| Ophiocordyceps lanpingensis is belonged to Ophiocordyceps (Ophiocordycepiacae) as a sibling species to Ophiocordyceps sinensis, is widely used as a folk tonic in the northwestern Yunnan. The taxonomic status, ecological characteristics, active ingredient and pharmacological action of O. lanpingensis have been detailedly studied by our group. However, little is known about population genetic diversity and genetic structure of O. lanpingensis, its host insects and its suitable area. In this study, the representative distribution areas were sampled, population genetic diversity and genetic structure of O. lanpingensis and its host insects were analyzed using molecular biological methods, its suitable area was predicted on the basis of niche model. The major findings were as following.Based on nrITS gene fragments of 83 samples in 8 populations and tefl-a gene fragments of 65 samples in 8 populations, population genetic diversity and genetic structure of O. lanpingensis was revealed that a high haplotype diversity (nrITS:Hd= 0.727; tefl-a:Hd= 0.561) and lower total nucleotide diversity (nrITS:Pi= 0.0023; tefl-a:P/=0.0007) were existed at the species level. At the population level, O. lanpingensis was showed a higher total genetic diversity (nrlTS:Hj= 0.751; tefl-a: HT= 0.677) and genetic diversity (nrITS:Hs= 0.594; tefl-a:Hs= 0.635) within populations. AMOVA analysis was showed that some genetic differentiation was resided among populations, i.e., the Fst values of nrlTS and tefl-a genes were 12.59% and 14.84% among populations, respectively. Permut analysis was also consistent with the above results, i.e., the Gst values of wITS and tefl-a genes were 0.21 and 0.063 among populations, respectively, and the Nst values were 0.261 and 0.09 among populations, respectively. The geographical distance had little relation with the genetic differentiation. The gene flow (Nm) of nrITS and tef1-a genes were 1.736 and 1.435 among populations, respectively. It was suggested that gene flow between populations might prevent genetic drift caused by genetic differentiation among populations, resulting in a significantly lower genetic differentiation and genetic polymorphism within populations. Mantel test analysis was showed that there was no significant correlation between genetic distance and geographic average distance for O. lanpingensis populations (nrITS:R=-0.07, P>0.05; tef-Iα: R= 0.051, P>0.05). In addition, neutral test and mismatch analysis were both showed that the O. lanpingensis populations were recently in a state of expansion.Molecular clock based on nrITS and tefl-α genes was estimated that Ophiocordyceps lanpingensis might originated from the regions of the Biluoxueshan Mountains and the Xuebang Mountains in Lanping and the Laojunshan Mountains in Jianchuan around the Hengduan Mountains at least from 18.4 Mya BP, its genetic differentiation became stronger coupling with the geological events of the Hengduan Mountains. According to researching nrITS haplotype sequences, the genetic differentiation of Ophiocordyceps lanpingensis should be occurred about 3.76 MyaBP, in which time the Qingzang Movement A of the Qinghai-Tibet Plateau was rapidly uplifted (3.6 Mya BP), corresponding with the Hengduan Movement of the Hengduan Mountains. Ophiocordyceps lanpingensis subsequently was further differentiated in 2.45-2.16 Mya BP, at that time the Qinghai-Tibet Plateau rapid uplift(2.5-1.8 Mya BP). The population genetic pattern of today had been formed in 1.54-1.26 Mya BP, corresponding to the Poyang inter-glacial(1.50-1.37 Mya BP), which is the beginning of Quaternary glacial and inter-glacial climate cycles. The population genetic differentiation was occurred about 1.8 Mya BP based on molecular clock of tefl-a gene, the further genetic differentiation was occurred in 1.3-0.5 Mya BP,0.384-0.126 Mya BP, corresponding the Qinghai-Tibet Plateau rapid uplifting, namely the Kunhuang Movement(1.2-0.6 Mya BP, i.e., the Yuanmou Movement in the Hengduan Mountains), and the Lij ing glacial (0.31-0.13 Mya BP), respectively.Based on Co I gene fragments of 42 samples in 7 populations and Cytb gene fragments of 43 samples in 7 populations, the population genetic diversity and genetic structure of host insects were revealed a high genetic diversity and lower nucleotide diversity, respectively, at both species and population levels. The Co I and Cytb gene fragments of host insects were rich in private haplotype, with being 86.4%and 78.6%, respectively. It was suggested that the complex and diverse ecological environments of the Hengduan Mountains caused environmental fragmentation and a limited gene flow. Both of Permut and AMOVA analyses were showed that a significant genetic differentiation was located among host insect populations and genetic polymorphism was rich within populations, indicating the gene flow was a limited and a few genes was exchanged among populations. Mantel test analysis on the basis of Co I gene was showed that there was no significant correlation between genetic distance and geographic average distance of host insects populations (P> 0.05). However, there was a significant correlation between these two (R= 0.515, P< 0.05), and this might be caused by different evolutionary rates. Neutral test and mismatch analysis were both showed that the hosts of O. lanpingensis populations were recently in a state of dynamic equilibrium.Estimates of differentiation time based on Co I and Cytb gene fragments was showed that the genetic differentiation time of host insect was coupled with the geological and climate events. The first differentiation was occured during 3.668-3.126Mya BP, coupling to the Qingzang Movement A, and then differentiation was occured during 1.175 Mya BP-0.972 Mya BP (namely, the Kunhuang Movement) after a period of stable long-term adaptable phase. This differentiation was formed into two branches and might be the present two species, i.e., Ahamus yunnanensis (Clade I) and A. jianchuanensis (Clade II). Subsequently, each time node of haplotype differentiation was coupled with the Ganhaizi glacial period (0.53-0.45 Mya BP), the Lijiang glacial period (0.31-0.13Mya BP), the Gonghe Movement (0.15 Mya BP to prsent) and the Dali glacial period (0.13-0.01 Mya BP), respectively. The local geological and climate events, especially the last glaciation period and interglaciation cycle, had been greatly impacting the genetic differentiation of host insects, accompanying a massive contraction and expansion. Therefore, it is inferred that the Biluoxueshan Mountains and the Xuebang Mountains in Lanping and the Laojunshan Mountains in Jianchuan around the Hengduan Mountains might be the refuge of host insects in the last glacial period.The suitable area of O. lanpingensis was predicted by employing MaxEnt model, it was showed that its potential suitable area was up to 208,077.2 KM2. The main areas were included the regions around the Hengduan Mountains, the central region of Yunnan, and the southern Tibetan of China, Bhutan, Sikkimin of India and north-central Nepal in Himalayas. Among these areas, the middle-high suitable area (0.6-0.8) was up to 37,218.9 KM2, ideal suitable area (0.8-1.0) was up to 6,421.7 KM2. Temperature, humidity and altitude were the main factors influencing the distribution of O. lanpingensis, especially the temperature variance and the rainfall of the wettest month was greatly impacted its distribution pattern. Lanping county, Yulong county and Jianchuan county in the Hengduan Mountains are now centre distributed of O. lanpingensis. Herein presented a high genetic diversity of O. lanpingensis in these regions, and should provide scientific basis for protecting O. lanpingensis. |
