Bailinggu is one of the most widely cultivated mushrooms in China. Recently, this species has been involved in the researches of genetic diversity evaluation15,16, temperature response mechanism17,18,19,20, fructification mechanism21, and bioactive substance exploitation22. However, the most essential information on the taxonomic status of Bailinggu and its phylogenetic relationships with its sibling species remain uncertain.

The mushrooms from Pleurotus genus that grow on the roots and stems of Umbelliferae plants belong to the P. eryngii species complex. The morphological characteristics of Bailinggu from western China conform to those of the P. eryngii species complex23. The morphological differences between Bailinggu and its related species are shown in Table 1. The pileus color of P. eryngii var. eryngii ranges from brown and beige-brown to light beige, whereas the pileus color of P. eryngii var. ferulae from Europe ranges from grey-brown to slate grey to beige brown. The macro-morphological characteristics of Bailinggu are similar to those of P. nebrodensis, but the basidiospores of Bailinggu are slightly smaller than those of P. nebrodensis. The pileus color of P. eryngii var. ferulae from China is brown to white, therefore, it is not possible to distinguish Bailinggu from P. eryngii var. ferulae from China based exclusively on their macroscopic and microscopic characteristics.

Table 1 The distinctive discriminating morphological characters for the Pleurotus eryngii species complex. Full size table

The intersterility criterion is a derivative of the biological species criteria. Many cryptic species, such as the Armillaria mellea (Vahl) P. Kumm.24, have recently been recognized using this criterion. Previous mating compatibility tests of the P. eryngii species complex did not indicate any complete reproductive isolation among the genetic groups within the P. eryngii species complex. The mating rate between P. eryngii var. eryngii and P. eryngii var. ferulae was the highest, with a value of 98%8 or 93%10, but those between P. nebrodensis and P. eryngii var. eryngii and between P. nebrodensis and were significantly lower, with values of 6–18%25. Very few mating tests have been performed between Bailinggu and other genetic groups. According to the previous studies, Chinese Bailinggu showed much higher compatibility with P. eryngii var. eryngii (65%) and P. eryngii var. ferulae (82%) than with P. nebrodensis (15%) and P. ferulaginis (11%)8,11. This indicates that Bailinggu might be closer to P. eryngii var. eryngii and P. eryngii var. ferulae. However, some evidence that many fungi genetically isolated in nature retain the ancestral character of interbreeding14. Hilber26 found that P. eryngii var. eryngii and P. eryngii var. ferulae could mate with each other in the laboratory, but they appear to be reproductively isolated in the field and are associated with specific host plants.

The results of this study based on molecular data showed that Bailinggu is a separate phylogenetic species instead of a variety or subspecies of the P. eryngii complex according to the GCPSR criterion, although this mushroom retains high intercompatibility with P. eryngii var. eryngii and P. eryngii var. ferulae in the laboratory. A similar observation was found in a study of the P. ostreatus complex. Three intersterility groups or biological species (I, II, and VI) in the P. ostreatus complex were found to contain more than one phylogenetic species27. A phylogenetic reconstruction based on the ITS dataset and the combined dataset revealed that the genetic distance between Bailinggu and P. eryngii (var. eryngii, var. ferulae, var. elaeoselini, var. thapsiae, var. tingitanus) was greater than those between Bailinggu and P. ferulaginis and between Bailinggu and P. nebrodensis. This result is in conflict with the previous findings in the mating tests. Considering the geographical isolation of Bailinggu in nature, the results inferred from molecular data are more acceptable because DNA sequence divergence, be it allopatric or sympatric, might occur much earlier than the evolution of intersterility28,29.

Previous research using sequence analyses of ITS and IGS1 showed that Bailinggu is a phylogenetic sister group to P. eryngii11. However, our study indicates that P. ferulaginis is much more similar to P. eryngii in terms of not only morphology, distribution, and ecology but also DNA divergence. The phylogenetic analysis revealed that Bailinggu is a sister group to the eryngii-ferulaginis-nebrodensis clade and is not closely related to the other genetic groups of the P. eryngii species complex.

Reproductive isolation caused by host specialization is often observed in basidiomycetes, particularly plant pathogenic fungal species30. To the best of our knowledge, the P. eryngii species complex has developed a certain degree of host specificity. To detect whether the relationships among the genetic groups of the species complex correlate with those among their hosts, the phylogeny of the relevant hosts was reconstructed based on ITS1 and ITS2 sequences retrieved from GenBank (Fig. S4). The results showed that the eryngii, ferulae, elaeoselini, thapsiae, and tingitanus varieties are so closely related genetically that they could not be distinguished by ITS analysis, but the relationship among hosts of P. eryngii var. eryngii, P. ferulaginis, and P. nebrodensis is markedly closer. In contrast, the genetic relationships of Bailinggu with the ferulae, elaeoselini, thapsiae, and tingitanus varieties are distant, but the genetic relationships among their hosts are close, indicating that hosts might not be the main reason for the divergence of Bailinggu from other genetic groups. Its long geographical isolation might be the main reason for the distant genetic relationship among Bailinggu and other genetic groups.

Pleurotus eryngii, P. ferulaginis and P. nebrodensis are mainly distributed in the Mediterranean and surrounding areas, whereas recent studies found that P. eryngii and P. nebrodensis also occur in Asia11. The distributions of the two mushrooms are wide and continuous, but there is very limited information on the distribution of Bailinggu. The samples of Bailinggu used in the present study were mostly from western China, and partly from Iran11,31. The main distribution area of Bailinggu in China is located far from the distribution areas of other genetic groups with the exception of P. eryngii var. ferulae from China. There are no obvious differences in morphological characteristics or habitat between Bailinggu and P. eryngii var. ferulae from China. However, a sequence analysis showed a remarkable difference between them in terms of DNA sequence, which is consistent with previous results6. What efficient prezygotic barriers that maintain the separation of both gene pools will require further study. The pileus color of P. eryngii var. ferulae from China is different from that of P. eryngii var. ferulae from Europe. Moreover, the phylogenetic analysis showed that they cluster according to their geographical origins even though they still belong to the same genetic group. Geographical isolation and differences in biotope would likely lead to increasing divergence of an individual population to enhance differentiation32,33.