Zhi-Rong Zhang

Comparative phylogeography of two sympatric beeches in subtropical China: Species‐specific geographic mosaic of lineages

In subtropical China, large-scale phylogeographic comparisons among multiple sympatric plants with similar ecological preferences are scarce, making generalizations about common response to historical events necessarily tentative. A phylogeographic comparison of two sympatric Chinese beeches (Fagus lucida and F. longipetiolata, 21 and 28 populations, respectively) was conducted to test whether they have responded to historical events in a concerted fashion and to determine whether their phylogeographic structure is exclusively due to Quaternary events or it is also associated with pre-Quaternary events. Twenty-three haplotypes were recovered for F. lucida and F. longipetiolata (14 each one and five shared). Both species exhibited a species-specific mosaic distribution of haplotypes, with many of them being range-restricted and even private to populations. The two beeches had comparable total haplotype diversity but F. lucida had much higher within-population diversity than F. longipetiolata. Molecular dating showed that the time to most recent common ancestor of all haplotypes was 6.36 Ma, with most haplotypes differentiating during the Quaternary. [Correction added on 14 October 2013, after first online publication: the timeunit has been corrected to ‘6.36’.] Our results support a late Miocene origin and southwards colonization of Chinese beeches when the aridity in Central Asia intensified and the monsoon climate began to dominate the East Asia. During the Quaternary, long-term isolation in subtropical mountains of China coupled with limited gene flow would have lead to the current species-specific mosaic distribution of lineages.

Taxonomic status, phylogenetic affinities and genetic diversity of a presumed extinct genus, Paraisometrum W.T. Wang (gesneriaceae) from the karst regions of Southwest China.

Background The karst regions in South China have an abundance of endemic plants that face high extinction risks. The Chinese Gesneriaceae endemic Paraisometrum mileense ( = Oreocharis mileensis), was presumed extinct for 100 years. After its re-discovery, the species has become one of five key plants selected by the Chinese forestry government to establish a new conservation category for plants with extremely small populations. For conservation purposes, we studied the phylogenetic and population genetic status of P. mileense at the three only known localities in Guangxi, Guizhou and Yunnan. Methodology/Principal Findings We collected 64 samples (52 species) of Oreocharis and 8 samples from three provinces of P. mileense and generated molecular phylogenies, and inferred that P. mileense represents a relatively isolated and derived taxonomic unit within Oreocharis. Phylogeographic results of 104 samples of 12 populations of P. mileense indicated that the populations in Yunnan have derived from those in Guangxi and Guizhou. Based on AFLP data, the populations were found to harbor low levels of genetic diversity (He = 0.118), with no apparent gradient across the species’ range, a restricted gene flow and significant isolation-by-distance with limited genetic differentiation among the populations across the three provinces (F ST = 0.207, P<0.001). The 10 populations in Yunnan were found to represent two distinct lineages residing at different altitudes and distances from villages. Conclusion/Significance The low levels of genetic diversity found in P. mileense are perhaps a consequence of severe bottlenecks in the recent past. The distribution of the genetic diversity suggests that all populations are significant for conservation. Current in situ and ex situ measures are discussed. Further conservation actions are apparently needed to fully safeguard this conservation flagship species. Our work provides a model of an integrated study for the numerous endemic species in the karst regions with extremely small populations.

Molecular phylogenetic reconstruction of Osmanthus Lour. (Oleaceae) and related genera based on three chloroplast intergenic spacers

Although polyphyly of Osmanthus has been suggested by different authors, the conclusions of previous studies have lacked robust support due to limited sampling or a paucity of phylogenetic characters. In this study, the phylogeny of Osmanthus was explored using sequences of three informative chloroplast regions (psbJ-petA, rpl32-trnL and rps16-trnQ), including all the five sections of the genus and eight closely related genera. The results confirm that Osmanthus, as presently circumscribed, is a polyphyletic group, containing three or four distinct lineages, i.e. sect. Leiolea (lineage I), sect. Notosmanthus (lineage III), sects. Osmanthus (excluding O. decorus), Siphosmanthus and Linocieroides (lineage IV), and an uncertain lineage including only O. decorus (lineage II). These results emphasize that the generic delimitation within subtribe Oleinae is in need of revision. In addition, this study found that the four cultivar groups of sweet osmanthus formed a paraphyletic clade, implying that cultivated sweet osmanthus might originate from several species.

A SET OF NOVEL MICROSATELLITE MARKERS DEVELOPED FOR THE TRADITIONAL TIBETAN MEDICINAL PLANT HALENIA ELLIPTICA (GENTIANACEAE)

PREMISE OF THE STUDY Microsatellite primers were developed in the traditional Tibetan medicinal plant Halenia elliptica D. Don to investigate its genetic diversity and population genetic structure. METHODS AND RESULTS Using the Fast Isolation by AFLP of Sequences Containing (FIASCO) repeats protocol, 24 primer sets were identified in two wild populations. Of these primers, 12 displayed polymorphisms and 12 were monomorphic. The number of alleles per locus ranged from 2 to 6, with a mean of 3.9. The expected (H(E)) and observed (H(O)) heterozygosities ranged from 0.191 to 0.784 and from 0.417 to 0.917, respectively. All these primers successfully amplified in two close relatives of H. elliptica, Swertia bimaculata (Siebold & Zucc.) Hook. f. & Thomson ex C. B. Clarke and S. tetraptera Maxim. CONCLUSIONS These markers will facilitate further studies on the population genetics of Halenia elliptica and its allied species.

Sixteen novel microsatellite markers developed for Dendrocalamus sinicus (Poaceae), the strongest woody bamboo in the world

PREMISE OF THE STUDY Microsatellite primers were developed in the strongest and most economically important bamboo species, Dendrocalamus sinicus, to investigate its genetic variability. METHODS AND RESULTS Using the Fast Isolation by AFLP of Sequences COntaining Repeats (FIASCO) protocol, 16 polymorphic primer sets were identified within 24 individuals from two geographic locations. The number of alleles per locus ranged from two to four, with a mean of 2.6. The observed and expected heterozygosities varied from 0 to 1.000 and from 0.311 to 0.754, respectively. The cross-amplification of the 16 primer pairs was tested in four other Dendrocalamus species, and nine to 13 (56.3-81.3%) primer pairs were successfully amplified. CONCLUSIONS These markers will be useful for studies on the genetic diversity of D. sinicus, which may facilitate conservation of this species.

Development of 29 microsatellite markers for Osmanthus fragrans (Oleaceae), a traditional fragrant flowering tree of China

PREMISE OF THE STUDY Microsatellite markers were developed for a traditional fragrant flowering tree of China, Osmanthus fragrans, to investigate the genetic diversity of its wild populations and to facilitate the classification and identification of O. fragrans cultivars. METHODS AND RESULTS Using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol, 29 primer sets were identified in two wild populations. All primer pairs displayed polymorphism. The number of alleles per locus ranged from two to eight, with a mean of 3.9. The expected and observed heterozygosities ranged from 0.125 to 0.932 and from 0.083 to 0.917, respectively. The transferability of the 29 primer pairs was tested on O. serrulatus, O. delavayi, and O. yunnanensis (three individuals for each species). Eighteen (62.1%), 16 (55.2%), and 21 (72.4%) of them were successfully amplified in O. serrulatus, O. delavayi, and O. yunnanensis, respectively. CONCLUSIONS These markers will facilitate further studies on the population genetics of O. fragrans and the classification and identification of O. fragrans cultivars.

Isolation and characterization of 18 microsatellites for Aconitum vilmorinianum Kom. (Ranunculaceae) using next-generation sequencing technology

Aconitum vilmorinianum is a very important Chinese traditional medicinal plant, which is one of the main raw materials in Yunnan Baiyao as an analgesic and anti-inflammatory agent. Recently, it has been threatened by overexploitation and human disturbances. Genetic background of this species is essential for the efficient conservation and rational utilization. Therefore, a total of 18 novel microsatellite markers were developed for A. vilmorinianum using next-generation sequencing technology. The number of alleles per locus ranged from 1 to 5, with a mean of 2.560, and the observed and expected heterozygosities ranged from 0 to 1.000 and from 0 to 0.733, respectively. These polymorphic microsatellite loci will be especially useful for genetics studies of this important medicinal plant.

Microsatellite markers for Duperrea pavettifolia (Rubiaceae)

PREMISE OF THE STUDY The development of microsatellite primers for Duperrea pavettifolia will be the foundation for mating system analysis and conservation research. METHODS AND RESULTS Nineteen microsatellite markers were developed and characterized in two wild populations by using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol. Polymorphisms were evaluated in 24 individuals from two natural populations. Eleven of these primers generated polymorphic loci. CONCLUSIONS These microsatellite markers will be useful in future investigations into the population genetics and mating system of D. pavettifolia.

Microsatellite markers developed for Corallodiscus lanuginosus (Gesneriaceae) and their cross-species transferability

UNLABELLED PREMISE OF THE STUDY Corallodiscus is widespread across China and in adjacent countries to the west and south, with C. lanuginosus covering the entire range of the genus. Microsatellite markers will be useful to address within-complex taxonomic and biogeographic structures of this species. • METHODS AND RESULTS Fourteen markers were developed using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol. Polymorphism was assessed in 12 individuals each from two populations from West Yunnan, China. Nine primers displayed polymorphisms. The number of alleles per locus ranged from one to six (mean: 2.7). The observed and expected heterozygosities ranged from 0 to 1 and from 0 to 0.772 (average: 0.487 and 0.448). Only one of the polymorphic loci deviated from Hardy-Weinberg equilibrium. Among the four congeneric species, cross-amplification success ranged from two to eight markers. • CONCLUSIONS The markers will be useful for population genetic and evolutionary history studies for C. lanuginosus and the allied species C. bhutanicus, C. cooperi, and C. kingianus.

Isolation and characterization of microsatellite markers from Clematoclethra scandens (Actinidiaceae)

PREMISE OF THE STUDY Microsatellite makers were developed for Clematoclethra scandens to investigate its population genetics and speciation. METHODS AND RESULTS A total of 36 microsatellite markers were isolated using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) method. Their polymorphisms were assessed in two natural populations. The results showed that 30 markers displayed prominent polymorphisms and six markers were monomorphic. CONCLUSIONS These microsatellite loci will facilitate further studies on population genetics and speciation of C. scandens.