| Elymus L. is a genus that contains many high quality forage species. There are about 12 perennial Elymus species distributed across northern China which play an important role in pasture and livestock production and ecological environment improvement. Previous studies have showed that some of Elymus species were affected with Epichloe endophytes, however, little is known about the phylogenetic relationship of these endophytes and related Elymus species. In the present study, nine Elymus species containing 871 samples were collected in northwest China, including the provinces of Gansu, Ningxia, Qinghai, Xinjiang, Sichuan and Tibet. The origin time of host-Elymus species and taxonomy and phylogenetics of related fungal Epichloe endophytes were analyzed based on molecular technology. The main results are as follows:1. Phylogenetic trees and network were constructed using sequences of the nuclear ribosomal internal transcribed spacer (nrITS) and non-coding chloroplast DNA (trnL-F) sequences from 871 samples of nine polyploid Elymus species and some accessions from GenBank. The results showed that tetraploid(Elymus dahuricus, E. dolichatherus, E. sibiricus, E. antiquus and E. tibeticus) and hexaploid (E. tangutorum, E. dahuricus var. cylindricus, E. excelsus and E. nutans) Elymus species from northwest China had an independent origin, which started diverging 17 and 8.5 million years ago, respectively. The genetic diversity cluster of Elymus species corresponded to geographic regions of origins.2. There were sixteen ecotype populations from Ningxia, Gansu and Qinghai in total 871 samples which were affected with asexual Epichloe endophytes using aniline blue staining method. The Epichloe endophytes were isolated from 6 E. tangutorum populations and the fungal morphology was observed. The results showed that there was correlation between endophyte growth and altitude of host samples. The growth speed of the asexual endophytes from regions below 3000 m altitude was (0.45-0.70) mm/day which was fast than that of the asexual endophytes from regions above 3000 m altitude which was (0.43-0.58) mm/day. Except with the growth speed, the colonial morphology of the endophytes from the two regions was also totally different. These differences were also observed in other endophytes isolated from other tested Elymus species. These results of morphology observation were supported by the molecular data. The phylogenetic trees constructed from sequences of β-tubulin (tubB) and translation elongation factor 1-α (tefA) indicated that endophytes from regions above 3000 m altitude clustered together and endophytes from regions below 3000 m altitude yielded a star-like topology.3. There are 19 Epichloe species in Europe and 11 Epichloe speices in Asia which counting for 70% of the all named 43 Epichloe species. This is consistent with network topology study which suggest that diversity of Epichloe species from Europe and Asia is greater that that of Epichloe species from other continent. In tubB and tefA phylogenetic tree, Epichloe species from Europe and Asia place near the root of the clades, indicating they are diverged early in the phylogeny. In conclusion, Epichloe species likely originated in Eurasia.4. No gene duplication was found based on tubB, tefA and actin (actG) genes through PCR and sequencing. The tubB, tefA and actG phylogenetic study revealed that these asexual Epichloe endophytes from northwest Chinese Elymus species were derived from Epichloe bromicola, which isolated from Hordeum bogdanii or H. brevisubulatum. In addition, the phylogenetic relationship suggested that asexual Epichloe endophytes from northwest Chinese had different origination from the North American sexual Epichloe species from E. villosus, E. virginicus and E. hystrix species.5. Compared to the sequences of Epichloe endophyte from Elymus species and diploid Hordeum species, Hordeum species likely contain two species of Epichloe species. One is also found in North American Elymus species and the other one is found in Chinese Elymus species. |
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