Patterns of hybrid formation among cryptic species of bird‐nest fern,Asplenium nidus complex (Aspleniaceae), in West Malesia

In order to clarify patterns of hybrid formation in the Asplenium nidus complex, artificial crossing experiments were performed between individuals of genetically differentiated groups based on the sequence of the rbcL gene, including A. australasicum from New Caledonia, A. setoi from Japan and several cryptic species in the A. nidus complex. No hybrid plants were obtained in crosses between nine of the 16 pairs. Even for pairs that generated hybrids, the frequency of hybrid formation was lower than expected given random mating, or only one group was able to act as the maternal parent, when the genetic distance (Kimura's two parameter) between parental individuals was at least 0.006. Sterile hybrids were produced by three pairs that were distantly related but capable of forming hybrids. Considering the results of the crosses together with the genetic distance between the parental individuals, it seems that the frequency of hybrid formation decreases rapidly with increasing divergence. The frequency of hybrid formation has not been previously examined in homosporous ferns, but it seems that a low frequency of hybrid formation can function as an important mechanism of reproductive isolation between closely related pairs of species in the A. nidus complex in addition to hybrid sterility. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 42–63.     

Molecular systematics of the Asplenium nidus complex from Mt. Halimun National Park, Indonesia: evidence for reproductive isolation among three sympatric rbcL sequence types

Asplenium nidus is an epiphytic fern with large simple leaves. Because A. nidus lacks the good taxonomic characters available for species recognition, multiple cryptic species may exist within A. nidus. In Mt. Halimun National Park, West Java, Indonesia, three rbcL sequence types of A. nidus were recorded. All plants regardless of rbcL sequence were 2n = 144. Crossing experiments among these rbcL types were conducted, and it was observed that the molecularly distinct types were reproductively isolated because hybrids failed to form between at least two pairs of rbcL types. These results suggest that these rbcL types are cryptic species because they are reproductively isolated but they are difficult to distinguish morphologically. Thus, the characters of DNA sequences information are useful in the discovery of cryptic species in ferns.     

Molecular Taxonomic Study and Revision of the Three Japanese Species of Asplenium sect. Thamnopteris

Asplenium sect. Thamnopteris or A. nidus L. complex is defined by the synapomorphic character peculiar to Aspleniaceae, an anastomosing vein near the margin of the simple lamina. Thus, it is easily recognizable and its monophyly is quite clear. In spite of its naturalness as the whole group, species delimitation is very confusing. Three species of sect. Thamnopteris, A. antiquum Makino, A. australasicum (J. Smith) Hooker and A. nidus L. have been recognized in Japan, but the naturalness of each species is still not clear because their morphology is too simple to find good qualitative taxonomical characters. In the present work, we examined the intraspecific variation of allozymes and rbcL sequences in the Japanese plants of sect. Thamnopteris and compared them with those from other paleotropical localities in order to recognize natural units in such morphologically simple plants. We found a large amount of genetic variation in this section and inferred that A. antiquum is a species of ancient origin, though morphologically it is not so different from other species of the sect. Thamnopteris. It was also discovered that the so called “A. australasicum” in Japan has a very different rbcL sequence from A. australasicum sensu Holttum, which is distributed in Australia and South Pacific Islands. Based on these molecular data, we described the Japanese “A. australasicum” as a new species, Asplenium setoi N. Murak. et Seriz. Received 9 September 1998/ Accepted in revised form 22 December 1998     

Factors Affecting the Distribution and Abundance of Asplenium nidus L. in a Tropical Lowland Rain Forest in Peninsular Malaysia

Asplenium nidus is an abundant epiphytic fern of tropical rain forests in the Old World, where it plays an important ecological role in the forest canopy as host to diverse arthropod communities. We investigated the factors that determine the distribution and abundance of A. nidus in the canopy of an aseasonal lowland dipterocarp forest at Pasoh Forest Reserve, Malaysia. We found that A. nidus was more abundant in the understory, and on hosts with smooth bark and relatively flat branch angles. Ferns were found on a wide diversity and size range of host taxa. However, both host taxa and host diameter at breast height had a significant effect on A. nidus occupancy. Asplenium nidus had an aggregated spatial distribution at all scales within the study area. Spatial aggregation at larger scales appears to be driven by habitat preference, as A. nidus abundance was positively associated with swampy areas and negatively associated with hilly areas. At smaller scales, limited dispersal of their wind-dispersed spores most likely explains the aggregated distribution. Larger individuals occurred higher in the canopy and were more common in the hilly area. Thus, the distribution of A. nidus may represent a trade-off between the availability of suitable microsites for establishment in the understory and better growth conditions higher in the canopy. However, A. nidus is known to comprise a complex of cryptic species, and future studies should incorporate molecular techniques to elucidate the potential role of speciation in explaining these patterns.     

Two new species of the cryptic chrysidid parasitoid subfamily Loboscelidiinae: The second species in Rhadinoscelidia and the first Loboscelidia for the Indonesian fauna

Two new species in the cryptic chrysidid wasp subfamily Loboscelidiinae are described based on males from Gunung Halimun National Park, West Java, Indonesia. One is the second species in Rhadinoscelidia Kimsey, and the other, belonging in Loboscelidia Westwood, is the first species of the genus for the Indonesian fauna.     

Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii.

Summary We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of nonphotosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.     

Distributional Patterns of Epiphytic Ferns are Explained by the Presence of Cryptic Species

In a recent article in this journal Zhang and colleagues investigated the factors affecting the distribution of a species of epiphytic fern Asplenium nidus L. in rain forest in Peninsular Malaysia. Here we suggest that their findings may be interpreted in the light of there being two cryptic species present, each with different ecologies, as is the case in Malaysian Borneo. We also discuss the implications of the existence of cryptic species when attempting to conserve forest diversity in the face of climate change and habitat conversion. Abstract in Malay is available at http://www.blackwell‐synergy.com/loi/btp .     

Discriminating plants using the DNA barcode rbcLb: an appraisal based on a large data set

The ideal DNA barcode for plants remains to be discovered, and the candidate barcode rbcL has been met with considerable skepticism since its proposal. In fact, the variability within this gene has never been fully explored across all plant groups from algae to flowering plants, and its performance as a barcode has not been adequately tested. By analysing all of the rbcL sequences currently available in GenBank, we attempted to determine how well a region of rbcL performs as a barcode in species discrimination. We found that the rbcLb region was more variable than the frequently used rbcLa region. Both universal and plant group‐specific primers were designed to amplify rbcLb, and the performance of rbcLa and rbcLb was tested in several ways. Using blast , both regions successfully identified all families and nearly all genera; however, the successful species identification rates varied significantly among plant groups, ranging from 24.58% to 85.50% for rbcLa and from 36.67% to 90.89% for rbcLb. Successful species discrimination ranged from 5.19% to 96.33% for rbcLa and from 22.09% to 98.43% for rbcLb in species‐rich families, and from 0 to 88.73% for rbcLa and from 2.04% to 100% for rbcLb in species‐rich genera. Both regions performed better for lower plants than for higher plants, although rbcLb performed significantly better than rbcLa overall, particularly for angiosperms. Considering the applicability across plants, easy and unambiguous alignment, high primer universality, high sequence quality and high species discrimination power for lower plants, we suggest rbcLb as a universal plant barcode.     

Cryptic species in the fern Ceratopteris thalictroides (L.) Brongn. (Parkeriaceae). I. Molecular analyses and crossing tests

For the taxonomic revision of the problematic species Ceratopteris thalictroides, molecular analyses and crossing tests were conducted for 16 sources in the world. An analysis of allozyme composition of five enzymes revealed the presence of three intraspecific entities, which were called the south type, the north type, and the third type. An analysis of the nucleotide sequences of chloroplast DNA also distinguished the same entities. Crossing tests showed that the south type was completely cross-sterile with the other two types, and that the other two were considerably cross-sterile with each other. These results suggest that the three entities should be regarded as different biological species. Although the south type and the other two meet in several regions, complete cross-sterility between them seems to sustain their genetic distinctiveness in spite of occasional crossing. The results from the present study suggest that widely distributed fern species are apt to comprise several cryptic species. Received: August 1, 2001 / Accepted: November 1, 2001     

EVOLUTIONARY ANALYSIS OF THE LARGE SUBUNIT OF CARBOXYLASE (rbcL) NUCLEOTIDE SEQUENCE AMONG THE GRASSES (GRAMINEAE)

The full nucleotide sequences of the chloroplast encoded large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) are available for nine grass species and partial sequence data for one species. Relative rate tests of the “molecular clock” hypothesis suggest that rbcL evolved more rapidly in the lineage leading to Zea than in those leading to the other species. The estimated overall substitution rate for rbcL among these grasses is about 5 times 10^?10 substitutions per site per year, or about one-half the synonymous rate. The nine full sequences were analyzed by the UPGMA, Wagner parsimony, maximum likelihood, and Fitch-Margoliash methods. The latter three methods produced trees with the same topology. This topology largely agrees with current taxonomic evidence regarding the relationships among these grasses. UPGMA produced a topology that conflicts more substantially with available taxonomic evidence. Statistical comparison of the three alternative topologies for the subfamilies Panicoideae, Pooideae and Bambusoideae failed to support one of these topologies over the others, reflecting the taxonomic ambiguities surrounding the relationships among these taxa. Phylogenetic analyses based on the partial sequences of all 10 species gave conflicting results with regard to the relationship between Hordeum and Triticum, both members of the tribe Triticeae. This indicates that rbcL sequences contain too little information to resolve relationships among genera within this tribe. Overall, the results suggest that rbcL sequence data can provide some new information concerning grass phylogeny, but that the amount of available data from this gene is too small to differentiate statistically among alternative topologies for the grasses. Conflicting results from parsimony, maximum likelihood, and Fitch-Margoliash methods proved useful in exploring the validity of assumptions underlying these methods.     

Prevalence,geographic distribution and phylogenetic relationships among cryptic species of Plasmopara viticola in grape‐producing regions of Georgia and Florida,USA

Previous phylogenetic studies of the grape downy mildew pathogen, Plasmopara viticola, revealed five cryptic species in eastern North America that differed in their host range and geographic distribution. Preliminary comparative studies also documented differences in temperature responses during infection between certain cryptic species, indicating the biological relevance of knowing which cryptic species of the pathogen are present in a given region. However, limited information is available regarding the presence, prevalence and dynamics of cryptic species of P. viticola in the southeastern United States. Here, 301 P. viticola isolates obtained from cultivated grape species in five distinct grape‐growing regions of Georgia and Florida were subjected to cleaved amplified polymorphic sequence analysis and multilocus sequencing (internal transcribed spacer region of the rDNA, actin and β‐tubulin) to identify cryptic species and infer phylogenetic relationships. Three cryptic species, P. viticola clade aestivalis (Pva), clade vinifera (Pvv) and clade vulpina (Pvu), were identified in Georgia, whereas two, Pva and Pvv, were found in Florida; all three cryptic species are reported here for the first time in Georgia, whereas Pva is reported for the first time in Florida. Pva was the most prevalent cryptic species (72.1% of isolates) and was distributed widely from the North Georgia Mountains to Mid‐Florida, whereas Pvv (27.2%) and Pvu (0.7%) were found only in the Coastal Plain region of the two states. Interestingly, Pvu was obtained from French American hybrid Blanc du Bois and could be subcultured on Vitis vinifera Chardonnay, suggesting a broader host range than only the wild species Vitis vulpina reported previously.     

Genetic variation in populations of the morphologically and ecologically variable fern <Emphasis Type="Italic">Stegnogramma pozoi</Emphasis> subsp. <Emphasis Type="Italic">mollissima</Emphasis> (Thelypteridaceae) in Japan

In Japanese Stegnogramma pozoi subsp. mollissima (Fisher ex Kunze) K. Iwats. there is the intrasubspecific variation among rbcL sequences. Northern and southern plants are genetically differentiated for maternally inherited cpDNA. In the present study we examined allozyme polymorphisms to test the hypothesis that northern and southern plants may be separate species. Based on allozyme data, the degree of gene flow among populations was estimated to be large. The artificial crossing experiments between cpDNA haplotypes also suggested that isolation has not developed among these cpDNA haplotypes. However, interpopulation genetic differentiation in cpDNA was observed even in the small area at the foot of Mt. Hakone, and the cpDNA haplotypes appear to have different habitat preferences. Received: May 1, 2001 / Accepted: September 30, 2001     

Comparison of the evolution of ribulose-1, 5-biphosphate carboxylase (rbcL) and atpB-rbcL noncoding spacer sequences in a recent plant group,the tribe Rubieae (Rubiaceae)

Plastid sequences of the atpB-rbcL spacer and rbcL gene itself were used to evaluate their respective potential in reconstructing the phylogeny of 15 taxa from the tribe Rubieae (Rubiaceae). From our previous analyses using the atpB-rbcL spacer, the 15 selected taxa represent most of the variability of the tribe. Since this group is considered to be relatively recent (Upper Tertiary), it should allow the study of early dynamics of nucleotide substitutions in recent divergences. The results show that the spacer and rbcL inferred phylogenies are not totally congruent; the spacer trees are more similar to interpretations of morphological data. A comparative analysis of the pattern of nucleotide substitution of these two sequences in the Rubieae shows that (1) the overall rate of substitution is similar in the spacer and in rbcL, and the rate of synonymous substitution in rbcL is much higher; (2) the level of homoplasy is higher in rbcL than in the spacer matrix which shows a higher phylogenetic structure; and (3) the pattern of transition and transversion substitutions is different in the two sequences, and is not linear in rbcL. As a result of these observations, we suggest that (1) the spacer is evolving relatively slowly because of unsuspected, and phylogenetically important; selective constraints on its sequence; and (2) in the rbcL sequence, many sites, free of constraint, are changing at high rate, and some of these sites seem to have undergone multiple substitutions even in this recent tribe. This could explain the high level of homoplasy found in Rubieae rbcL sequences. Correspondence to: J.-F. Manen     

Testing DNA barcodes in closely related species of Curcuma (Zingiberaceae) from Myanmar and China

The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear‐cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH‐psbA and trnL‐F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra‐ and inter‐specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH‐psbA (100%), trnL‐F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH‐psbA and trnL‐F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers.     

Importance of drought on the distribution of the birds nest fern,Asplenium nidus,in the canopy of a lowland tropical rainforest in north‐eastern Australia

Abstract The birds nest fern, Asplenium nidus, contributes greatly to the epiphytic biomass in the canopies of both south‐east Asia and tropical north Queensland rainforests. It is generally believed that their abundance and their capacity to store water is an important feature for habitat fragmentation in the canopy. We investigated the microclimate of A. nidus and the effects of severe drought periods on the A. nidus population over a 20‐year period, hypothesizing that water availability is the most important factor controlling the population under drought conditions. One of two neighbouring A. nidus plants of the same size and age was irrigated artificially before, during and after a significant dry period in 2000. By monitoring the microclimate within and around both ferns we were able to estimate that four continuous weeks of rainless weather completely dried out the accumulated humus of the non‐irrigated A. nidus fern. Prolonged dry periods were shown to kill the roots of A. nidus, which attach the fern to the bark and eventually the affected A. nidus on verticals stems fell to the ground. Periods longer than 8 weeks may even kill adult plants sitting in more protected branch forks. Analysis of the whole A. nidus population within the 1‐ha Canopy Crane plot and the determination of the morphological age of all plants enabled an evaluation of the historical development of the population. The oldest plant originated in 1985, just 1 year after the longest recorded drought for the site. We suspect that the 1984 drought killed every A. nidus plant within the study plot. Years with low recruitment coincide with years with long drought periods.     

Hemiboea guangdongensis comb. & stat. nov., a cryptic species segregated from H. subcapitata (Gesneriaceae) based on morphological and molecular data

The taxonomic problem of cryptic species has long been recognized. Hemiboea subcapitata C. B. Clarke is a widespread and morphologically diverse species including two varieties, H. subcapitata var. subcapitata C. B. Clarke and H. subcapitata var. guangdongensis (Z. Y. Li) Z. Y. Li. However, genetic distance and molecular phylogenetic analyses based on nuclear ITS and four plastid DNA sequences (atpB‐rbcL, matK, rbcL, rpS16 intron) revealed that H. subcapitata var. guangdongensis is sister to H. subacaulis, and separated from H. subcapitata var. subcapitata, suggesting that it should be raised to the rank of an independent species as H. guangdongensis (Z. Y. Li) X. Q. Li & X. G. Xiang, comb. & stat. nov. Morphologically, the new species can be distinguished from H. subacaulis by its calyx 5‐sect from base, segments narrowly oblong‐lanceolate (versus calyx 5‐sect from base or 2‐lipped and adaxial lip 2‐lobed from below middle, segments ovate), peduncle glabrous (versus pubescent), and vermiform sclereids dispersed in leaf mesophyll (versus without sclereids).     

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.     

DNA-Barcoding of Some Medicinal Plant Species in Saudi Arabia Using <i>rbcL</i> and <i>matK</i> Genes

In the Kingdom of Saudi Arabia (KSA), thousands of plants are considered to have therapeutic value. The ambiguous use of identification mainly morphological characteristics of many plants has resulted in the adulteration and displacement of plant products which undermine their therapeutic value and weak documentation of plant resources. The aims of this study were therefore to evaluate genetic variability and explore the phylogeographic architecture for Saudi medicinal plant samples using rbcL and matK genes as barcodes for genomic identification. The matK and rbcL sequences collected for these samples were used as key markers for examining the relationship between Saudi medicinal plant species based on genetic diversity. During our study we were successful in identifying and documenting 4 different species (Foeniculum vulgare, Nitraria retusa, Dodonaea viscosa, and Rumex nervosus) located in Saudi Arabia using DNA barcoding technique. A total number of 8 sequences were obtained with a total sequence length of 6176 bp, where it ranged from 617 bp to 878 bp with an average length of 772 bp. The total number of rbcL sequences length is 2801 bp, where it ranges from 617 bp to 807 bp with an average length of 700.2 bp. Out of the 4 plant samples used, only three samples were identified correctly on the species level with an identity percentage higher than 95% using rbcL gene. Additionally, 4 matK sequences have been retrieved belong to 4 species. The total number of matK sequences length is 3375 bp, where it ranges from 819 bp to 878 bp with an average length of 843.8 bp. Out of the 4 plant samples used, only two samples were identified correctly on the species level with an identity percentage higher than 98% using matK gene. Both rbcL and matK have been able to identify most of our collected plant samples by genus, and some by species. Using only one DNA-barcoding technique was not reliable for plant identification, where matK and rbcL must be used as a dual DNA-barcoding procedure.     

Molecular phylogeny in the Lardizabalaceae

Eleven species belonging to seven genera in the Lardizabalaceae were analyzed in terms of restriction fragment length polymorphism (RFLP) of chloroplast DNA and the sequence of the chloroplast gene,rbcL, of Lardizabalaceae and its related families. Phylogenetic trees inferred from parsimony, neighbor joining and maximum likelihood methods based on RFLP data showed that two South American genera,Boquila andLardizabala, and three East Asian genera,Akebia, Holboellia andStauntonia are closely related to each other, respectively. On the other hand, the parsimony, neighbor joining and maximum likelihood trees constructed using sequence data of therbcL gene showed thatAkebia, Stauntonia, Boquila andLardizabala clustered as(((Akebia, Stauntonia), Boquila), Lardizabala). This difference may be attributable to fewer informative sites inrbcL genes than in RFLP in this family.Decaisnea diverges at the very base of the Lardizabalaceae.