Species and speciation in the Hebeloma crustuliniforme complex.

Research output: Thesisinternal PhD, WU


<p>This thesis deals with species delimitation and speciation in the ectomycorrhizal <em>Hebeloma crustuliniforme</em> complex. The species concept traditionally used in this complex is based on morphology of basidiocarps. However, species delimitation has been controversial. One of the best known names is <em>H. crustuliniforme</em> (Bull.) Quél., a name regularly encountered in the mycorrhizal literature. However, this name has been used for a species complex of taxa with relatively pale (but sometimes more brown-tinged), veilless carpophores with weeping lamellae. In this thesis the basal criterion to delimit species within the <em>Hebeloma crustuliniforme</em> complex is sexual intercompatibility. Therefore, speciation is here defined as the origin of sexual interincompatibility. The strategy that has been used in this thesis to address the questions of species delimitation and speciation in the <em>Hebeloma crustuliniforme</em> complex can be summarized as follows:</p><OL><LI>Define InterCompatibility Groups (ICGs) and look for examples of partial incompatibility (Chapter 2);<LI>Determine phylogenetic relationships between those ICGs (Chapter 3);<LI>Focus on closely related, and preferentially partially incompatible, ICGs to study speciation (Chapters 4, 5);<LI>Derive an operational species concept for the <em>Hebeloma crustuliniforme</em> complex that is based on morphological recognition of (combinations of) biological species within an explicitly phylogenetic framework (Chapter 6).</OL>In Chapter 2 the results are described of intercompatibility tests in this species complex. In a sample of 110 collections 20 InterCompatibility Groups (ICGs) were found. Partial compatibility was found between ICGs 3 and 4, between 2 and 3/4, between 1 and 2 and between 16 and 17. One strain (605) was compatible with all strains of ICGs 3 and 4. In all other cases, however, assignment of isolates to a single ICG was unambiguous. Individual compatible combinations between members of the partially compatible ICG 1 and 2 and between members of the partially compatible ICG 16 and 17 showed signs of reduced compatibility. This was reflected by: (i) no or unidirectional nuclear migration, (ii) reduced growth rate of the dikaryon and (iii) aberrant morphology of hyphae.</p><p>In Chapter 3 phylogenetic relationships were studied between the ICGs of the <em>H. crustuliniforme</em> complex and between them and the other main groups in the genus <em>Hebeloma</em> based on nuclear ribosomal ITS sequences, using cladistic methods. The 20 ICGs of the <em>H. crustuliniforme</em> complex do not form a monophyletic group, but instead form two distinct clades, one consisting of three ICGs (clade I) and the other of 17 ICGs (clade II). Most of the ICGs in the latter clade were very closely related as suggested by a low sequence divergence. The majority of ICGs of this clade showed a preference for <em>Salicaceae</em> , but the basal ICG (ICG 21) did not. The host tree switch to <em>Salicaceae</em> has probably been followed by extensive and rapid speciation.</p><p>Several other well supported clades were found in the genus <em>Hebeloma</em> , but the basal relationships between them were not well resolved. It is therefore impossible to propose a new infrageneric division for <em>Hebelama</em> .</p><p>In Chapter 4 a subclade of clade II (subclade IIa) was studied in detail. In this subclade nine ICGs were found, four of which were partially compatible. This partial intercompatibility was organised hierarchically with an intermediate level of compatibility between ICGs 3 and 4 and very limited compatibility between 2 and 3/4 and between 1 and 2. The single strain (605) that was intercompatible with all strains of ICGs 3 and 4 was a member of subclade IIa as well. A mitochondrial and a nuclear phylogeny of strains belonging to these partially compatible populations were reconstructed. For ICGs 2, 3 and 4 a positive correlation was found between the level of interincompatibility and the relative age of the most recent common ancestor. ICGs generally formed monophyletic groups, ICG 3 and 4 (15% partial intercompatibility) together formed a monophyletic group and the sister group of (3,4) was ICG 2 (0.4% intercompatible with (3,4)). This is consistent with a gradual origin of sexual incompatibility (divergence-first). ICG 1 had a different position in the nuclear and mitochondrial phylogeny. In the nuclear phylogeny it was the sister taxon of ICG 5, and in the mitochondrial the sister group of ICG 2. A possible explanation is that ICG 1 has a hybrid origin, with the ancestor of ICG 5 as the nuclear donor and the ancestor of 2 as the mitochondrial donor.</p><p>The subject of Chapter 5 is a polymorphism in the ribosomal Internal Transcribed Spacer of ICG 17. Within this ICG of the morphospecies <em>Hebeloma velutipes</em> a dikaryotic strain (d504) was found with two divergent types ITS. These two types segregated in monokaryotic progeny of the same strain, showing that the different ITS types represent different alleles at homologous rDNA loci. RFLP analysis of more strains of ICG 17 showed that the polymorphism is widespread, with both types occurring in Europe as well as in America. Cladistic analyses of the two ITS sequences showed that they did not form a monophyletic group. One of the types belonged to a clade together with the single ITS type found in the partially compatible ICG 16 and the other to a clade together with the single ITS type found in the fully incompatible ICG 18. RFLP analysis of the mitochondrial ribosomal SSU showed that there were fixed differences between the mitochondria of ICG 16 and 17. Several lines of evidence were described that the ITS polymorphism in ICG 17 is not the result of actual hybridisation between 16 and 17. The polymorphism within ICG 17 must therefore be of a different origin. The lack of recombinants, neither within the rDNA locus nor between ITS 1 and 2, suggests that the two types have come together relatively recently. The ITS polymorphism described in this Chapter clearly showed the potential danger of using single ribosomal sequences for reconstructing species phylogenies and the potential problems for molecular identification of species.</p><p>In Chapter 6 a method is presented to derive an operational species concept for the <em>Hebeloma crustuliniforme</em> complex that is based on (combinations of) biological species within an explicitly phylogenetic framework. Crucial in this analysis is a reliable estimate of the phylogeny of biological species in the <em>H. crustuliniforme</em> complex. Based on two nuclear sequences, we presented a best estimate of the phylogeny of biological species within the <em>H. crustuliniforme</em> complex. Using this phylogeny, on the basis of (strict) monophyly only two species could be recognised among 20 biological species, viz. <em>H. velutipes</em> and <em>H. helodes</em> . An earlier phylogenetic analysis indicated that these two morphological species are not sister taxa. Relaxing the criterion of monophyly and allowing paraphyletic groupings of biological species as a morphospecies resulted in the recognition of three morphospecies, viz. <em>H. velutipes</em> , <em>H. incarnatulum</em> and <em>H. helodes</em> . A tree, with the five ICGs of the previously defined morphospecies <em>Hebeloma crustuliniforme</em> (1, 2, 3, 4 and 5) constrained as a monophyletic group could not be rejected. This constrained tree, together with the relaxed criterion, allowed the recognition of four species, viz. <em>H. helodes</em> , <em>H. crustuliniforme</em> , <em>H. velutipes</em> and <em>H. incarnatulum</em> . The limited ability to translate a biological species concept into an operational species concept was explained by the lack of qualitative characters and the plasticity of quantitative characters. Based on the close relationship between the ICGs in the two clades of the <em>H. crustuliniforme</em> complex, it was shown that a good correspondence between a biological species concept and a morphological species concept is not likely to be forthcoming.</p><p>In the final Chapter the results found in this study were integrated and discussed in a broader context and directions for future research were suggested. Future phylogenetic studies should consider the possibility of genetic exchange between divergent populations more explicitly.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Hoekstra, R.F., Promotor, External person
  • Kuijper, Thomas, Promotor
Award date17 Nov 1999
Place of PublicationS.l.
Print ISBNs9789064641176
Publication statusPublished - 1999


  • hebeloma crustuliniforme
  • taxonomy
  • speciation
  • evolution


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