The caterpillar fungus Ophiocordyceps sinensis is one of the most valuable medicinal fungi in the world, and it requires host insects in family Hepialidae (Lepidoptera) to complete its life cycle. However, the genetic diversity and phylogeographic structures of the host insects remain to be explored. We analyzed the genetic diversity and temporal and spatial distribution patterns of genetic variation of the host insects throughout the O. sinensis distribution. Abundant haplotype and nucleotide diversity mainly existed in the areas of Nyingchi, ShangriLa, and around the edge of the Qinghai-Tibet Plateau, where are considered as the diversity center or micro-refuges of the host insects of O. sinensis. However, there was little genetic variation among host insects from 72.1% of all populations, indicating that the host species composition might be relatively simple in large-scale O. sinensis populations. All host insects are monophyletic except for those from four O. sinensis populations around Qinghai Lake. Significant phylogeographic structure (NST>GST, P<0.05) was revealed for the monophyletic host insects, and the three major phylogenetic groups corresponded with specific geographical areas. The divergence of most host insects was estimated to have occurred at ca. 3.7 Ma, shortly before the rapid uplift of the QTP. The geographical distribution and star-like network of the haplotypes implied that most host insects were derived from the relicts of a once-widespread host that subsequently became fragmented. Neutrality tests, mismatch distribution analysis, and expansion time estimation confirmed that most host insects presented recent demographic expansions that began ca. 0.118 Ma in the late Pleistocene. Therefore, the genetic diversity and distribution of the present-day insects should be attributed to effects of the Qinghai-Tibet Plateau uplift and glacial advance/retreat cycles during the Quaternary ice age. These results provide valuable information to guide the protection and sustainable use of these host insects as well as O. sinensis.

To elucidate reactive oxygen species (ROS) metabolism of cotton cytoplasmic male sterility and the effects of restorer gene on the metabolism of ROS, the metabolism changes in the production and scavenging of ROS and gene expression related to ROS-scavenging enzymes were investigated in the anther mitochondria of CMS line, maintainer line and hybrid F(1). During the abortion preliminary stage (sporogenous cell division stage), anthers of CMS line had a little higher superoxide (O(2)(-)) production rate and hydrogen peroxide (H(2)O(2)) and malondialdehyde (MDA) contents than those of maintainer or hybrid F(1. )Simultaneously, a little higher ROS contents might serve as a signal to increase the activity of superoxide dismutase (SOD) in anthers of CMS line to reduce the ROS damage to the anther development. But at the abortion peak (pollen mother cell meiosis stage), anthers of CMS line had extraordinarily higher ROS contents and lower ROS-scavenging enzymic activities compared with the hybrid F(1), during which the ROS contents and ROS-scavenging enzymic activities in hybrid F(1) were approximate to those of maintainer line. The expression of Mn-sod and apx mRNA in anther of CMS line was obviously inhibited when ROS produced with a great deal during anther abortion, however the gene expression in hybrid F(1) kept normal with the maintainer. Excessive accumulation of O(2)(*-) , H(2)O(2 )and MDA, significant reduction of ROS-scavenging enzymic activities and lower gene expression level of ROS-scavenging enzyme were coinstantaneous with male cells death in anthers of CMS line. But when the restorer gene was transferred into CMS line, excessive production of ROS could be eliminated in the anthers of hybrid F(1). The restorer gene likely plays an important role in keeping the dynamic balance between the production and elimination of ROS.

Job’s tears (Coix lachryma-jobi L.) is an important crop used as food and herbal medicine in Asian countries. A drug made of Job’s tears seed oil has been clinically applied to treat multiple cancers. In this study, the genetic diversity of Job’s tears accessions and the fatty acid composition, triglyceride composition, and anti-proliferative effect of Job’s tears seed oil were analyzed using morphological characteristics and ISSR markers, GC-MS, HPLC-ELSD, and the MTT method. ISSR analysis demonstrated low genetic diversity of Job’s tears at the species level (h = 0.21, I = 0.33) and the accession level (h = 0.07, I = 0.10), and strong genetic differentiation (GST = 0.6702) among all accessions. It also clustered the 11 accessions into three cultivated clades corresponding with geographical locations and two evidently divergent wild clades. The grouping patterns based on morphological characteristics and chemical profiles were in accordance with those clustered by ISSR analysis. Significant differences in morphological characteristics, fatty acid composition, triglyceride composition, and inhibition rates of seed oil were detected among different accessions, which showed a highly significant positive correlation with genetic variation. These results suggest that the seed morphological characteristics, fatty acid composition, and triglyceride composition may be mainly attributed to genetic factors. The proportion of palmitic acid and linoleic acid to oleic acid displayed a highly significant positive correlation with the inhibition rates of Job’s tears seed oil for T24 cells, and thus can be an important indicator for quality control for Job’s tears.

Ophiocordyceps sinensis (Ascomycota: Ophiocordycipitaceae) is a native fungal parasite of Hepialidae caterpillars and one of the most economically important medicinal caterpillar fungi in China. However, little is known about the phylogenetic and evolutionary relationships between O. sinensis and its host insects. In this study, nuclear ITS and β-tubulin sequences from O. sinensis and mitochondrial COI, COII, and Cytb sequences from its hosts were analyzed across 33 populations sampled from five regions in China. Phylogenetically, both O. sinensis and its hosts were divided into three geographically correlated clades, and their phylogenies were congruent. Analysis of molecular variance and calculated coefficients of genetic differentiation revealed significant genetic divergence among the clades within both O. sinensis (F(ST)= 0.878, N(ST)=0.842) and its hosts (F(ST)=0.861, N(ST)=0.816). Estimated gene flow was very low for O. sinensis (Nm=0.04) and the host insects (Nm=0.04) among these three clades. Mantel tests demonstrated a significant correlation (P<0.01) between the genetic distances for O. sinensis and its hosts, as well as a significant association (P<0.05) between geographic and genetic distances in both. The similar phylogenetic relationships, geographic distributions, and genetic structure and differentiation between O. sinensis and its hosts imply that they have coevolved.