Conservation Biology Of The Wild Plants With Extremely Small Population Rhododendron Protistum Var. Giganteum

Big tree rhododendron, Rhododendron protistum var. giganteum (Forrest) D. F. Chamberlain, belongs to the family Ericaceae and subgenus Hymenanthes. This species is one of the tallest and most ancient rhododendron tree, reaching30m in height and1m in basal diameter. It exhibits a very limited distribution, with only two populations found in the Gaoligong Mountains of Northwestern Yunnan Province in China. In addition, only approximately2,000individual plants have been found. Considering its habitat specialization and extremely limited distribution, big tree rhododendron has been protected under the Conservation Program for Wild Plants with Extremely Small Population in China (2012-2015Operational Plan) and included on the Red List of Critically Endangered Species in China. However, there are few research on conservation biology of this species. Thus, basic data on big tree rhododendron is urgently needed to inform current and future conservation activities. In order to elucidate the causes of endangered of big tree rhododendron, field investigations and laboratory experiments, population structure and dynamics, ecophysiological of seed and conservation genetics were investigated in this thesis. Furthermore, the corresponding conservation strategies were also proposed according to the above-mentioned. The major results and conclusions are given below.1. Population structure and dynamics of big tree rhododendronWe investigated the size structure, life table and dynamics of big tree rhododendron populations by using spatial series rather than time series and substituting trunk size structure for age structure. According to field investigation, only two remaining big tree rhododendron populations are distributed in the Gaoligong Mountain National Nature Reserve, Tengchong County. Maintenance of big tree rhododendron populations was largely dependent on middle trees and adult because of low numbers of seedlings. The survival curves for the big tree rhododendron populations were classified as Deevey-III type and showed declining trends. Time-series prediction showed that big tree rhododendron population will be eventually able to keep stable because the amount of population will have a light rising tendency in the coming2and5age classes. We suggest that management policies should be improved to maintain the appropriate effective population size of big tree rhododendron and to protect its natural habitats.2. Ecophysiological characteristics of big tree rhododendron seedWe studied the effects of light, temperature, GA3, soil substrates types, site condition and storage on germination of big tree rhododendron seeds under laboratory conditions. The results showed that seeds of big tree rhododendron were non-dormant and temperature significantly affected germination percentage (GP). The optimal temperatures were relatively lower of15and20℃. High temperature had negative effects on seed germination. The seeds have less than5%of GP when incubated in the complete darkness, suggesting that darkness significantly inhibited seeds germination. GA3treatment increased the GP and facilitated the germination speed.The seeds exhibited orthodox seed storage behavior according to the international seed storage behavior classification. Storage periods, temperature and their interaction have significantly effect on start germination time (SGT), time to obtain50%germination (T50), mean germination time (MGT) and GP of big tree rhododendron seeds except for their interaction on the GP. Soil substrates had no significantly influence on the seedling emergency, but native soil is beneficial to the seedling growth. When the seeds were sowed on the soil surface, the seedling emergency reached over72.67%, but then decreased with sowing depth increasing, and no seedling emerged when at the depth of3cm, suggesting that site condition has led to a bottleneck in population natural regeneration.3. Genetic diversity and structure analysis of big tree rhododendron based on AFLP markerWe conducted amplified fragment length polymorphism (AFLP) analysis to characterize the genetic diversity and variation of this species within and between remaining populations. Twelve primer combinations of AFLP produced447unambiguous and repetitious bands. Among these bands,298(66.67%) were polymorphic. We found high genetic diversity at the species level (PPB=66.67%, h=0.240,/=0.358) and low genetic differentiation (GST=0.110) between the two populations. Gene flow between populations (Nm) was relatively high at4.065. Analysis of molecular variance results revealed that22%of the genetic variation was partitioned between populations, and78%of the genetic variation was within populations. The presence of moderate-to-high genetic diversity and low genetic differentiation in the two populations can be explained by life history traits, pollen dispersal, and high gene flow (Nm=4.065). Bayesian structure and principal coordinate analysis revealed that56sampled trees were clustered into two groups. Our results suggest that some rare and endangered species are able to maintain high levels of genetic diversity even at small population sizes.4. Conservation genetic analysis based on microsatellite markerIn this study, we used data from fourteen polymorphic microsatellite loci to assess the levels of genetic diversity, genetic structure and demographic history for60individuals from2populations of big tree rhododendron. A total of81alleles were identified in60individuals across the fourteen microsatellites analysed with a mean value of5.786alleles per locus. We found a modrate high level of genetic diversity at the species level (Ne=2.863, AR=5.726, Ho=0.510, He=0.602,I=1.174, PPB=100%), which corresponded with the results based on AFLP marker. STRUCTURE and PCoA analyses indicated that60sampled trees were clustered into two distinct lineages. POPGENE v.1.32, Migrate-n v3.6.4, GeneClass v2.0and BayesAss v3.0software revealed high historieal and low contemporary gene flow between two remnant populations. Under the TPM model, BOTTLENECK analysis revealed that big tree rhododendron could have experienced recent historic bottleneck. LDNe analysis found that the effective population sizes in two populations are smaller than30. Our results suggest that in situ conservation should take priority for this species, complemented by ex situ and near situ conservation approaches, so that maintain the appropriate effective population size and to protect its natural habitats and promote gene flow.5. Endangered mechanism and conservation strategiesThe presence of moderate to high genetic diversity in big tree rhododendron indicated that the current endangered status of this species is not caused by genetic factors (e.g. genetic diversity decline, genetic drift and inbreeding) and biological characteristics. The main threat to this plant species may be the extremely limited distribution, and rarity individuals. In addition, site condition has led to a bottleneck in population natural regeneration, and colonization of new populations is lack of effective ways. We suggest that management policies should be improved to maintain the appropriate effective population size of big tree rhododendron and to protect its natural habitats. Furthermore, adult big tree rhododendrons are critical resources, not only to maintain current genetic diversity but also to provide provenance for its future recovery. Thus, protecting adult trees should be the priority in conservation to ensure ongoing recruitment. Meanwhile, appropriate human disturbance can promote population natural regeneration due to site condition. In addition, we recommend that seeds be collected for germplasm storage and artificial seedlings for ex situ conservation. Supporting the recovery of wild populations, the population recover and reintroductions of big tree rhododendron should also be carried out.