Conservation Biology Of The Plant Species With Extremely Small Population Rhododendron Longipedicellatum

About one-fifth of the world’s plants risk extinction.Some species have small populations,limited distribution,and endure enormous pressure from habitat destruction.There is an urgent need for research on conservation biology of these most threatened groups,among which,Rhododendron longipedicellatum L.Cai & Y.P.Ma is a typical group.R.longipedicellatum is a narrow endemic species and a subject of urgent demand in the domestic market and overseas,belongs to the evergreen shrubs of the genus Rhododendron,subg.Rhododendron,sect.Vireya and subsect.Pseudovireya,and wasn’t published as a new species until 2016.Its fascinating shapes,brilliantly gilvous flowers,and unusual flowering time endow this species with extremely high ornamental value.Since 2014,our study group has found 5 wild populations of 80-350 plants each surviving in limestone habitat in Babu Community,Malipo County,Yunnan Province,through multiple professional field investigations.The seedlings in each population are scare and relatively poor in natural regeneration.The remaining partial populations have been seriously damaged by human activity;these wild resources urgently need to be protected,as their status is of grave concern.According to IUCN Red List Categories and Criteria(version3.1),R.longipedicellatum is a critically endangered [CR B1ab(i,iii,v)] species.This article systematically studied the general situation of its wild resource,biological properties,pollen morphology and characteristics,reproductive biology,conservation genetics,introduction and domestication etc.It aims to provide theoretical basis for effective protection and reasonable development and utilization of R.longipedicellatum and serves as a reference for the protection of other plant species with extremely small populations in China.The major results and conclusions are given below.1.Biological characteristics and current situation of wild resourcesR.longipedicellatum are is a multi-branched evergreen shrub,1.0–3.3 m high with beautiful flowers and leaves.The number of flowers ranges from 1 to 12 per inflorescence with pedicels 3.0–5.5 cm in length;the flowers are bright yellow without spots.Surprisingly,unlike other wild rhododendrons whose flowering times occur between March and June,the natural flowering time of R.longipedicellatum lasts from the last ten-day period of November to the first ten-day period of February and the fruiting period was from March to May;only 5 wild populations were found remnant in limestone habitats of Malipo County through a long field surveys combined with field visits to Max Ent ecological niche model to predict its suitable growth area.The number of plants in the population is relatively small and under serious anthropogenic interference with a few plants attached to the trunk;moreover,there are few seedlings in all populations with relatively weak ability for natural regeneration,and some plants are dominated by clonal propagation.2.Study on pollen morphology and its characteristicsThe pollen grains mature gradually from the alabastrum intumescence to the petal full bloom stage,and are arranged in tetrahedral tetrad.The tetrad diameter ranges from 43.0 μm to 65.4 μm with an average 51.3 μm.A single pollen is subspheroidal with tricolporate aperture,and viscin threads were seen on the pollen grains surface.The pollen grains exine sculpture is uniform granular in SEM,and the granulate around the colpus are more compact.Exine is composed with two layers in LM.The highest germination rate of R.longipedicellatum pollen grains at 9:00 on the first day of anthesis was 90.26% when cultured in the medium for 10 g · L-1 agar + 100 g · L-1 sucrose + 200 mg · L-1 H3BO3 + 0 mg · L-1 Ca Cl2.Range analysis showed that the important order of influence on pollen germination rate was sucrose,Ca Cl2 and H3BO3,a certain concentration of sucrose and H3BO3 had a good promote sprouting effect,while the addition of Ca Cl2 significantly inhibited the pollen grains germination.Furthermore,the appropriate low temperature was helpful to storage of R.longipedicellatum pollen,the germination rate maintained a certain level after 48 days storage with﹣18℃.3.Study on reproductive biologyArched styles not only create obvious herkogamy that avoide self-pollination,but also effectively reduce rain damage to the intrinsic characteristics of the stigma surface secretions,promoting the female fitness of R.longipedicellatum in poor weather.Pollen viability maintained a high level over the flowering period.The reflectance spectrum of petals had two peaks at wavelengths of 360 nm and 580 nm.Tests of OCI,P/O and artificial pollination all indicated that R.longipedicellatum was self-compatible and that the breeding system was mixed mating.Geitonogamy mediated by Bombus braccatus was the primary pollination route in the natural environment,which suggested that the breeding system of R.longipedicellatum might be evolving from selfing to outcrossing.The pollination vector of R.longipedicellatum was very specific,in that only B.braccatus was confirmed to deliver pollen to the stigmas.Visitation frequency was influenced by the activity rhythms and resource requirements of the different castes(i.e.,sex).B.braccatus workers were the most effective pollinators because of higher visitation frequency and more effective contribution to fruit production,whereas the presence of B.braccatus males might enhance pollen flow within the population to a certain extent.4.Transcriptome analysisThe transcriptome was sequenced by using Illumina Hiseq 4000,in total,74092 Unigenes with an average length of 938 nt,N50 of 1616 nt,Q20 of 98.22%,Q30 of 95.20% and GC content of 43.24% were obtained by de novo assembly and cluster with filtered data.Among them,there were 23879 Unigenes with more than 1 kb.Then the Unigenes were annotated by 7 functional databases,and finally,39876(NR: 53.82%),38065(NT: 51.38%),27384(Swissprot: 36.96%),16099(COG: 21.73%),30401(KEGG: 41.03%),17518(GO: 23.64%)and 29676(Interpro: 40.05%)Unigenes were annotated.The Unigenes were roughly divided into three functional categories(i.e.biological processes,cellular components and molecular function)and 56 sub-categories according to GO function.Most of the genes performed biological processes.KEGG functional annotation analysis showed that Unigenes could be grouped into six categories,32 metabolic pathways.176 Unigenes relating to human diseases were detected,including endocrine and metabolic diseases(167)and antimicrobial resistance(9).5.Characteristic analysis and primer development of EST-SSRA total of 23192 SSRs were identified in 17354 unigenes,with an average density of one SSR per 3 kb.Dinucleotide and trinucleotide repeat were the main SSR types,accounting for 69.25% and 15.07% of all SSRs,respectively.Among all the 187 repeat motifs,(AG/CT)n was the most frequent repeat motif(62.01%),followed by the(A/T)n(12.34%)、(AC/GT)n(4.52%)and(AAG/CTT)n(4.23%).A total of 15908 SSRs occurred in the intersection of SSR and CDS,only 2792 of which occurred in protein-coding regions of these sequences.The density of SSRs was 0.076 SSR/kb in coding regions which was significantly lower than in non-coding regions of 0.344 SSR/kb.Moreover,trinucleotide repeat was the most abundant in coding regions(1356,48.57%).In terms of different length repeat units,the variation of the length of dinucleotide repeat SSR was the most abundant,followed by the mononucleotide.Based on the above results,17 pairs of highly polymorphic primers were screened from 102 pairs of EST-SSR primers,The number of alleles per polymorphic locus ranged from two to 13,and the observed and expected heterozygosities for the 17 loci varied from 0.235 to 0.943 and from 0.271 to 0.800,respectively.All 17 loci were found to amplify in R.molle(Blume)G.Don and R.simsii Planch.6.Research on conservation genetics based on EST-SSR markersThe genetic characteristics of 150 individual R.longipedicellatum samples of 5 remnant populations were studied by marking 17 pairs of highly polymorphic EST-SSR.The average data deletion rate of 17 pairs of markers was 1%,and a total of 162 alleles were amplified with the average number of alleles per site reaching 9.529.R.longipedicellatum maintained a high genetic diversity(He = 0.559,PPB = 100%)and a gene flow capable of homogenization(Nm = 2.763),despite the limited number of field resources and severe anthropogenic interference.It is speculated that it may be related to the limestone habitats,outcrossing breeding system,life forms and ancestral populations with rich genetic basis.AMOVA analysis showed that 91.25% of the total genetic variation was from within the population,the variation between populations accounting for only 8.75%.The STRUCTURE analysis clearly divided 150 individuals from 5 existing populations into 2 gene pools.Population NJ trees and PCo A analysis further subdivided them into 3 major groups.In addition,the bottleneck effect analysis based on SMM and TPM model showed that R.longipedicellatum experienced a genetic bottleneck at the species level.7.Endangered mechanism and protection strategiesBased on the results of this study,the early stage of sexual reproduction of R.longipedicellatum has not yet been blocked,and the main factors that threaten the survival of various populations are ecological factors(including small population size,narrow habitat,isolation of populations,etc.);moreover,the natural regeneration of populations are weak and lack of effective migratory routes.Although R.longipedicellatum still maintains a high level of genetic variation and gene flow,it is experiencing the effects of genetic bottleneck.Therefore,its protection should firstly focus on in-situ protection of the 5 remnant populations,strictly prohibit indiscriminate felling,and protect it from anthropogenic interference;in particular,the most severely damaged ZWL population should be regarded as an evolutionarily significant unit(ESU)with conservation priority.At the same time,provenance collection,artificial propagation and regression tests should be carried out to promote the study of near-field protection and exsitu conservation.In addition,taking into account that cross-pollination of the same plant with B.braccatus as the medium is the main mode of pollination for R.longipedicellatum under natural conditions,and the remnant populations are isolated from each other with habitats being severely fragmented.The protection of this species also requires artificial strengthening of pollen flow among populations,such as long-distance pollination assisted by humans,introducing plants from other populations to the same population in the wilderness,sampling multiple populations when practicing introduction and domestication etc.,all made to ease the genetic pressure on the species caused by geitonogamous pollination.