Shiitake mushrooms and molecular clocks:historical biogeography of LentinulaDavid S. Hibbett Harvard University Herbaria, 22 Divinity Avenue, Cambridge, Massachu-setts 02138, USA; Present address: Department of Biology, Clark University, 950 MainStreet, Worcester, MA 01610-1477, USA. E-mail: [email protected] Fungi make up a large, ecologically important group of eukaryotes that has beenneglected in historical biogeography. In this study, molecular phylogenies and molecularclock dating were used to infer historical biogeography of the shiitake genus, LentinulaEarle, which occurs in both the Old World and New World. The major goals of this studywere to determine if the Old World and New World groups are monophyletic, and whetherthe Old World/New World disjunction is the result of vicariance or long distance dispersal.Location Collections studied are from Asia, Australasia, the Gulf Coast region of NorthAmerica, the Caribbean, Central America, and northern South America.Methods Phylogenetic trees were inferred with nuclear large subunit (nuc-Isu) ribos-omal DNA (rDNA) and internal transcribed spacer (ITS) sequences. Molecular clockdating was performed using trees derived from nuc-Isu rDNA and mitochondrial small-subunit (mt-ssu) rDNA sequences. Rate constancy was tested with maximum likelihood,and clocks were calibrated using evidence from fossils or other molecular clock studies,or according to expected ages under different historical biogeographic scenarios.Results Analyses of ITS and nuc-Isu rDNA sequences suggest that there are sevenspecies of Lentinula worldwide, which occur in two main clades, one in the New Worldthe other in the Old World. Rate constancy cannot be rejected in nuc-Isu rDNA, but canbe rejected in mt-ssu rDNA. Both genes suggest that the Old World/New Worlddisjunction could be due to fragmentation of an ancient Laurasian range. An alternativeGondwanan hypothesis is not supported by the molecular clock age estimates.Main conclusions Only one long distance dispersal event must be invoked in Lentinula,that being between Australia and New Zealand. Despite having airborne spores, itappears that long distance dispersal is rare in Lentinula. This may also be true in otherfungi, which are therefore excellent candidates for historical biogeographic studies usingmolecular characters.KeywordsBeringia, biogeography, fungi, Lentinula, maximum likelihood, molecular clock,phylogeny.INTRODUCTIONOne of the major goals of biogeography is to demonstratecommon patterns in the historical movements of organisms.By integrating results from diverse taxa, it is possible to gaininsight into the formation of ecological communities and theorigins of global patterns of biodiversity. Plants and animalshave been well studied by historical biogeographers, butfungi have been largely neglected. This is unfortunate notonly because fungi make up a major clade of eukaryotes(with an estimated 1.5 million extant species; Hawksworth,1991) but also they play pivotal ecological roles. Lack ofknowledge regarding the movements of mycorrhizal, patho-genic, and saprotrophic fungi limits understanding of theevolution of terrestrial ecosystems.Impediments to fungal historical biogeography haveincluded poor knowledge of fungal phylogeny, the scantyfungal fossil record, and the perception that fungi, with theirJournal of Biogeography, 28, 231±241Ó 2001 Blackwell Science Ltdairborne spores, have few barriers for dispersal. However,prospects are improving for historical biogeography of fungi(as the existence of this symposium indicates). This isprimarily because of the growth of molecular systematics,which has provided phylogenetic resolution at all taxonomiclevels in the fungi, as well as insight into dispersal abilitiesand patterns of gene ¯ow. Finally, in conjunction with theslowly improving fungal fossil record, molecular charactershave provided clues to the absolute ages of the major groupsof fungi (Berbee & Taylor, 1993; Hibbett et al., 1997a).Recently, there has been an upsurge in the number of fungalphylogenetic studies that have a biogeographic focus, inclu-ding work on Pleurotus (Vilgalys & Sun, 1994), andGibberella (O'Donnell et al., 1998). In the present study,molecular characters were used to infer phylogenetic andhistorical biogeographic relationships in Lentinula Earle(Agaricales, Tricholomataceae, Collybieae), which includesshiitake mushrooms.Shiitake mushrooms have been cultivated for manycenturies in China and Japan (Chang & Miles, 1987) andare now grown on a commercial scale worldwide. Wildpopulations of shiitake mushrooms occur in the Old Worldand New World. In the Old World, Lentinula ranges from anorthern limit in Japan and the Russian Far East, to South-east Asia and Australasia (including New Guinea, Australia,Tasmania, and New Zealand), and west to Nepal and India,in the foothills of the Himalayas. In the New World,Lentinula occurs in the Gulf Coast region of North America,the Caribbean, Central America, and northern South Amer-ica (Pegler, 1983). Lentinula has not been recorded inEurope or Africa.Broad disjunct distributions, like that of Lentinula, canresult from long distance dispersal (LDD), fragmentation ofancestral ranges, or both. Lentinula reproduces by micro-scopic airborne spores, which at ®rst glance might suggest thatLDD could be common. However, several aspects of theecology and life cycle of Lentinula suggest that LDD should berare. Lentinula is heterothallic, meaning that it is obligatelyoutcrossing, and it does not produce asexual spores or othermitotic propagules. In addition, Lentinula grows only onhardwood logs, which are patchy discrete resources, and itdoes not produce rhizomorphs or other somatic structuresthat would enable it to bridge gaps between logs. Conse-quently, for LDD of Lentinula to occur via spores, two matingcompatible spores would have to colonize the very same log.Alternatively, Lentinula could be dispersed as dikaryons (theeffectively diploid phase of the life cycle) by rafting insidehardwood logs that were already colonized.Shiitake mushrooms are homobasidiomycetes. In additionto gilled mushrooms, homobasdiomycetes include puffballs,polypores, and other ¯eshy macrofungi. Homobasidiomyce-tes are nested within a more inclusive clade, the basidiomy-cetes, which also includes smuts, rusts, jelly fungi, andcertain yeasts. The oldest unambiguous fossil basidiomyceteis Palaeancistrus martinii, which is a hyphal fossil from