Yu Chung Chiang

Organelle DNA phylogeography of Cycas taitungensis, a relict species in Taiwan.

The phylogegraphic pattern of Cycas taitungensis, an endemic species with two remaining populations in Taiwan, was investigated based on genetic variability and phylogeny of the atpB‐rbcL noncoding spacer of chloroplast DNA (cpDNA) and the ribosomal DNA (rDNA) internal transcribed spacer (ITS) of mitochondrial DNA (mtDNA). High levels of genetic variation at both organelle loci, due to frequent intramolecular recombination, and low levels of genetic differentiation were detected in the relict gymnosperm. The apportionment of genetic variation within and between populations agreed with a migrant‐pool model, which describes a migratory pattern with colonists recruited from a random sample of earlier existing populations. Phylogenies obtained from cpDNA and mtDNA were discordant according to neighbour‐joining analyses. In total four chlorotypes (clades I–IV) and five mitotypes (clades A–E) were identified based on minimum spanning networks of each locus. Significant linkage disequilibrium in mitotype–chlorotype associations excluded the possibility of the recurrent homoplasious mutations as the major force causing phylogenetic inconsistency. The most abundant chlorotype I was associated with all mitotypes and the most abundant mitotype C with all chlorotypes; no combinations of rare mitotypes with rare chlorotypes were found. According to nested clade analyses, such nonrandom associations may be ascribed to relative ages among alleles associated with the geological history through which cycads evolved. Nested in networks as interior nodes coupled with wide geographical distribution, the most dominant cytotypes of CI and EI may represent ancestral haplotypes of C. taitungensis with a possible long existence prior to the Pleistocene glacial maximum. In contrast, rare chlorotypes and mitotypes with restricted and patchy distribution may have relatively recent origins. Newly evolved genetic elements of mtDNA, with a low frequency, were likely to be associated with the dominant chlorotype, and vice versa, resulting in the nonrandom mitotype–chlorotype associations. Paraphyly of CI and EI cytotypes, leading to the low level of genetic differentiation between cycad populations, indicated a short period for isolation, which allowed low possibilities of the attainment of coalescence at polymorphic ancestral alleles.

Phylogeography of Kandelia candel in East Asiatic mangroves based on nucleotide variation of chloroplast and mitochondrial DNAs.

Vivipary with precocious seedlings in mangrove plants was thought to be a hindrance to long‐range dispersal. To examine the extent of seedling dispersal across oceans, we investigated the phylogeny and genetic structure among East Asiatic populations of Kandelia candel based on organelle DNAs. In total, three, 28 and seven haplotypes of the chloroplast DNA (cpDNA) atpB‐rbcL spacer, cpDNA trnL‐trnF spacer, and mitochondrial DNA (mtDNA) internal transcribed spacer (ITS) were identified, respectively, from 202 individuals. Three data sets suggested consistent phylogenies recovering two differentiated lineages corresponding to geographical regions, i.e. northern South‐China‐Sea + East‐China‐Sea region and southern South‐China‐Sea region (Sarawak). Phylogenetically, the Sarawak population was closely related to the Ranong population of western Peninsula Malaysia instead of other South‐China‐Sea populations, indicating its possible origin from the Indian Ocean Rim. No geographical subdivision was detected within the northern geographical region. An analysis of molecular variance (amova) revealed low levels of genetic differentiation between and within mainland and island populations (ΦCT = 0.015, ΦSC = 0.037), indicating conspicuous long‐distance seedling dispersal across oceans. Significant linkage disequilibrium excluded the possibility of recurrent homoplasious mutations as the major force causing phylogenetic discrepancy between mtDNA and the trnL‐trnF spacer within the northern region. Instead, relative ages of alleles contributed to nonrandom chlorotype–mitotype associations and tree inconsistency. Widespread distribution and random associations (χ2 = 0.822, P = 0.189) of eight hypothetical ancestral cytotypes indicated the panmixis of populations of the northern geographical region as a whole. In contrast, rare and recently evolved alleles were restricted to marginal populations, revealing some preferential directional migration.

Contrasting phylogeographical patterns between mainland and island taxa of the Pinus luchuensis complex

Species whose geographical distribution encompasses both mainland and island populations provide an ideal system for examining isolation and genetic divergence. In this study, paternally transmitted chloroplast DNA (cpDNA) and maternally transmitted mitochondrial DNA (mtDNA) were used to estimate population structure and phylogeography of Pinus luchuensis, a species found in eastern China (ssp. hwangshanensis), Taiwan (ssp. taiwanensis), and the Ryukyu Archipelago (ssp. luchuensis). Gene genealogies of both mtDNA and cpDNA reveal two major lineages. Molecular dating indicates that these lineages diverged before the colonization of P. luchuensis subspecies in Taiwan and the Ryukyu Archipelago. Both mtDNA and cpDNA show a lack of correspondence between molecular phylogeny and subspecies designation. Phylogeographical analysis suggests that paraphyly of the subspecies is the result of recent divergence rather than secondary contacts. In spite of the short divergence history of P. luchuensis on islands, the island populations show the same degree of genetic divergence as mainland populations. Low levels of genetic diversity in the mainland ssp. hwangshanensis suggest demographic bottlenecks. In contrast, the high heterogeneity of genetic composition for island populations is likely to be associated with a history of multiple colonization from the mainland. The spatial apportionment of organelle DNA polymorphisms is consistent with a pattern of stepwise colonization on island populations.

Contrasting selection modes at the Adh1 locus in outcrossing Miscanthus sinensis vs. inbreeding Miscanthus condensatus (Poaceae)

We estimated DNA sequence variation of the Adh1 locus in the outcrossing Miscanthus sinensis (Poaceae) and its close selfing relative, M. condensatus. Tajimas test of selection is significantly negative for both overall exons and replacement sites in M. sinensis. Among its entire sample, nucleotide diversity of nonsynonymous sites is higher than that of synonymous sites. A McDonald and Kreitman test of neutrality indicates an excess of intraspecific replacement polymorphisms, suggesting possible directional selection toward advantageous mutants. However, frequent intragenic recombination suggests both purifying and positive selection is unlikely. Recent demographic expansions coupled with relaxation of purifying selection may have resulted in elevated genetic diversity at the Adh1 locus as well as the trnL-trnF intergenic spacer of cpDNA in this outcrossing species. In contrast, low levels of genetic diversity were detected at both the Adh1 locus and the cpDNA spacer in M. condensatus, consistent with bottlenecks associated with selfing in all populations. While Tajimas D and Fu and Lis F statistics did not reveal deviation from neutrality at the Adh1 locus in M. condensatus, 12 replacements vs. 10 synonymous changes were detected. Based on pairwise comparisons of the d(N)/d(S) ratio, lineages of closely related populations of the species distributed along saline habitats appeared to be under directional selection.

Nested clade analysis of Dunnia sinensis (Rubiaceae), a monotypic genus from China based on organelle DNA sequences

Dunnia sinensis, a monotypic genus of theRubiaceae endemic to the southeast mainlandChina, is an endangered species due to habitatdestruction over the past decades. Informationon levels and apportionment of geneticvariation across populations and geographicalregions is fundamental to conservation. In thepresent study, we used organelle DNA variationand nested phylogeographic analyses to test theisolation-by-distance model in this specieswith wind-mediated seed-dispersal and todistinguish ongoing gene flow from historicalprocesses. As expected, low levels of geneticvariation were detected at the ribosomal ITSregion of mtDNA (θ = 0.0019± 0.0002) and the atpB-rbcLintergenic spacer of cpDNA (θ =0.0022 ± 0.0009) in the rare species. Sixand seven haplotypes of mt- and cpDNA wereidentified from 125 individuals, respectively,according to the reconstructed neighbor-joiningtrees. Both data sets suggested consistentphylogenies that recovered two differentiatedlineages corresponding to western (Yangchun)and eastern (four others populations) portionsof the range. Hierarchical analyses of themolecular variance (AMOVA) of mt- and cpDNAindicated that molecular variance wasattributable to the difference between regions(Φct = 0.911 and 0.771 for mt- and cpDNA,respectively) in D. sinensis. Based ongeographic distributions of haplotypes in thehaplotype networks, significant geneticdifferentiation between the two geographicregions, which can be seen as evolutionarilyconservation units, was associated withhistorical fragmentation. In contrast, limitedgene flow with occasional long-range dispersalshaped the apportionment of organelle DNAalleles among populations of the easternregion, within which two incompletely isolatedphylogeographic groups can be recognized asconservation units for management.

Molecular phylogeny of Phalaenopsis Blume (Orchidaceae) on the basis of plastid and nuclear DNA

The internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA) and plastid DNA, including the trnL intron, the trnL-F spacer, and the atpB-rbcL spacer, from most of the living species in the genus Phalaenopsis were sequenced. The monophyly of the genus described by Christenson (Christenson EA (2001) Phalaenopsis. Timber Press, Portland, p 330), that Doritis and Kingidium are synonyms of Phalaenopsis, was supported by these molecular data. Within the genus, subgenus Polychilos was monophyletic, and the species were divided into two subclades. The subgenus Phalaenopsis was shown to be non-monophyletic, because the sections Esmeralda and Deliciosae appeared separated the from sections Phalaenopsis and Stauroglottis. Meanwhile, subgenera Aphyllae and Parishianae were also shown to be non-monophyletic on the basis of the molecular data. Furthermore, the monotypic species of subgenus Proboscidioides, P. lowii, formed a clade with subgenus Aphyllae. In accordance with geographical distribution, the historical geography of Southeast Asia due to the periodic glacial epochs, and molecular phylogeny, two evolutionary trends of Phalaenopsis from the original center in South China to the Philippines, Indonesia, and Malaysia were suggested. First, using Indochina and some older parts of the Philippines (e.g., Mindoro and Palawan) as stepping stones, Phalaenopsis species dispersed from South China to the Philippines, where the sections Phalaenopsis and Stauroglottis of subgenus Phalaenopsis developed. Second, using the Malay Peninsula as a stepping stone, Phalaenopsis species dispersed from South China to Indonesia and Malaysia, where the subgenus Polychilos developed.

Patterns of Genetic Variation and Demography of Cycas taitungensis in Taiwan

Cycas taitungensis Shen, Hill, Tsou & Chen is an endemic species remaining in two remnant populations in southeastern Taiwan. Ecological studies showed that the sex ratio between female and male of the main population is approximately 1.7:1. Leaf production was found to be correlated with tree height before reaching 1 m in length (r = 0.95). The annual reproduction rate of female plants is highly variable, with seed numbering between 80 and 400 in each tree. The site study revealed a significant difference in vegetative growth and age structure between the subpopulations collected in two opposite microhabitats. Genetic studies using isozyme analysis showed low genetic variability (HE = 0.039) and little genetic differentiation between the populations (FST = 0.051). The genetic data are well correlated with the ecological observation that the differences reflect various microhabitat effects within a very local environment and that the impact influenced the extent of the degree of genetic differentiation within local populations. This work presents extensive genetic information forC. taitungensis that give rise to more ecological and genetic insights into the plant for better establishment of in situ and ex situ conservation programs.

Characterization and adaptive evolution of α-tubulin genes in the Miscanthus sinensis complex (Poaceae)

To investigate the organization of and mode of selection in the α-tubulin genes, full-length α-tubulin genes were cloned from four intraspecific taxa of Miscanthus sinensis and its close relatives M. floridulus and M. condensatus using standard polymerase chain reaction (PCR) and rapid amplification of genomic ends (RAGE)-PCR strategies. Genealogical analysis of angiosperms recovered a monophyletic group of Miscanthus α-tubulin genes, which is homologous to the tua5 locus of maize. Two clusters of nearly equal frequency revealed paraphyly within each Miscanthus taxon. Between-cluster recombination was frequent. Additional evidence for co-occurrence of two haplotypes within individuals and a large-scale crossover all suggested a likely allelic relationship between the Miscanthus clusters. Given a long between-species divergence time in Miscanthus, wide occurrence of the trans-species polymorphisms in α-tubulin genes and the approximately equal frequency of each allelic type make it extremely unlikely that α-tubulin diversity has been maintained under neutrality. Balancing selection may have contributed to such an apportioning of genetic variability as well as to high levels of genetic variation in α-tubulin and higher substitution rates at synonymous sites of exons than at intron bases of M. sinensis. In addition, certain effects of demographic oscillation may have distorted the scenario of a functional locus operating under balancing selection.

Possible consequences of fungal contamination on the RAPD fingerprinting in Miscanthus (Poaceae)

Fungal contamination has been frequently reported in higher plants. In Miscanthus species, a wide range of fungal flora has also been recorded previously, including an investigation based on nrITS amplification. In order to understand the effects of the fungal genomes on the random amplified polymorphic DNA (RAPD) fingerprinting, callus specimens were obtained from the tissue culture of shoot apices of Miscanthus. RAPD fingerprinting with 60 oligoprimers was conducted with genomic DNA extracted from leaf tissue collected in the field and from the greenhouse, as well as callus derived from the same individuals. Extra bands were detected in the RAPD fingerprints amplified with 44 primers (84.6%) from the genomic DNA of both the field and greenhouse leaf tissue of most Miscanthus taxa examined, except for M. sinensis var. condensatus. Positive PCR amplification of organelle DNA non-coding spacers with both leaf and callus DNA ruled out the possibility that such DNA fingerprinting discrepancies were due to loss of organelles in the callus after consecutive subcultures. Among the 44 primers, one yielded no amplified fragments from the callus DNA, indicating that the amplified DNA fragments from leaf-tissue DNA were likely to be derived from fungi. The contaminating fungal DNA not only caused the overestimation of genetic diversity in the host plants, but also interfered with the phylogenetic inference. Systematic inconsistency occurred between the UPGMA dendrograms of leaf and callus DNA fingerprints. The detection of contaminating fungal DNA suggested that precautions are required for PCR-based fingerprinting when field materials are used for DNA resources. A method for quick screening of the contaminating fungal DNA with universal primers for the nrITS (internal transcribed spacer) region is suggested.

Isolation and characterization of eight polymorphic microsatellite loci from Ludwigia polycarpa (Onagraceae), a threaten herb in North America

Ludwigia polycarpa is the only species of section Microcarpium occurring north of 37°N in the central midwest of the United States. Recently, the loss of wetlands in North America reduced the population number and size of L. polycarpa dramatically. In this study, for the purpose of the conservation of the endangered species, we described eight microsatellite DNA loci. High variabilities enable these molecular markers to assess the population structure. The number of alleles per locus ranged from 8 to 23. The expected (HE) and observed (HO) heterozygosities ranged from 0.86 to 0.96 and 0.00 to 1.00, respectively. Seven of the eight microsatellite loci displayed significant departures from Hardy–Weinberg expectations, likely due to the loss of habitats and the small population size. No linkage disequilibrium was observed in the pairwise comparisons of loci. The application of these microsatellite loci in L. polycarpa may provide a tool for understanding its demography and population structure.