Plastid DNA sequence data help to clarify phylogenetic relationships and reticulate evolution in Lycoris (Amaryllidaceae)

The temperate East Asian genus Lycoris is a well known lineage of ornamental geophytes consisting of at least 20 species, some of which are thought to be of natural hybrid origin. Previous genetic studies have supported this hypothesis, but these have relied solely on the use of karyology and/or nuclear ribosomal ITS sequences. No plastid DNA data have been available to address interspecific relationships within Lycoris until now. In this study, 500 individuals from 29 populations representing 16 of the 20 published Lycoris spp. were sampled, and DNA sequences were generated for two plastid markers (trnS‐trnfM and trnC‐ycf6). From these data we inferred phylogenetic relationships among the sampled taxa at the species and population levels using concatenated phylogenetic methods. A well resolved and strongly supported phylogenetic reconstruction for Lycoris was obtained. Although the plastid DNA topology differs from that derived previously using ITS, both genomes produce trees that cluster Lycoris spp. into three clades. One of these, containing polyploid taxa such as L. albiflora, L. caldwellii, L. straminea and L. houdyshelii, shows strong evidence of reticulation, and we discuss the identity of potential parents of these allopolyploids. In contrast, we offer evidence that challenges the hypothesis that triploid individuals of L. radiata are the result of hybridization. Instead, they appear to be autotriploids that have arisen in more than one location. By comparing the phylogenetic results obtained using nuclear genomic data to those from the plastid genome, a much clearer picture of the role that hybridization and reticulation have played in the evolution of Lycoris is emerging. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 115–126.     

Isozyme evidence for the allotriploid origin of Lycoris flavescens (Amaryllidaceae)

 Variation in isozyme patterns from ten populations of the Korean endemic Lycoris species was used to test the hypothesis that L. flavescens originated from natural hybridization between diploid L. chinensis and L. sanguinea var. koreana. Lycoris sanguinea var. koreana shows fixed heterozygosity at four of nine loci assayed, suggesting that this species is an allotetraploid instead of a diploid. Electrophoretic data suggest that Lycoris flavescens is an allotriploid species derived from the hybridization between diploid L. chinensis and tetraploid L. sanguinea var. koreana. The patterns of allelic distribution in populations of L. flavescens suggest multiple origins of the allotriploid. Within the L. flavescens complex, our isozyme data support the recognition of two taxa, L. flavescens and a recently recognized species, L. uydoensis. Received August 28, 2000 Accepted December 27, 2000     

Comparison of the photosynthetic characteristics of four Lycoris species with leaf appearing in autumn under field conditions

The diurnal trends of gas exchange and chlorophyll fluorescence parameters in four Lycoris species (L. houdyshelii, L. aurea, L. radiata var. pumila and L. albiflora) were determined and compared with a portable photosynthesis analysis system. Our study revealed that L. houdyshelii had the lowest light compensation point (LCP), while the other three species had higher LCP (12.37–14.99 μmol m^?2 s^?1); L. aurea had the highest light saturation point (LSP) (1,189 μmol m^?2 s^?1), and L. houdyshelii and L. albiflora had lower LSP with the values being 322 and 345 μmol m^?2 s^?1, respectively, and L. radiata var. pumila showed the intermediate LSP. Both the species L. houdyshelii and L. albiflora exhibited a typical and obvious decline in net photosynthetic rate (P _N) during midday, which was not observed in L. aurea. This indicated a possible photoinhibition in L. houdyshelii and L. albiflora as the ratio of variable to maximum fluorescence (Fv/Fm) values were higher in these two species. The minimal fluorescence (F₀) values were lower in L. aurea and L. radiata var. pumila. The diurnal changes of transpiration rate (E) in all four species presented only one peak, appearing between 11:00 h or 13:00 h. By using simple correlation analyses, it was observed that the environmental factors affecting P _N were different among four species and the main factors were photosynthetic photon flux density (PPFD) and relative humidity especially for L. aurea and L. radiata. The results of studying indicated that the four species could be divided into two groups. The species L. radiata var. pumila and L. aurea were more adapted to a relatively high irradiance, and L. houdyshelii and L. albiflora could be grown in moderate-shade environment in order to scale up their growth and productivity.     

换锦花和中国石蒜对干旱胁迫的生理响应

以2种春出叶石蒜属植物中国石蒜和换锦花为材料,通过盆栽控水试验,以适宜水分(最大持水量的75%~80%)为对照,设置干旱胁迫(最大持水量的35%~40%)处理,研究干旱胁迫对其幼苗生理生化指标的影响,以明确2种植物的耐旱特性。结果显示:(1)换锦花和中国石蒜幼苗叶片相对含水量(RWC)和叶绿素a、b含量均随着干旱胁迫时间的延长而降低。(2)换锦花可溶性糖含量和脯氨酸含量均随着干旱时间的延长表现出持续增加的趋势,而中国石蒜则表现出先升高后降低的趋势。(3)换锦花和中国石蒜幼苗叶片TBARS含量和相对电导率总体上呈增大趋势,并在干旱末期达到最大值;超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均呈现出先上升后下降趋势。(4)换锦花和中国石蒜幼苗叶片净光合速率(Pn)、胞内二氧化碳浓度(Ci)和蒸腾速率(Tr)随着干旱胁迫时间延长均有不同程度下降。研究表明,在土壤干旱胁迫条件下,换锦花和中国石蒜幼苗叶片在水分生理、光合特性、渗透调节物质和抗氧化酶活性等方面表现出一定的差异,其中换锦花较中国石蒜表现出较强的耐旱性,且具有明显的优势。     

Diverse chromosome complements in the functional gametes of interspecific hybrids of MT- and A-karyotype Lycoris spp.

The karyotype and numeric changes in chromosomes among taxa of Lycoris (spider lilies) have been attributed to whole-arm rearrangements; however, the history of karyotype evolution of Lycoris is still ambiguous. In the natural habitat, one-third of Lycoris taxa are interspecific hybrids that are mainly sterile and extremely diverse in morphologies. Lycoris are geophytes with the reproductive stage initiated inside the bulbs during the storage period, which brings some inconveniences in collecting meiotic materials for studying chromosome pairing. The partial fertility of an artificial F1 interspecific hybrid between L. aurea (2n = 14) and L. radiata (2n = 22) provides an alternative option for tracing the meiotic process in F1 hybrids. The chromosome compositions of those functional gametes generated by the F1 hybrid could be recovered according to the chromosome complements of backcross progenies. We perform genomic in situ hybridization (GISH) analysis on somatic chromosomes of 34 BC1 plants (2n = 14–22) to reveal chromosomal divergences in number and composition of those functional gametes. GISH results also indicated a high homology between the MT- and A-genomes of Lycoris, reflecting on the partial fertility and frequently homoeologous recombination at meiosis of the F1 interspecific hybrids. The diverse chromosome complements and recombinant patterns presented in these functional gametes suggested that interspecific hybridization is an important force in driving diversification among Lycoris species. We suggest that the MT-karyotype genome may be the ancestral type in Lycoris, and some other chromosomal rearrangements in addition to centromeric fission may have played roles in the karyotype evolution of Lycoris.     

Electrophoretic evidence for the origins of tetraploids and hybrids ofSphenomeris chinensis sensu lato (Lindsaeaceae) in Japan

Hypotheses for the origins of tetraploids and hybrids ofSphenomeris chinensis sensu lato in Japan were tested using electrophoretic analysis of isozymes. Comparison of banding patterns supported the hypothesis that plants having abortive spores had resulted following hybridization between diploidS. chinensis var.chinensis andS. biflora and between diploid var.chinensis andS. gracilis. Results from isozymic analyses also revealed thatS. intermedia probably originated through allotetraploid speciation following hybridization between var.chinensis andS. biflora. There were two electrophoretic variants among tetraploids ofS. chinensis. One variant, restricted to the southern Ryukyu Islands, is probably an allotetraploid of hybrid origin between diploidS. chinensis andS. gracilis. It has been described as a new species,S. yaeyamensis Lin, Kato & K. Iwatsuki. The other variant, morphologically corresponding to var.chinensis, is distributed at least throughout southern Japan and also in northern Thailand. Its origin is uncertain, but one of the parents is probably diploidS. chinensis. Sphenomeris intermedia andS. yaeyamensis occur inland, whereas the parentalS. biflora andS. gracilis are halophilous and rheophllous, respectively.     

Analysis of genetic diversity and relationships among genus Lycoris based on start codon targeted (SCoT) marker

Genetic diversity and relationship of Lycoris species were investigated using SCoT marker analysis. Of 57 SCoT primers screened, 23 SCoT primers were identified to be high polymorphism. A total of 154 DNA bands with size varied from 0.2 kb to 2.5 kb were amplified, and 131 (82.5%) of them were polymorphic. The average number of polymorphic DNA band per primer was 5.7. Based on Nei's similarity coefficients and genetic distances, total of 43 accessions from 14 species of the genus Lycoris tested were clustered into four groups. Group I consisting of 17 accessions was further divided into two subgroup (Ia and Ib). Subgroup Ia included four species with red flower and 22 (2n) chromosomes. Subgroup Ib contained Lycoris haywardii and Lycoris albiflora which were natural hybrids with oyster white flower. Group II consisted of three species with yellow flower and 16 (2n) chromosomes. Group III was composed of Lycoris Squamigera, Lycoris incarnata and all of hybrids whose flower color was variegated. Group IV only has one species (Lycoris sprengeri) whose petal was a mixture of pink and blue. Notably, the polymorphism generated by SCoT was associated with flower color and chromosome number in this genus plants. The present data provide high-valued information for the management of germplasm, genetic improvement, and conservation of the genetic resources of Lycoris species, important horticulture and medical plants.     

Confirmation of natural hybrids between Gentiana straminea and G. siphonantha (Gentianaceae) based on molecular evidence

A few individuals with intermediate morphology always appeared in the sympatric distributions of Gentiana straminea and G. siphonantha. These intermediate individuals were hypothesized to be the hybrids of two species after a careful evaluation of their morphological characteristics. To test this hypothesis, sequence comparison of the internal transcribed spacer (ITS) regions of the nuclear ribosomal and trnS (GCU)-trnG (UCC) intergenic spacer region of the chloroplast DNA from Gentiana straminea, G. siphonantha and the putative hybrids was performed. The results suggest that most intermediate individuals were the natural hybrids between G. straminea and G. siphonantha. In addition, we examined the sequence variation among the individuals of both parent species and analyzed the possibility leading to the incongruent identification in some individuals based on morphologic and molecular evidences, respectively. The intraspecific diversification of DNA fragments within both parent species and their high variability in hybrid swarms probably resulted from chloroplast genome recombination and incomplete lineage sorting during the early stages of speciation origin of the parent species. __________ Translated from Acta Botanica Yunnanica, 2007, 29 (1): 91–97 [译自:云南植物研究]     

Airborne‐pollen pool and mating pattern in a hybrid zone between Pinus pumila and P. parviflora var. pentaphylla

The reproductive isolation barriers and the mating patterns among Pinus pumila, P. parviflora var. pentaphylla and their hybrids were examined by flowering phenology and genetic assays of three life stages: airborne‐pollen grains, adults and seeds, in a hybrid zone on Mount Apoi, Hokkaido, Japan. Chloroplast DNA composition of the airborne‐pollen was determined by single‐pollen polymerase chain reaction. Mating patterns were analysed by estimating the molecular hybrid index of the seed parent, their seed embryos and pollen parents. The observation of flowering phenology showed that the flowering of P. pumila precedes that of P. parviflora var. pentaphylla by about 6 to 10 days within the same altitudinal ranges. Although this prezygotic isolation barrier is effective, the genetic assay of airborne‐pollen showed that the two pine species, particularly P. pumila, still have chances to form F₁ hybrid seeds. Both parental species showed a strong assortative mating pattern; F₁ seeds were found in only 1.4% of seeds from P. pumila mother trees and not at all in P. parviflora var. pentaphylla. The assortative mating was concluded as the combined result of flowering time differentiation and cross‐incompatibility. In contrast to the parental species, hybrids were fertilized evenly by the two parental species and themselves. The breakdown of prezygotic barriers (intermediate flowering phenology) and cross‐incompatibility may account for the unselective mating. It is suggested that introgression is ongoing on Mount Apoi through backcrossing between hybrids and parental species, despite strong isolation barriers between the parental species.     

Impact of Asymmetrical Hybridization Followed By Sterile F1 Hybrids on Species Replacement in Pseudorasbora

Pseudorasbora pumila, one of the endangered freshwater minnows in eastern Japan, has been largely replaced by the accidentally introduced species, P. parva, which originated from western Japan. In the contact zone, P. pumila and P. parva have hybridized intensively, producing sterile F₁ hybrids. The present study determined the maternal parent of F₁ hybrids using mtDNA haplotypes to investigate the mating system between P. parva and P. pumila in the hybrid zone. We also pursued the successive changes in the genetic structures of hybridizing populations over a 5-year period using allozymes. A total of 100 natural F₁ hybrids collected from six different populations had P. pumila mtDNA without exception, suggesting that sterile F₁ hybrids resulted from mating only between P. pumila females and P. parva males. Such asymmetrical hybridization implies that P. pumila females waste considerably greater reproductive efforts compared with P. parva males. The data suggest that the rapid replacement of P. pumila by P. parva has been promoted by asymmetrical hybridization, resulting in sterile F₁ hybrids.     

火炭母的一个新异名及其系统位置初探

该文将铺地火炭母(Polygonum chinense L. var. procumbens Z. E. Zhao et J. R. Zhao)处理为火炭母原变种(Persicaria chinensis var. chinensis)的异名,并讨论了火炭母在命名上的问题,及其系统学位置。     

A revision of the Verticillium fungicola species complex and its affinity with the genus Lecanicillium

Verticillium fungicola, the type species of Verticillium sect. Albo-erecta, and related taxa were studied using morphological and molecular techniques. Sequences of the ITS region and SSU rDNA suggest that V. fungicola is very close to members of the genus Lecanicillium and unrelated to other species that were originally accommodated in the same section. New combinations in Lecanicillium are proposed for Verticillium fungicola and its var. flavidum at species rank, L. f. var. aleophilum is retained as a variety. These taxa can be distinguished from each other by optimum and maximum temperatures for growth, in addition to ITS sequence differences. Morphologically, L. flavidum is also distinct by repeated branching of the conidiophores, whereas the two varieties of L. fungicola have a simple conidiophore axis.     

Ribosomal DNA evidence for the diversification of Tropaeolum sect. Chilensia (Tropaeolaceae)

Maximum parsimony and Bayesian likelihood analysis of ribosomal DNA internal transcribed spacer (ITS) sequences from 55 samples representing 25 species, subspecies, and/or hybrids of Tropaeolum sect. Chilensia yielded a tree with a large proportion of well-supported nodes at the interspecific and intersubspecific level. However, the tree shows samples from two species, T.azureum and T. brachyceras, arising in two different positions. Samples representing subspecies of T. hookerianum and T. leptophyllum suggest that these species are polyphyletic. The data corroborate evidence for hybridization between T. brachyceras and T. tricolor. Consideration of interfertility data, past and present Chilean ecology, and empirical evidence for the behavior of phenotypic and genotypic characters in known hybrids suggest a high likelihood that reticulate evolution has played a role in the diversification of T. sect. Chilensia. This reticulate evolution may explain the discordance between the ITS tree and the conventional taxonomy. High divergence in ITS sequences between T. sect. Chilensia and other members of Tropaeolum prohibits reliable outgroup-rooting, but midpoint rooting places the root at a partition comprising taxa whose distribution conforms to a relictual eastern-western South American disjunction described for other taxa. Within the Chilean taxa, the analysis suggests that biogeographic diversification has been from the mesophytic southern habitats northward to central mediterranean and northern arid zones.     

Hybridization between the threatened plant, <Emphasis Type="Italic">Lespedeza leptostachya</Emphasis> Englem. and its co-occurring congener <Emphasis Type="Italic">Lespedeza capitata</Emphasis> Michx.: morphological and molecular evidence

Natural hybridization is common in the genus Lespedeza. No hybrids between Lespedeza leptostachya Englem. and Lespedeza capitata Michx. are formally recognized in any of the current floras, however observations in the field suggest that hybridization might occur in many of their shared habitats. Putative hybrids were compared to L. leptostachya and L. capitata using morphological measurements and screened for the presence of species-specific trnL-F gene region (cpDNA) and the ITS gene region (nrDNA). A discriminate analysis of 10 morphological measurements identified the hybrids as intermediate to both parents with two PCA axes explaining 99% of the variation between taxa. The presence of hybrids was confirmed by genetic markers with individuals morphologically identified as hybrids having cpDNA trnL-F genotypes identical to L. leptostachya and the ITS (nrDNA) phenotypes in most cases contain the ITS genotype of both parents, however, some putative hybrid individuals contained the ITS genotype of only one parents. Those individuals with L. leptostachya ITS and trnL-F could be a case of misclassification, but the presence of both L. capitata ITS genotypes and L. leptostachya trnL-F genotypes suggest segregation has occurred, which may result from either selfing or backcrossing.     

Genetic structures of allopatric and sympatric populations in Pseudorasbora pumila pumila and Pseudorasbora parva

Pseudorasbora pumila pumila was collected to investigate its genetic relationships from 11 localities in Hokkaido and eastern Honshu of Japan with Pseudorasbora parva from 6 localities in Hokkaido, eastern Honshu, and Polovynka River (a tributary of the Amur River in eastern Russia) in 1996–1998. In Hokkaido and Akita prefectures (Honshu), three P. pumila pumila populations were distributed with P. parva sympatrically. Samples of P. pumila pumila and P. parva were analyzed based on 27 allozyme loci. Genetic differentiation between P. pumila pumila and P. parva (D = 0.522–0.622, average 0.601) was almost equal to or greater than that between other congeneric fish species previously reported. In the three sympatric populations of two species, which had arisen from artificial transplantation of the latter into localities of the former, the individual frequency composition of genetic types (P. pumila pumila, P. parva, F₁ hybrid, and later filial generation hybrid) were significantly different from each other. Differing from the previous studies, F₁ hybrids were fertile and coexisting populations of the two species included the F₁ hybrid and the later filial generation hybrid. These results suggest that genetic population dynamics of sympatric populations of the two species would be influenced not only by hybridization but also by ecological and environmental factors. Alternatively, the existence of populations that possessed both the alleles specific to P. pumila pumila and those specific to P. parva may indicate that introgressive hybridization, instead of species displacement, has occurred between the two species under a certain condition.     

Extensive Mitochondrial Introgression from Pinus pumila to P. parviflora var. pentaphylla (Pinaceae)

Pinus species exhibit a paternal chloroplast inheritance and a maternal mitochondrial inheritance. The levels and patterns of cpDNA and mtDNA introgression between the two pine species, P. pumila and P. parviflora var. pentaphylla, were examined at three mountain sites in Japan. The pine species were examined by using PCR-based diagnostic genetic markers of cpDNA and mtDNA. The survey which was carried out in multiple hybrid zones demonstrated a generality in the uni-directional pattern of cytoplasmic gene flow between the two pine species, i.e. paternal cpDNA flowed from P. parviflora var. pentaphylla to P. pumila, and in contrast, maternal mtDNA flowed from P. pumila to P. parviflora var. pentaphylla. Whenever plants which had a non-native combination of cpDNA and mtDNA were observed, they always had the cpDNA haplotype of P. parviflora var. pentaphylla and the mtDNA haplotype of P. pumila. The existence of only this type of cytoplasmic chimera may suggest that F₁ hybrids are successfully produced only in the crossing of P. pumila as the maternal parent and P. parviflora var. pentaphylla as the paternal parent. The present study also detected extensive mtDNA capture in populations of P. parviflora var. pentaphylla located in the southern and middle parts of the Ohu Mountains, Tohoku, Japan. In that area, nearly all of the plants examined had the mtDNA haplotype of P. pumila. The extensive mtDNA introgression suggests that seed flow could be an effective medium for interspecific gene exchange. Received 17 August 1998/ Accepted in revised form 7 January 1999     

基于低拷贝核基因GBBSI的禺毛茛复合体系统进化研究

禺毛茛多倍体复合体及其近缘种系统进化关系复杂,杂交与多倍化现象同时存在。该复合体内高倍性植物的形成及扩散过程仍需进一步研究。首次克隆了毛茛属植物低拷贝核基因颗粒结合型淀粉合成酶 I (GBBSI )基因,并利用其构建禺毛茛多倍体复合体及其近缘种的系统进化树和网状进化关系,进而证明其适合于研究毛茛属植物种下系统发育研究。结果表明：匍枝毛茛与多倍体复合体关系密切,参与了该多倍体复合体的起源和进化;禺毛茛起源于茴茴蒜和卷喙毛茛,扬子毛茛起源于茴茴蒜和匍枝毛茛;在该类群中茴茴蒜是个关键种,它在多倍体复合体中可能起着枢纽基因组的重要作用。     

Length variation in the internal transcribed spacers of ribosomal DNA in Picea abies and related species

The structure and variation of nuclear ribosomal DNA (rDNA) units of Picea abies, (L.) Karst. was studied by restriction mapping and Southern hybridization. Conspicuous length variation was found in the internal transcribed spacer (ITS) region of P. abies, although the length of this region is highly conserved both within and among most of the plant species. Two types of ITS variants (A and B), displaying a size difference of 0.5 kb in the ITS2 region, were present within individuals of P. abies from Sweden, Central Europe and Siberia. A preliminary survey of 14 additional Eurasian and North American species of Picea suggested that length variation in the ITS region is widespread in this genus. Alltogether three length variants (A, B and C) were identified. Within individuals of eight Picea species, two length variants were present within the genome (combinations of A and B variants in P. glehnii, P. maximowiczii, P. omorika, P. polita and P. sitchensis and variants B and C in P. jezoensis, P. likiangensis and P. spinulosa). Within individuals from five species, however only one rDNA variant was present in their genome (variant A in P. aurantiaca, P. engelmannii, P. glauca, P. koraiensis and P. koyamai; variant B in P. bicolor). The ITS length variation will be useful as a molecular marker in evolutionary studies of the Picea species complex, whose phylogeny is controversial. The presence of intraindividual variation in, and shared polymorphism of the, ITS length variants raises questions about the occurrence of interspecific hybridization during the evolutionary history of Picea.     

Taxonomic status of Monotropastrum humile, with special reference to M. humile var. glaberrimum (Ericaceae, Monotropoideae)

Taxonomic treatment of the achlorophyllous monotropoid plant Monotropastrum humile is still unclear and confusing because of the lack of detailed morphological analyses and molecular phylogeny. In particular, the taxonomic status of a glabrous variety, M. humile var. glaberrimum, is under debate. Our detailed examination of the morphological characteristics of living plants revealed that M. humile var. glaberrimum can be easily distinguished from the putative conspecific taxon M. humile var. humile by characteristics not previously recognized, namely the shape and color of the floral disc. Most morphological features characterizing Cheilotheca were also found in M. humile var. glaberrimum. Moreover, there was considerable nucleotide differentiation in the internal transcribed spacer (ITS)2 sequences of M. humile var. humile and var. glaberrimum. Molecular analysis of the phylogenetic relationship of M. humile var. humile, var. glaberrimum, and other monotropoids using ITS2 sequences showed that two varieties of M. humile formed a monophyletic clade with a member of a different genus, Monotropa L., but obvious phylogenetic relationships among these three taxa were not obtained. Thus we conclude that Monotropastrum humile var. glaberrimum should be treated as a distinct species. However, the generic affiliation of M. humile var. glaberrimum could not be determined because of its intermediate character state combination and the insufficient characterization of related species. We strongly suggest that Monotropastrum as a whole needs re-evaluation.