atp6 seems to be linked to these mtDNA genes.     

Chloroplast DNA variation in Populus. III. Novel chloroplast DNA variants in natural Populus x canadensis hybrids

A rare phenomenon of the occurrence of novel non-parental chloroplast DNA (cpDNA) variants in natural sexual interspecific hybrids between Populus deltoides var deltoides and P. nigra, P. x canadensis is described. Restriction fragment variation of cpDNA in 17 P. x canadensis cultivars was examined and compared with that of representative samples of P. deltoides and P. nigra using 83 combinations of 16 restriction enzymes and six Petunia hybrida cpDNA probes. Twelve cultivars had one to five novel non-parental cpDNA fragments in the chloroplast genome region homologous to the 9.0-kb PstI cpDNA fragment of Petunia from the large single-copy region.     

Chloroplast DNA assorts randomly in intraspecific somatic hybrids of Nicotiana debneyi

Summary Plants were regenerated following intraspecific fusion of leaf protoplasts from two naturally occurring genotypes of Nicotiana debneyi. The two genotypes differed in the EcoRl fragmentation pattern of chloroplast DNA and in the nuclear-coded phosphoglucomutase (Pgm) isozymes. There was no conscious selection for hybrid genotypes during protoplast culture or plant regeneration. Among 225 plants screened for Pgm, six were identified as nuclear hybrids. Restriction endonuclease and filter hybridisation analysis revealed that the cytoplasms of the hybrids contained one or other but never both parental chloroplast DNAs. The sorting out of chloroplasts was random and complete; the limit of detecting a rare chloroplast-DNA type in a mixture was 0.1%.     

Paternal inheritance of chloroplast DNA in interspecific hybrids in the genus Larrea (Zygophyllaceae)

The mode of chloroplast DNA (cpDNA) inheritance was investigated in the genus Larrea (Zygophyllaceae) by polymerase chain reaction (PCR) amplification of cpDNA fragments using three pairs of chloroplast universal primers. A total of 20 F(1)s from interspecific crosses among five different taxa in the section Bifolium was examined. Twelve F(1)s were from six crosses between L. cuneifolia (4x) and L. divaricata (2x) (Peru or Argentina) or L. tridentata (2x or 4x). Eight F(1)s were from two sets of reciprocal crosses between L. divaricata (2x) (Argentina) and L. tridentata (2x). Length polymorphism was observed in all three regions of cpDNA that separated L. cuneifolia parents from L. divaricata and L. tridentata parents and in one of the three cpDNA regions that differentiated L. divaricata (Argentina) parents from L. tridentata (2x) parents. In each case, it was the paternal cpDNA marker that appeared in the F(1) individuals. This was further confirmed by restriction fragment length polymorphism (RFLP) analysis of the amplified cpDNA fragments. Larrea may be the fifth genus reported in angiosperms with a paternal bias in cpDNA transmission. Possible mechanisms that may result in paternal cpDNA inheritance were briefly reviewed. Based on the observed uniparental paternal inheritance of cpDNA, restriction analysis of the three cpDNA regions and previous cytogenetic studies, L. divaricata was probably the maternal progenitor of L. cuneifolia.     

云南铁角蕨与泸山铁角蕨的关系:来自叶绿体rbcL、trnL-F和rps4-trnS序列的证据

云南铁角蕨Asplenium yunnanense与泸山铁角蕨Asplenium lushanense的分类是一个悬而未决的问题。本文对云南铁角蕨与泸山铁角蕨及近缘类群的叶绿体rbcL基因和trnL-F、rps4-trnS基因间隔区序列进行PCR扩增和序列分析;并与已经发表的铁角蕨属植物的相应序列进行比较,发现云南铁角蕨与泸山铁角蕨的rbcL基因和trnL-F、rps4-trnS基因间隔区序列之间均未表现出差异,因此叶绿体DNA序列的证据不能将云南铁角蕨与泸山铁角蕨分开,从而从分予系统学方而证明云南铁角蕨与泸山铁角蕨是一对亲缘关系非常近的物种。     

Spore morphology as a taxonomic tool in the delimitation of three Asplenium L. species complexes (Aspleniaceae: Pteridophyta) in Cuba

As a contribution to the taxonomic study of Asplenium L. for the new "Flora de la República de Cuba", the spore morphology was studied into three complexes of hardly delimited species. Twenty-two collections have been studied by SEM and eighty-nine by LM. Shape, number of spores per sporangium and perispore ornamentation, specially fine details revealed by scanning electron microscopy, were the most valuable characters in the recognition of taxa. Irregularities in spore shape and size, unusual development of perispore wall surface elements, and collapsed protoplast characterize aborted spores and suggest the occurrence of a possible hybrid species.     

Chloroplast DNA variation in the grass tribe Festuceae

Summary Six grasses, Hordeum sativum, Dactylis glomerata, Festuca arundinacea, F. pratensis, F. rubra and Lolium multiflorum were subjected to chloroplast DNA analysis based on restriction endonuclease digestion fragments and end labeling with ³⁵S nucleotides. This method is compared with others in general use. The results indicate that Lolium multiflorum is closely affiliated with Festuca pratensis and F. arundinacea; in fact much closer than F. rubra is to any of them.     

Chloroplast DNA differences between cultivated hop,Humulus lupulus and the related species H. japonicus

Chloroplast DNA (cpDNA) of Humulus Lupulus and H. japonicus was examined by restriction endonuclease analysis with BamHI, BanI, BclI, BstEII, DraI, EcoRI, EcoRV, HindIII, KpnI, PaeR7I, PstI, PvuII, SalI and XhoI. The restriction fragment patterns showed that the cpDNAs shared a large number of restriction sites. However, the chloroplast genomes of the two species could be distinguished by differences in restriction site and restriction fragment patterns in the PstI, PvuII, BclI, EcoRV, DraI and HindIII digests. On the basis of the complexity of restriction enzyme patterns, the enzymes PstI, PvuII, SalI, KpnI and XhoI were selected for mapping the chloroplast genomes. Single and double restriction enzyme digests of cpDNA from the two species were hybridized to cpDNA probes of barley and tobacco. The data obtained from molecular hybridization experiments were used to construct the cleavage site maps. Except for the PstI digest, the arrangement of cpDNA restriction sites was found to be the same for both species. An extra PstI site was present in H. lupulus. Three small insertions/deletions of about 0.8 kbp each were detected in the chloroplast genomes of the two species. Two of these insertions/deletions were present in the large and one in the small singlecopy region of the chloroplast genome. The cpDNA of Humulus was found to be a circular molecule of approximately 148 kbp that contains two inverted repeat regions of 23 kbp each, a small and a large single -copy region of approximately 20 kbp and 81 kbp, respectively. The chloroplast genome of hop has the same physical and structural organization as that found in most angiosperms.