Using DNA‐based techniques to identify hybrids among linear‐leaved Potamogeton plants collected in China

Abstract It is well known that interspecific hybrids occur in the genus Potamogeton. The linear‐leaved Potamogeton species commonly have highly variable morphological characteristics. Their hybrids often show similar vegetative characters to their parental species and their identification based solely on morphology is not always conclusive. In order to clarify whether there are any hybrids from the linear‐leaved Potamogeton plants collected in China, we used internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast rbcL gene sequences to identify the hybrids. Using ITS sequence additivity, we identified four hybrids, namely P. orientalis (P. pusillus×P. oxyphyllus), P. pusillus×P. berchtoldii, P. foliosus×P. octandrus, and P. cristatus×P. octandrus. The latter three hybrids should be considered as new hybrids in Potamogeton. The maternal parents of the four hybrids were confirmed using chloroplast rbcL gene sequences.     

Molecular evidence for a natural primary triple hybrid in plants revealed from direct sequencing

BACKGROUND AND AIMS: Molecular evidence for natural primary hybrids composed of three different plant species is very rarely reported. An investigation was therefore carried out into the origin and a possible scenario for the rise of a sterile plant clone showing a combination of diagnostic morphological features of three separate, well-defined Potamogeton species. METHODS: The combination of sequences from maternally inherited cytoplasmic (rpl20-rps12) and biparentally inherited nuclear ribosomal DNA (ITS) was used to identify the exact identity of the putative triple hybrid. KEY RESULTS: Direct sequencing showed ITS variants of three parental taxa, P. gramineus, P. lucens and P. perfoliatus, whereas chloroplast DNA identified P. perfoliatus as the female parent. A scenario for the rise of the triple hybrid through a fertile binary hybrid P. gramineus x P. lucens crossed with P. perfoliatus is described. CONCLUSIONS: Even though the triple hybrid is sterile, it possesses an efficient strategy for its existence and became locally successful even in the parental environment, perhaps as a result of heterosis. The population investigated is the only one known of this hybrid, P. x torssanderi, worldwide. Isozyme analysis indicated the colony to be genetically uniform. The plants studied represented a single clone that seems to have persisted at this site for a long time.     

Ability of the endangered species Papaver fauriei to produce hybrids with a cultivated poppy (Papaver sp.)

Papaver fauriei is an endemic and endangered species that grows only on the gravelly alpine slopes of Mt. Rishiri, Japan. Cultivated poppy (Papaver sp.), the species name of which is unknown, has been introduced to the natural habitats of P. fauriei through human activities. Because the appearance and internal transcribed spacer (ITS) sequences of these two poppies are highly similar, it is of concern that they could produce hybrids in their natural habitats. Thus, first, the ability of these two poppies to produce hybrids was analyzed by artificial fertilization in this study. A large number of seeds were produced by reciprocal crosses between P. fauriei and the cultivated poppy, comparable with the number of seeds obtained by self‐ or cross‐fertilization of P. fauriei or the cultivated poppy. In addition, high germination was observed for seeds obtained from crosses between the two poppies, and deleterious phenotypes, such as albinism and dwarfism, were not detected in the F₁ generation. These results indicate that after pollination, there is no reproductive isolation between the two poppies. Second, we sequenced the internal transcribed spacer (ITS) region of 240 poppy individuals collected from the gravelly alpine slopes of Mt. Rishiri, and 66 showed the sequence of P. fauriei, whereas 174 showed the sequence of the cultivated poppy. However, the ITS sequence that confirms hybridism between the two poppies was not detected in these individuals, indicating that hybridization of P. fauriei and the cultivated poppy rarely occurs under natural conditions. Unknown mechanism(s) appear to prevent cross‐pollination between the two poppies.     

Testing four candidate barcoding markers in temperate woody bamboos (Poaceae: Bambusoideae)

Abstract Bambusoideae is an important subfamily of the grass family Poaceae that has considerable economic, ecologic and cultural value. In addition, Bambusoideae species are important constituents of the forest vegetation in China. Because of the paucity of flower‐bearing specimens and homoplasies of morphological characters, it is difficult to identify species of Bambusoideae using morphology alone, especially in the case of temperate woody bamboos (i.e. Arundinarieae). To this end, DNA barcoding has shown great potential in identifying species. The present study is the first attempt to test the feasibility of four proposed DNA barcoding markers (matK, rbcL, trnH–psbA, and internal transcribed spacer [ITS]) in identifying 27 species of the temperate woody bamboos. Three plastid markers showed high levels of universality, whereas the universality of ITS was comparatively low. A single plastid marker provided low levels of discrimination success at both the genus and species levels (<12%). Among the combinations of plastid markers, the highest discriminatory power was obtained using the combination of rbcL+matK (14.8%). Using a combination of three markers did not increase species discrimination. The nuclear region ITS alone could identify 66.7% of species, although fewer taxa were included in the ITS analyses than in the plastid analyses. When ITS was integrated with a single or combination of plastid markers, the species discriminatory power was significantly improved. We suggest that a combination of rbcL+ ITS, which exhibited the highest species identification power of all combinations in the present study, could be used as a potential DNA barcode for temperate woody bamboos.     

Phylogeography of the marine macroalga Sargassum hemiphyllum (Phaeophyceae,Heterokontophyta) in northwestern Pacific

Sargassum hemiphyllum is commonly found in Japan and Korea, with a variety, var. chinense, that is found distributed in the southern Chinese coast. We previously reported distinct genetic differentiation between the two taxa based on the PCR‐RFLP data of plastid RubiscoL‐S spacer. The present study aims at elucidating the phylogeographic pattern of S. hemiphyllum based on more markers in the nuclear and extranuclear genomes, with a view to reveal the occurrence of hybridization. The two allopatrically distributed taxa were found to be genetically distinct in nuclear ITS2, plastidial Rubisco (Rbc) and mitochondrial TrnW_I (Trn) spacers. Their divergence was postulated to be attributable to the vicariant event which resulted from the isolation of the Sea of Japan during the late Miocene (6.58–11.25 Mya). Divergence within both S. hemiphyllum and the chinense variety was observed based on Trn spacer, while the divergence in S. hemiphyllum was further confirmed in Rbc spacer. This divergence appears to correspond to the separation of the Japanese populations between the Sea of Japan and the Pacific that occurred around 0.92–2.88 Mya (the early Pleistocene). The presence of an ITS2 clone resembling var. chinense sequences in a Japanese population of S. hemiphyllum (JpNS) raises the possibility of the introgression of var. chinense individuals into S. hemiphyllum population. Compared to that between S. hemiphyllum and the chinense variety, hybridization among the Japanese and Korean populations of S. hemiphyllum is highly probable as all these individuals share a pool of nuclear ITS2 sequences, possibly attributable to incomplete concerted evolution of ITS2.     

Molecular criteria for determining new hybrid species--an application to the Sonneratia hybrids

The possible hybrid origin of new species can usually be corroborated by molecular means. Here, we suggest that the segregation patterns of the molecular markers be further analyzed. A true hybrid species should show the patterns under continuous breeding among its members, at least beyond the F2 generation. We applied the guidelines to the putative hybrid species of Sonneratia, a widespread mangrove genus, and concluded that all the observed hybrids in this genus are simple F1's. Thus, S. x gulngai and S. x hainanensis are not true hybrid species. The segregation patterns of molecular markers should be heeded in interpreting the existence of hybrid species.     

Testing four barcoding markers for species identification of Potamogetonaceae

The pondweeds (Potamogetonaceae) are among the most important plant groups in the aquatic environment. Owing to their high morphological and ecological diversity, species identification of this aquatic family remains problematic. DNA barcoding involves sequencing a standard DNA region and has been shown to be a powerful tool for species identification. In the present study, we tested four barcoding markers (rbcL, matK, internal transcribed spacer (ITS), and trnH-psbA) in 15 Potamogeton species and two Stuckenia species, representing most species of the Potamogetonaceae in China. The results show that all four regions can distinguish and support the newly proposed genera of Stuckenia from Potamogeton. Using ITS and trnH-psbA, significant interspecific genetic variability was shown. However, intraspecific genetic variability of trnH-psbA is high and so it is not suitable for barcoding in Potamogetonaceae. The ITS and matK regions showed good discrimination. However, matK was not easy to sequence using universal primers. The best performing single locus was ITS, making it a potentially useful DNA barcode in Potamogetonaceae.     

Molecular characterisation and development of rapid molecular methods to identify species of Gracilariaceae from the Atlantic coast of Morocco

In Gracilariaceae, species identification is traditionally based on gross morphology; therefore the taxonomic status of terete individuals remains frequently problematic due to the lack of diagnostic characters to identify specimens. Different morphospecies have been recorded along the Atlantic coast of Morocco; however, no clear diagnostic characters were available to discriminate between terete species. Rapid molecular techniques have been developed recently to resolve many taxonomic problems and to re-assess the global diversity and biogeography in algae. In this study, molecular markers were used as DNA barcoding to characterise species. The sequence of the Rubisco spacer allowed identification of six species of Gracilariaceae: Gracilaria gracilis, Gracilaria dura, Gracilaria conferta, Gracilaria vermiculophylla, Gracilaria multipartita and Gracilariopsis longissima. In order to identify species with certainty, two simple and rapid methods based on the amplification of rDNA ITS and PCR-RFLP of the large subunit of the Rubisco were developed.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

Molecular characterization of fungal endophytes from Calotropis procera plants in Taif region (Saudi Arabia) and their antifungal activities

Fungal endophytes were isolated from the leaves of Calotropis procera (Apocynaceae) collected from Taif region (Saudi Arabia). Thirty-three different taxa were recovered. The overall foliar colonization rate was 35.1%. A total of 161 isolates were obtained and identified into 33 distinct operational taxonomic units based on the sequencing of the internal transcribed spacer regions of the rRNA gene. The most prevalent fungi were Aspergillus flavus, Chaetomium globosum, Cochliobolus lunatus, Fusarium dimerum, F. oxysporum, and Penicillium chrysogenum. A total of 161 isolates were tested for antifungal activities against four plant pathogenic fungi (Alternaria alternata, Fusarium oxysporum, Botrytis cinerea, and Pythium ultimum), of which 33 isolates showed antifungal activity against at least one plant pathogenic fungi. Four isolates of Chaetomium globosum and three isolates of Myrothecium verrucaria showed the strongest antifungal activity. This study reported the occurrence of a much wider spectrum of fungi, when compared with previous work. Also, it confirmed the variation of different isolates from the same species in terms of antifungal activity.     

Research note: First report of the Japanese species Grateloupia lanceolata (Halymeniaceae, Rhodophyta) from California, USA

The Japanese red alga Grateloupia lanceolata (Okamura) Kawaguchi was discovered in southern California at Santa Catalina Island in spring 2003 and April 2008 and in central California at the mouth of the Elkhorn Slough in Moss Landing in May, June and July of 2008. The morphology of thalli from both localities agrees with published figures. Sequences from the rbc L gene and the nuclear marker, internal transcribed spacer-1 from Californian G. lanceolata were identical to those from two specimens of G. lanceolata introduced to the Thau Lagoon, Mediterranean France and a specimen from Japan. It is likely that the import of oysters for mariculture played a role in its introduction into California.     

植物DNA条形码技术

DNA条形码技术是利用标准的、具有足够变异的、易扩增且相对较短的DNA片段在物种内的特异性和种间的多样性而创建的一种新的生物身份识别系统,从而实现对物种的快速自动鉴定。尽管这一技术在理论上和具体应用上仍存在很多争论。但DNA条形码概念自2003年由加拿大分类学家Paul Hebert首次提出后就在世界范围内受到了广泛关注。在植物类群中条形码的研究和应用尚处于探索阶段,稍落后于对动物类群的研究,这主要表现在：（1）DNA条形码的选择及其评价仍没有统一的标准：（2）对类群较全面的形态分类学修订和植物DNA条形码研究的结合十分缺乏：（3）以往研究在取样上尺度较大,而对具体类群的研究较少,一个科或一个属只用有限的种类作为代表,同一种内的取样个体数量也不足,这样虽然表面上看来利用选定的DNA条形码可以较容易地把代表物种区分开,但实际上目前建议的植物DNA条形码（例如由生命条形码咨询委员会植物工作组最近提出的rbcL和matK）由于其分子进化速率较慢,在种级水平上,特别是对于那些经历了适应辐射或快速进化的属来说,分辨率较低。而DNA条形码的应用主要集中在属内物种水平的鉴别,因此只有针对具体类群进行探索研究,发现进化速率较快、分辨率高且通用性好的条形码,才可能为建立完整的条形码数据库起到积极有效的作用。     

DNA barcodes for Mexican Cactaceae, plants under pressure from wild collecting

DNA barcodes could be a useful tool for plant conservation. Of particular importance is the ability to identify unknown plant material, such as from customs seizures of illegally collected specimens. Mexican cacti are an example of a threatened group, under pressure because of wild collection for the xeriscaping trade and private collectors. Mexican cacti also provide a taxonomically and geographically coherent group with which to test DNA barcodes. Here, we sample the matK barcode for 528 species of Cactaceae including approximately 75% of Mexican species and test the utility of the matK region for species-level identification. We find that the matK DNA barcode can be used to identify uniquely 77% of species sampled, and 79-87% of species of particular conservation importance. However, this is far below the desired rate of 95% and there are significant issues for PCR amplification because of the variability of primer sites. Additionally, we test the nuclear ITS regions for the cactus subfamily Opuntioideae and for the genus Ariocarpus (subfamily Cactoideae). We observed higher rates of variation for ITS (86% unique for Opuntioideae sampled) but a much lower PCR success, encountering significant intra-individual polymorphism in Ariocarpus precluding the use of this marker in this taxon. We conclude that the matK region should provide useful information as a DNA barcode for Cactaceae if the problems with primers can be addressed, but matK alone is not sufficiently variable to achieve species-level identification. Additional complementary regions should be investigated as ITS is shown to be unsuitable.     

Testing four proposed barcoding markers for the identification of species within Ligustrum L. (Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, matK, and trnH–psbA), were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH–psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH–psbA, and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%, respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH–psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH–psbA as a candidate barcode sequence.     

Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms

We present two methods for DNA extraction from fresh and dried mushrooms that are adaptable to high-throughput sequencing initiatives, such as DNA barcoding. Our results show that these protocols yield ∼85% sequencing success from recently collected materials. Tests with both recent (<2 year) and older (>100 years) specimens reveal that older collections have low success rates and may be an inefficient resource for populating a barcode database. However, our method of extracting DNA from herbarium samples using small amount of tissue is reliable and could be used for important historical specimens. The application of these protocols greatly reduces time, and therefore cost, of generating DNA sequences from mushrooms and other fungi vs. traditional extraction methods. The efficiency of these methods illustrates that standardization and streamlining of sample processing should be shifted from the laboratory to the field.     

Chemical and genetic differentiation of Ligularia tsangchanensis in Yunnan and Sichuan provinces of China

The intraspecific diversity in L. tsangchanensis collected in the Chinese Provinces Yunnan and southwestern Sichuan was studied by chemical and genetic approaches. The samples collected in Yunnan were found to contain cacalol (1) as the sole major component, while samples from Sichuan contained 7alpha- and 7beta-eremophila-9,11-dien-8-one (5 and 6) as well as the 3alpha-angeloyloxy derivative 7 as major components. In addition, the sequences of the internal transcribed spacers (ITSs) of the ribosomal RNA gene indicated that the Yunnan and the Sichuan samples constitute separate clades. These results demonstrate that L. tsangchanensis in Yunnan and Sichuan are distinct.     

Cryptic diversity in the pen shell Atrina pectinata (Bivalvia: Pinnidae): high divergence and hybridization revealed by molecular and morphological data

Cryptic species have been increasingly revealed in the marine realm through an analytical approach incorporating multiple lines of evidence (e.g., mtDNA, nuclear genes and morphology). Illustrations of cryptic taxa improve our understanding of species diversity and evolutionary histories within marine animals. The pen shell Atrina pectinata is known to exhibit extensive morphological variations that may harbour cryptic diversity. In this study, we investigated A. pectinata populations along the coast of China and one from Japan to explore possible cryptic diversity and hybridization using a combination of mitochondrial (cytochrome c oxidase subunit I, mtCOI) and nuclear (ribosomal internal transcribed spacer, nrITS) genes as well as morphology. Phylogenetic analyses of mtCOI ‘DNA barcoding gene’ sequences resolved six divergent lineages with intralineage divergences between 0.4% and 0.8%. Interlineage sequence differences ranged from 4.3% to 22.0%, suggesting that six candidate cryptic species are present. The nrITS gene revealed five deep lineages with Kimura 2‐parameter distances of 3.7–30.3%. The five nuclear lineages generally corresponded to mtCOI lineages 1–4 and (5 + 6), suggestive of five distinct evolutionary lineages. Multiple nrITS sequences of significant variance were found within an individual, clearly implying recent hybridization events between/among the evolutionary lineages, which contributed to cytonuclear discordance. Morphologically, five morphotypes matched the five genetic lineages, although the intermediates may well blur the boundaries of different morphotypes. This study demonstrates the importance of combining multiple lines of evidence to explore species cryptic diversity and past evolutionary histories.     

Delineation of Chondrus (Gigartinales, Florideophyceae) in China and the origin of C. crispus inferred from molecular data

Complete nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequences were obtained for 18 Chondrus populations collected at 15 sites from eight countries worldwide. Pairwise comparisons with the multiple alignment revealed that intraspecific divergences of ITS sequences ranged from 0.3 to 1.8% in C. crispus Stackhouse (except for the entity SVLH from France) and from 0.0 to 0.6% in C. ocellatus Holmes, whereas interspecific divergences in Chondrus varied from 1.4 to 5.0%. Three phylogenetic methods (neighbour joining, maximum parsimony and maximum likelihood) confirmed three main lineages: the North Atlantic lineage containing entities of C. crispus from Canada, France, Germany, England, Portugal, Ireland and Wales; a second lineage comprising three species: C. sp. 1, C. armatus (Harvey) Yamada et Mikami, and C. pinnulatus (Harvey) Okamura from the Northern Pacific; and a third lineage containing just one species: C. ocellatus from the Northern Pacific. Chondrus yendoi Yamada et Mikami separated from other Chondrus species singly. nrDNA ITS data indicate that a previous assignment of C. sp. 2 to Mazzaella japonica (Mikami) Hommersand may be incorrect, and additional evidence is needed to resolve the generic placement of this entity. It is inferred from the nrDNA ITS data that three Chondrus species are presently known in China with two, C. ocellatus and C. nipponicus, in Qingdao and two, C. armatus and C. nipponicus, in Dalian. We hypothesize that the ancestor of North Atlantic C. crispus had a Pacific origin, and that the present distribution of C. crispus in the Atlantic Ocean correlates with a trans-Arctic dispersal and vicariance events associated with Pleistocene glaciation maxima.     

GENETIC DIVERSITY AND INTROGRESSION IN TWO CULTIVATED SPECIES (PORPHYRA YEZOENSIS AND PORPHYRA TENERA) AND CLOSELY RELATED WILD SPECIES OF PORPHYRA (BANGIALES,RHODOPHYTA)1

We investigated the genetic variations of the samples that were tentatively identified as two cultivated Porphyra species (Porphyra yezoensis Ueda and Porphyra tenera Kjellm.) from various natural populations in Japan using molecular analyses of plastid and nuclear DNA. From PCR‐RFLP analyses using nuclear internal transcribed spacer (ITS) rDNA and plastid RUBISCO spacer regions and phylogenetic analyses using plastid rbcL and nuclear ITS‐1 rDNA sequences, our samples from natural populations of P. yezoensis and P. tenera showed remarkably higher genetic variations than found in strains that are currently used for cultivation. In addition, it is inferred that our samples contain four wild Porphyra species, and that three of the four species, containing Porphyra kinositae, are closely related to cultivated Porphyra species. Furthermore, our PCR‐RFLP and molecular phylogenetic analyses using both the nuclear and plastid DNA demonstrated the occurrence of plastid introgression from P. yezoensis to P. tenera and suggested the possibility of plastid introgression from cultivated P. yezoensis to wild P. yezoensis. These results imply the importance of collecting and establishing more strains of cultivated Porphyra species and related wild species from natural populations as genetic resources for further improvement of cultivated Porphyra strains.