Application of DNA barcodes in Hedyotis L. (Spermacoceae,Rubiaceae)

The potential application of DNA barcodes of plastid (matK, trnH–psbA, petD, and rbcL) and nuclear (internal transcribed spacer (ITS) of rDNA) DNA regions was investigated for 25 Hedyotis taxa. The ITS showed the best species discrimination by resolving 23 of the species as exclusive lineages with no shared alleles between any of the 24 distinct species (H. assimilis and H. mellii are not supported as distinct species based on our molecular and morphological data). Conversely, rbcL performed the worst and only resolved 10 of the species as exclusive lineages, and 10 species with shared alleles. Using ITS has the advantage of high PCR amplification success and it provides good intra- and interspecific variation distribution patterns. The most powerful plastid markers were petD and trnH–psbA, but we could amplify and sequence trnH–psbA for only 83% of the accessions sampled. Combination of ITS and petD performed extremely well, with all 24 of the distinct species resolved as exclusive lineages and no shared alleles between any of the distinct species. We therefore recommend ITS, or a combination of ITS and petD, as the standard DNA barcode in Hedyotis, but acknowledge that there are no shared alleles between distinct species for matK and rbcL combined.     

Development of microsatellite markers in heterostylous Hedyotis chrysotricha (Rubiaceae)

? Premise of the study: Microsatellite primers were developed for a heterostylous herb, Hedyotis chrysotricha to investigate the effect of habitat fragmentation on its genetic diversity and population structure. ? Methods and Results: Twelve primer sets were developed and their polymorphisms were tested on 47 individuals from two island populations of H. chrysotricha in Thousand Island Lake of China. The number of alleles per locus ranged from five to 10, with an average of seven alleles. Expected heterozygosity per locus ranged from 0.284 to 0.821 and observed heterozygosity ranged from 0.191 to 0.851. ? Conclusions: We showed that all of the 12 microsatellite markers developed for H. chrysotricha are polymorphic within populations, which should provide a powerful tool for assessing population structure and genetic diversity across fragmented and continuous populations, and for studying the genetic effects of habitat fragmentation on this species.     

国产耳草属(茜草科)一新种——蕴璋耳草

报道了中国茜草科Rubiaceae耳草属耳草组Hedyotis sect. Hedyotis植物的一个新种——蕴璋耳草H. koiana R. J. Wang。本新种同丹草H. herbacea L.和松叶耳草H. pinifolia Wall. ex G. Don因均具有较窄的叶片和仅顶部开裂的蒴果而相似, 但本种因具线形的叶片以及顶生和上部腋生的聚伞花序而不同于丹草, 与松叶耳草的不同之处在于蕴璋耳草具有较长的花梗和疏松的花序以及花冠管内部无毛。     

Hedyotis koiana R. J. Wang,a new species of Rubiaceae from China

Hedyotis koiana R. J. Wang is described as a new species from Guangdong, Hunan, Jiangxi, Guangxi, Fujian, and Hainan provinces of China. It is most similar to H. herbacea L. and H. pinifolia Wall. ex G. Don. because they all have narrow leaves and their capsules are loculicidally dehiscent at the top, but it differs from H. herbacea by having termi- nal or upper axillary cyme and narrow leaves, and it can be distinguished from H. pinifolia by having long flowering pedicles, sparse inflorescence, and adaxially glabrous corolla.     

耳草属（茜草科）植物的两个新种

耳草属（茜草科）植物的两个新种=Two new species of the genus Hedyotis　(Rubiaceae) from China[刊,中]/ 王 瑞 江（中国科学院华南植物研究所 广州 510650）,邢 福 武//植物分类学报.—2002,41（1）.—85～88 摘要：描述了国产耳草属(茜草科)植物的两个新种,启无耳草Hedyotis wangii　R. J. Wang和崖州耳草H. yazhouensis　F. W. Xing et R. J. Wang。     

Cryptic self‐incompatibility and distyly in Hedyotis acutangula Champ. (Rubiaceae)

Distyly, floral polymorphism frequently associated with reciprocal herkogamy, self‐ and intramorph incompatibility and secondary dimorphism, constitutes an important sexual system in the Rubiaceae. Here we report an unusual kind of distyly associated with self‐ and/or intramorph compatibility in a perennial herb, Hedyotis acutangula. Floral morphology, ancillary dimorphisms and compatibility of the two morphs were studied. H. acutangula did not exhibit precise reciprocal herkogamy, but this did not affect the equality of floral morphs in the population, as usually found in distylous plants. Both pin and thrum pollen retained relatively high viability for 8 h. The pollen to ovule ratio was 72.5 in pin flowers and 54.4 in thrum flowers. Pistils of pin flowers remained receptive for longer than those of thrum flowers. No apparent difference in the germination rate of pin and thrum pollen grains was observed when cultured in vitro, although growth of thrum pollen tubes was much faster than that of pin pollen tubes. Artificial pollination revealed that pollen tube growth in legitimate intermorph crosses was faster than in either intramorph crosses or self‐pollination, suggesting the occurrence of cryptic self‐incompatibility in this species. Cryptic self‐incompatibility functioned differently in the two morphs, with pollen tube growth rates after legitimate and illegitimate pollination much more highly differentiated in pin flowers than in thrum flowers. No fruit was produced in emasculated netted flowers, suggesting the absence of apomixis. Our results indicate that H. acutangula is distylous, with a cryptic self‐incompatibility breeding system.     

Notes on Galianthe (Spermacoceae, Rubiaceae) in the Bolivian flora

Four new species ofGalianthe (G. boliviana, G. chiquitosana, G. sudyungesis, andG. kempffiana) for Bolivia are described and illustrated.Galianthe kempffiana is also known from Brazil.Galianthe laxa andG. bisepala are reported for the first time for Bolivia.     

Identification of herbal medicinal materials using DNA barcodes

Herbal medicinal materials have been used worldwide for centuries to maintain health and to treat disease. However, adulteration of herbal medicines remains a major concern of users and industry for reasons of safety and efficacy. Identification of herbal medicinal materials by DNA technology has been widely applied,started from the mid-1990s. In recent years, DNA barcoding of global plant species using four standard barcodes (rbcL, matK, trnH-psbA and ITS) has been a major focus in the fields of biodiversity and conservation. These DNA barcodes can also be used as reliable tools to facilitate the identification of herbal medicinal materials for the safe use of herbs, quality control, and forensic investigation. Many studies have applied these DNA barcodes for the identification of herbal medicinal species and their adulterants. The present article reviews efforts in the identification of herbal medicinal materials using the standard DNA barcodes and other DNA sequence-based markers.     

DNA条形码在鞘翅目昆虫分子系统学研究中的应用

近年来,DNA条形码(DNA Barcoding)技术已经成为生物分类学研究中备受关注的新型技术,并在鞘翅目昆虫系统发育研究中得到广泛应用。本文总结了鞘翅目昆虫DNA条形码研究所用COⅠ基因序列,概述了DNA条形码在鞘翅目昆虫的物种分类鉴定、发现新种和隐存种、系统发育关系研究等方面的应用,并对DNA条形码研究技术新进展和标准序列筛选需要注意的问题进行了讨论。     

艳丽耳草的二型花柱及异型自交不亲和系统

异型花柱是一种受遗传因素控制的花型多态性现象,包括二型花柱和三型花柱两种类型.本文以茜草科艳丽耳草(Hedyotis pulcherrima)为实验材料,通过对其野外居群的花型、花部形态及花粉特征等观察,发现艳丽耳草野外居群同时存在长花柱型花和短花柱型花,长/短花柱型花的数量比例为1∶1.两型花具有精确的交互式雌雄异位特征,并且该特征与花冠长度相关性显著.长/短花柱型花的柱头裂片长度、花粉大小及淀粉含量等具有二型性.花粉体外培养时花粉萌发率及花粉管生长速率无显著性差异.人工授粉后,艳丽耳草长/短花柱型花型间异交花粉管生长形态正常,授粉24 h后花粉管均已进入子房.而长/短花柱型花在自交及型内异交下均表现为不亲和,花粉管生长停止于柱头,花粉管顶端累积胼胝质并膨大.艳丽耳草没有无融合生殖现象,型间人工辅助异交授粉结实率为100％,显著高于自然结实率.本研究结果表明,艳丽耳草是典型的二型花柱植物,并具有异型自交不亲和系统.     

Efficiency of DNA barcodes for species delimitation: A case in Pterygiella Oliv.(Orobanchaceae)

DNA barcoding is becoming an increasingly popular means to identify species. The obscure discrimination in the genus Pterygiella calls into question the re-assessment of the criterion for species delimitation. We collected 20 individuals, representing all five described species of this genus in its distributional range. The aim was to use three proposed barcode DNA regions (rbcL, matK, and ITS) to diagnose Pterygiella species, and examine which barcode is more suitable for discerning the congeneric and related species. The results showed that the core barcodes matK and rbcL were comparatively less effective. However, the ITS region, especially ITS-1and ITS-2, successfully identified all species in the genus. Furthermore, the secondary structure of ITS-2 RNA, especially compensatory base changes, appears complementary to classical primary sequence analysis for DNA barcoding.     

DNA barcoding of Pedicularis L.(Orobanchaceae): Evaluating four universal barcode loci in a large and hemiparasitic genus

One application ofDNA barcoding is species identification based on sequences of a short and standardized DNA region.In plants,various DNA regions,alone or in combination,have been proposed and investigated,but consensus on a universal plant barcode remains elusive.In this study,we tested the utility of four candidate barcoding regions (rbcL,matK,trnH-psbA,and internal transcribed spacer (ITS)) as DNA barcodes for discriminating species in a large and hemiparasitic genus Pedicularis (Orobanchaceae).Amplification and sequencing was successful using single primer pairs for rbcL,trnH-psbA,and ITS,whereas two primer pairs were required for matK.Patterns of sequence divergence commonly showed a “barcoding gap”,that is,a bimodal frequency distribution of pairwise distances representing genetic diversity within and between species,respectively Considering primer universality,ease of amplification and sequencing,and performance in discriminating species,we found the most effective single-region barcode for Pedicularis to be ITS,and the most effective two-region barcode to be rbcL +ITS.Both discriminated at least 78％ of the 88 species and correctly identified at least 89％ of the sequences in our sample,and were effective in placing unidentified samples in known species groups.Our results suggest that DNA barcoding has the potential to aid taxonomic research in Pedicularis,a species-rich cosmopolitan clade much in need of revision,as well as ecological studies in its center of diversity,the Hengduan Mountains region of China.     

Applying plant DNA barcodes to identify species of Parnassia (Parnassiaceae)

DNA barcoding is a technique to identify species by using standardized DNA sequences. In this study, a total of 105 samples, representing 30 Parnassia species, were collected to test the effectiveness of four proposed DNA barcodes (rbcL, matK, trnH-psbA and ITS) for species identification. Our results demonstrated that all four candidate DNA markers have a maximum level of primer universality and sequencing success. As a single DNA marker, the ITS region provided the highest species resolution with 86.7%, followed by trnH-psbA with 73.3%. The combination of the core barcode regions, matK+rbcL, gave the lowest species identification success (63.3%) among any combination of multiple markers and was found unsuitable as DNA barcode for Parnassia. The combination of ITS+trnH-psbA achieved the highest species discrimination with 90.0% resolution (27 of 30 sampled species), equal to the four-marker combination and higher than any two or three marker combination including rbcL or matK. Therefore, matK and rbcL should not be used as DNA barcodes for the species identification of Parnassia. Based on the overall performance, the combination of ITS+trnH-psbA is proposed as the most suitable DNA barcode for identifying Parnassia species. DNA barcoding is a useful technique and provides a reliable and effective mean for the discrimination of Parnassia species, and in combination with morphology-based taxonomy, will be a robust approach for tackling taxonomically complex groups. In the light of our findings, we found among the three species not identified a possible cryptic speciation event in Parnassia.     

DNA条形码：物种分类和鉴定技术

当前,一项称为“生命的条形码”计划正在欧美等国展开,其目的是实现对地球上现存的约1000万物种进行快速和准确的鉴定。DNA条形码是一种利用短的DNA序列对物种进行鉴定的技术。对DNA条形码的概念和原理进行了介绍,举例说明了其在物种分类、遗传多样性及物种鉴定研究中广泛的利用价值,阐述了当前该领域的研究现状,对未来的发展方向进行了展望。     

Integration of cytochrome c oxidase I barcodes and geometric morphometrics to delimit species in the genus Gnopharmia (Lepidoptera: Geometridae,Ennominae)

We tested the hypothesis of species taxonomy in the genus Gnopharmia (Macariini, Ennominae) that was recently established in a review based on discrete morphological characters. For this objective we integrated both DNA‐based and morphometric approaches in order to infer species boundaries. A 658‐bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (CO1) (DNA barcode) was analysed from populations of five species distributed throughout the Middle East to assess their consistency with traditionally defined morphospecies. Signals in the morphological variation of the aedeagus of all relevant populations were evaluated using geometric landmarks. Consistent groupings compatible with the current taxonomic classification were found with both approaches. The results strongly support the distinction of seven closely related species. © 2013 The Linnean Society of London     

Systematic wood anatomy of the tribe Guettardeae (Rubiaceae)

Systematic wood anatomy of the tribe Guettardeae (Rubiaceae). The wood anatomy of nearly all genera of the Guettardeae (Rubiaceae, Guettardoideae) has been examined, and in this respect the tribe is heterogeneous. Suggestions are made for a delimitation of the tribe. Guettarda, Bobea, Antirhea, Malanea and Chomelia Jacq. are sufficiently similar in their wood anatomical characters to warrant retention in the same tribe. Machaonia, Timonius and Dichilanthe are anomalous. Suggestions are given for taxonomic revisions of some genera based on their wood anatomy.     

DNA barcodes for discriminating the medicinal plant Isatis indigotica Fort. (Cruciferae) and its adulterants

Isatis indigotica Fort. (Cruciferae) is a biennial medicinal plant. In order to protect the decreasing natural genetic resources of I. indigotica, three candidate DNA barcodes (ITS2, trnL-F and rbcL) were employed to establish an accurate and effective identification system for I. indigotica. The results demonstrated that all three candidate DNA barcodes have performed very well in I. indigotica. The interspecific genetic distances were obviously greater than the intraspecific distance among I. indigotica as indicated by ITS2, trnL-F and rbcL. Sequence alignment analysis of I. indigotica genotypes revealed that four SNPs (54, 108, 146 and 181 bp) located in ITS2, three (2, 30, 709 bp) in trnL-F and one (531 bp) in rbcL, respectively. UPGMA phylogenetic tree constructed from trnL-F and rbcL could allote I. indigotica to the correct corresponding genus, whereas rbcL could not distinguish I. indigotica from its adulterants. Meanwhile, UPGMA tree of ITS2 could accurately identify I. indigotica from its adulterants according to the corresponding species. Consequently, it can be concluded that ITS2 is a more suitable and accurate DNA barcode for identifying I. Indigotica and its adulterants than trnL-F and rbcL.