Genes 2022, 13, 1110. https://doi.org/10.3390/genes13071110 www.mdpi.com/journal/genes Article Genetic Analyses of Discrete Geographic Samples of a Golden Chanterelle in Canada Reveal Evidence for Recent Regional Differentiation Kuan Zhao 1,2 , Gregory A. Korfanty 1 , Jianping Xu 1, * and R. Greg Thorn 3, * 1 Department of Biology, McMaster University, Hamilton, ON L8S 4K1, Canada; zhaok35@mcmaster.ca (K.Z.); korfanga@mcmaster.ca (G.A.K.) 2 College of Life Science, Jiangxi Science and Technology Normal University, Nanchang 330013, China 3 Department of Biology, University of Western Ontario, London, ON N6A 5B7, Canada * Correspondance: jpxu@mcmaster.ca (J.X.); rgthorn@uwo.ca (R.G.T.) Abstract: The wild edible mushroom Cantharellus enelensis is a recently described species of the golden chanterelles found in eastern North America. At present, the genetic diversity and popula- tion structure of C. enelensis are not known. In this study, we analyzed a total of 230 fruiting bodies of C. enelensis that were collected from three regions of Canada: near the east and west coasts of Newfoundland (NFLD), with 110 fruiting bodies each, and around Hamilton, Ontario (10 fruiting bodies). Among the 110 fruiting bodies from each coast in NFLD, 10 from 2009 were without specific site information, while 100 sampled in 2010 were from each of five patches separated by at least 100 m from each other. Each fruiting body was genotyped at three microsatellite loci. Among the total 28 multilocus genotypes (MLGs) identified, 2 were shared among all three regions, 4 were shared between 2 of the t3hree regions, and the remaining 22 were each found in only 1 region. Minimal spanning network analyses revealed several region-specific MLG clusters, consistent with geo- graphic specific mutation and expansion. Though the most frequently observed MLGs were shared among local (patch) and regional populations, population genetic analyses revealed that both local and regional geographic separations contributed significantly to the observed genetic variation in the total sample. All three regional populations showed excess heterozygosity; for the eastern NFLD population, we reject the null hypothesis of Hardy–Weinberg equilibrium (HWE) at all three loci. However, the analyses of clone-corrected samples revealed that most loci were in HWE. Together, our results suggest that the three discrete regional populations of C. enelensis were likely colonized from a common refugium since the last ice age. However, the local and regional populations are diverging from each other through mutation, drift, and selection at least partly due to heterozygous advantage. Keywords: edible mushroom; microsatellite genotyping; population genetics; range expansion; Hardy–Weinberg equilibrium; excess heterozygotes 1. Introduction Cantharellus enelensis is a popular edible mushroom which is native to eastern North America. This golden chanterelle was historically regarded as part of the Cantharellus cibarius species complex until it was described in 2017 [1,2]. The species epithet “enelensis” in Latin refers to its presence in NL (the province of Newfoundland and Labrador, Can- ada), where it is the most common species of chanterelle, both on the island of Newfound- land (NFLD) and in mainland Labrador. However, C. enelensis has also been reported in several other parts of eastern North America, from Quebec, Canada to Wisconsin, USA [1,2]. At present, the genetic diversity and population structure of this edible mushroom are unknown. Indeed, little is known about population genetic variations of any Citation: Zhao, K.; Korfanty, G.A.; Xu, J.; Greg Thorn, R. Genetic Analyses of Discrete Geographic Samples of a Golden Chanterelle in Canada Reveal Evidence for Recent Regional Differentiation. Genes 2022, 13, 1110. https://doi.org/10.3390/ genes13071110 Academic Editor: Isabel Henriques Received: 20 May 2022 Accepted: 20 June 2022 Published: 21 June 2022 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Copyright: © 2022 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https://cre- ativecommons.org/licenses/by/4.0/).