Ectomycorrhizal mycelia reduce bacterial activity in a sandy soil

Abstract: Bacterial activity was studied in a growth system containing Pinus contorta seedlings inoculated with different mycorrhizal fungi. Nylon nets enabled separation of soil compartments with extramatrical mycorrhizal hyphae from soil compartments with roots and mycelium. In three separate experiments bacterial activity, estimated as thymidine incorporation, was reduced in soils with Paxillus involutus hyphae compared to controls without mycorrhizal hyphae. This effect was found irrespective of compartments with and without roots were compared. Laccaria bicolor only reduced the activity in one of these three experiments. Thelephora terrestris (tested in two experiments), Laccaria proxima, Suillus variegatus and Hebeloma crustuliniforme (one experiment), also reduced the thymidine and leucine incorporation rates of bacteria. The reduction for these fungi varied between 20% and 50% in all experiments. Numbers of viable bacteria appeared to be reduced by T. terrestris, L. proxima, S. variegatus and H. crustuliniforme in one experiment, while no effect was seen in the other experiments.     

Oxalate and ferricrocin exudation by the extramatrical mycelium of an ectomycorrhizal fungus in symbiosis with Pinus sylvestris

Accurate estimates of mycelial exudation in time and space are crucial for the assessment of ectomycorrhizal involvement in biogeochemical processes. Knowledge of exudation from mycelia of ectomycorrhizal fungi is still limited, especially for fungi in symbiosis with a host. Pinus sylvestris seedlings colonized by Hebeloma crustuliniforme were grown in aseptic multicompartment dishes. This novel system enabled identification of exudates originating only from extramatrical mycelium. At harvest, hyphal density and numbers were estimated using microscopic imaging. A fractal geometric approach was adopted for calculation of exudation rates. The main compounds identified were oxalate and ferricrocin. The exudation rate for oxalate was 19 +/- 3 fmol per hyphal tip h(-1) (mean +/- standard error of the mean) or 488 +/- 95 fmol hyphal mm(-2) h(-1). Ferricrocin rates were approx. 10 000 times lower. The fractal dimension (D) of the mycelia was 1.4 +/- 0.1, suggesting an explorative growth. Potassium nutrition was a significant regulatory factor for ferricrocin but not oxalate. The results suggest that hyphal exudation may alter the chemical conditions of soil microsites and affect mineral dissolution. Calculations also indicated that oxalate exudation may be a significant carbon sink.     

Element localization in ultrathin cryosections of high-pressure frozen ectomycorrhizal spruce roots

We present, for the first time, elemental mapping of ultra-thin cryosections from high-pressure frozen ectomycorrhizal roots of Picea abies–Hebeloma crustuliniforme. The maps provide interpretable information on the relationship between elements and the structure of inhomogeneous objects. Cryoultramicrotomy together with energy dispersive X-ray microanalysis (EDX) offers the potential to study the subcellular localization of specific ions and ecologically important tracers (Cs and Sr) in ectomycorrhizal roots under conditions resembling the natural slate as closely as possible. Structural changes of the ectomycorrhizal roots, in particular the absence of a Hartig net at high NH₄⁺ levels in the nutrient solution, were accompanied by elemental modification of Ca in cortical cell walls, where markedly higher concentrations of Ca were found. Cs and Sr applied to the nutrient solution were localized in root and fungal cells of the Hartig net. Cs accumulated mainly in the vacuoles of the Hartig net hyphae and its distribution was very similar to the distribution of K. In contrast to Cs, Sr was found to occur mainly in electron-opaque and P-rich granules. From this study, (here is no indication that Ca is the only ion accompanying P in the P-rich granules. Several elements including Ca, K, Cl, S, Cs and Sr, with highest concentrations for S, can occur together with P in these granules. The occurrence of the P-rich electron-opaque deposits in fungal cells might be the first evidence of polyphosphate granules in the native state, since our specimen preparation technique did not include chemical fixation.     

Draft Genome Sequence of the Soil Bacterium Burkholderia terrae Strain BS001, Which Interacts with Fungal Surface Structures

Burkholderia terrae BS001 is a soil bacterium which was originally isolated from the mycosphere of the ectomycorrhizal fungus Laccaria proxima. It exhibits a range of fungus-interacting traits which reveal its propensity to actively interact at fungal interfaces. Here, we present the approximately 11.5-Mb (G+C content, 61.52%) draft genome sequence of B. terrae BS001 with the aim of providing insight into the genomic basis of its ecological success in fungus-affected soil settings.     

Apatite as a P source in mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings

The objectives of the study are firstly to test the ability of ectomycorrhizal pine seedlings to use apatite as a P source in comparison with non-mycorrhizal pine seedlings and secondly, to determine if there is a relation between exudation of organic acids and the ability to use apatite as a P source. Non-mycorrhizal Pinus sylvestris (L.) seedlings and seedlings ectomycorrhizal with 4 different isolates of ectomycorrhizal fungi were grown for 220 days in sand/peat filled pots with apatite (Ca₅(F,OH)(PO₄)₃) as the sole P source. In an additional experiment, non-mycorrhizal Pinus sylvestris (L.) seedlings and seedlings ectomycorrhizal with 2 different isolates of ectomycorrhizal fungi were grown without any P source for 250 days. All other nutrients were supplied in a balanced nutrient solution.Ectomycorrhizal seedlings grew less than non-mycorrhizal seedlings but ectomycorrhizal seedlings produced a large external mycelium not included in the biomass estimates. All seedlings in the present study had low shoot:root ratios compared to seedlings growing under optimal conditions. All seedlings grown with apatite as P source had higher foliar P concentrations (0.71–2.11 mg/g) than seedlings growing without any P source (0.57–0.75 mg/g) indicating a significant ability to use apatite as a P source. Seedlings colonized by Suillus variegatus and Paxillus involutus had higher concentrations and total contents of P in shoots compared with non-mycorrhizal seedlings, indicating significant improvement of P uptake by these fungi in comparison with non-mycorrhizal seedlings or seedlings colonized Piloderma croceum.No clear relationship between exudation of organic acids and uptake of P was found. Seedlings colonized by S. variegatus reduced the pH of the soil more than seedlings colonized by P. involutus or non-mycorrhizal seedlings. It is suggested that S. variegatus colonization improves the P uptake by reducing the pH of the soil while P. involutus improves P uptake by having a greater ability to absorb dissolved phosphate than non-mycorrhizal roots or roots colonized by the other fungi used in the study.     

外生菌根对干旱胁迫的响应

从外生菌根真菌、外生菌根共生体以及外生菌根的间接作用等方面阐述外生菌根如何抵制干旱胁迫,并对未来我国外生菌根的研究提出了建议。干旱可以抑制外生菌根真菌的生长并降低其群落中真菌的多样性,干旱胁迫下外生菌根真菌子实体可以利用深度30cm以下的土壤水,子实体的表面积和体积比可作为筛选抗旱真菌的一个重要因子;在遭受干旱胁迫时,外生菌根共生体可以发生形态变化来应对干旱,同时增加了植株水分的吸收并改善了植物的光合作用、活性氧以及激素等相关代谢;外生菌根对植物生长的促进作用、增加土壤碳汇以及对其他根际微生物生长的促进作用等对宿主植物应对干旱胁迫有利。未来我国外生菌根研究应加强对干旱区优良菌-树组合的筛选工作,同时加大对乡土外生菌根真菌资源的调查力度,未来研究应重点向分子生物学领域推进。     

The effect of soil pH on the ability of ectomycorrhizal fungi to increase the growth of Eucalyptus globulus Labill.

We examined the effect of two levels of soil pH (5 and 6) on the ability (effectiveness) of ectomycorrhizal fungi to increase the growth of Eucalyptus globulus Labill. at a deficient supply of P. Plants were inoculated with one of six fungal isolates [Laccaria laccata (Scop. ex Fr.) Berk. and Br. (isolates A and B), Pisolithus tinctorius (Pers.) Coker and Couch (isolates A and B), Descolea maculata Bough. and Mal. and Setchelliogaster sp. nov.] and were grown in a P-deficient sand, in pots, in a temperature-controlled glasshouse. Seedlings were harvested 89 days after planting and were assessed for dry matter production, tissue P concentrations, ectomycorrhizal colonization of roots and hyphal development in soil.Uninoculated plants had less than 5% of their fine root length colonized by ectomycorrhizal fungi. In contrast, inoculated plants had 30% or greater of their fine root length ectomycorrhizal. Inoculation increased the uptake of P and growth of plants for all isolates and at both levels of soil pH, although growth responses to inoculation were greater at pH 6, particularly for the two L. laccata isolates. Isolates which colonized roots most extensively increased plant growth to the greatest extent. D. maculata was the most effective fungal isolate at pH 5, and both D. maculata and L. laccata A were most effective at pH 6. The effects of soil pH on plant growth were also related to some extent to the effects of soil pH on colonized root length. Growth responses to inoculation were related less well to hyphal development in soil. The L. laccata isolates formed more hyphae in soil (on a per pot, per m of fine root, and per m of colonized fine root basis) than other fungal isolates, but were not always more effective in increasing plant grown.     

接种彩色豆马勃对模拟酸沉降下马尾松幼苗生物量的影响

外生菌根能够提高宿主植物对外界环境胁迫的抵抗力,促进植物的生长,本文试图揭示外生菌根对酸雨胁迫下马尾松生长的保护作用。本研究采用盆栽试验,共设置四个处理：酸雨对照处理（Control check (CK), 约pH值5.5）不接种,酸雨对照处理接种,酸雨pH值3.5处理不接种,酸雨pH值3.5处理接种。pH值3.5的酸雨处理降低马尾松的生物量,在试验前期降低根冠比,试验中后期则提高根冠比,在试验初期增加了叶面积,但中后期显著降低了叶面积。接种外生菌根菌有利于马尾松幼苗的生长,pH值3.5处理下接种外生菌根菌能提高马尾松幼苗的生物量,外生菌根菌对生物量分配和叶面积的影响与酸雨胁迫的影响是相反的,即外生菌根菌抵消了酸雨胁迫对马尾松的影响。     

Gas exchange and growth responses of ectomycorrhizal Picea mariana, Picea glauca, and Pinus banksiana seedlings to NaCl and Na2SO4

Abstract: Black spruce (Picea mariana), white spruce (Picea glauca), and jack pine (Pinus banksiana) seedlings were inoculated with Hebeloma crustuliniforme or Laccaria bicolor and subjected to NaCl and Na₂SO₄ treatments. The effects of ectomycorrhizas on salt uptake, growth, gas exchange, and needle necrosis varied depending on the tree and fungal species. In jack pine seedlings, ectomycorrhizal (ECM) fungi reduced shoot and root dry weights and in the ECM white spruce, there was a small increase in dry weights. Sodium chloride treatment reduced net photosynthesis and transpiration rates in the three studied tree species. However, NaCl-treated black spruce and jack pine colonized by H. crustuliniforme maintained relatively high photosynthetic and transpiration rates and needle necrosis of NaCl-treated black spruce seedlings was reduced by the ECM fungi. Higher concentrations of Na+ were found in shoots compared with roots of the three examined conifer species. ECM fungi reduced the concentrations of Na+ mainly in the shoots and this reduction was greater in plants treated with NaCl compared with Na₂SO₄. Shoots contained generally higher concentrations of Cl^- compared with roots. In the NaCl-treated black spruce and white spruce, both ECM species significantly reduced Cl^- concentrations. Our results point to overall greater phytotoxicity of NaCl compared with Na₂SO₄ and support our earlier findings which demonstrated beneficial effects of ECM fungi for woody plants exposed to NaCl stress.     

Effect of ectomycorrhizal fungi on chestnut ink disease

 Seedlings of Castanea sativa were inoculated at transplanting time with four ectomycorrhizal (ECM) fungi, Laccaria laccata, Hebeloma crustuliniforme, H. sinapizans and Paxillus involutus. At the end of the first vegetative season, 7 months after sowing, half of the mycorrhizal and nonmycorrhizal seedlings were challenged with a zoospore suspension of Phytophthora cambivora and the other half with P. cinnamomi. Five months later, mycorrhizal plants infected with P. cambivora or P. cinnamomi showed no sign of pathogen infection. The ECM fungi increased plant biomass also in the presence of the pathogen. Mycorrhizal seedlings inoculated with the pathogens showed greater shoot and root development than nonmycorrhizal chestnut plants. All the fungi tested reduced the negative effect of the ink disease pathogens on the plant host in vivo. The mechanisms by which the ECM fungi protect chestnut seedlings are discussed. Accepted: 20 May 1999     

外生菌根研究及应用的回顾与展望

简要回顾了一百多年来外生菌根研究史;综述了外生菌根资源调查。分类鉴定,生态,生理,分子生物学以及生物技术研究与应用诸方面的阶段性成果和进展,通过对外生菌根研究与应用现状的归纳与分析,就我国菌根研究与应用等方面存在的主要问题提出了以下4点建议;1,加强菌根资源的系统调查;确认对应植物属种,进行编目与建立数据库以及菌根DNA文库。2,采集与收集菌根真菌,进行分离,培养,繁育与保藏,建立菌根真菌菌种库和DNA文库,3,加强菌根技术研究,侧重研究造林树种菌根化育苗和造林的新技术,新工艺,4,改革传统的育苗与造林的方式,方法,选育对应的优良菌根真菌菌株应用于育苗造林;制定相关的政策,法规以规范和推广菌根技术。     

From field sampling to pneumatic bioreactor mycelia production of the ectomycorrhizal mushroom Laccaria trichodermophora

In order to increase survival rates of greenhouse seedlings destined for restoration and conservation programs, successful mycorrhization of the seedlings is necessary. To reforest forest ecosystems, host trees must be inoculated with ectomycorrhizal fungi and, in order to guarantee a sufficient supply of ectomycorrhizal inoculum, it is necessary to develop technologies for the mass production of ectomycorrhizal fungi mycelia. We selected the ectomycorrhizal fungus Laccaria trichodermophora, due to its ecological traits and feasible mycelia production in asymbiotic conditions. Here, we report the field sampling of genetic resources, as well as the highly productive nutritional media and cultivation parameters in solid cultures. Furthermore, in order to achieve high mycelial production, we used strain screening and evaluated pH, carbon source concentration, and culture conditions of submerged cultures in normal and baffled shake flasks. The higher productivity culture conditions in shake flasks were selected for evaluation in a pneumatic bioreactor, using modified BAF media with a 10 g/L glucose, pH 5.5, 25 °C, and a volumetric oxygen transfer coefficient (K_La) of 36 h⁻¹. Under those conditions less biomass (12–37 %) was produced in the pneumatic bioreactor compared with the baffled shake flasks. This approach shows that L. trichodermophora can generate a large biomass concentration and constitute the biotechnological foundation of its mycelia mass production.     

Waiting for fungi: the ectomycorrhizal invasion of lowland heathlands

1.  In England, the loss of lowland heathland, a habitat of global conservation importance, is primarily due to the invasion of birch and pine. This encroachment has been researched in depth from a plant perspective but little is known about the role of mycorrhizal fungi. In lowland heathlands the resident dwarf shrubs form ericoid mycorrhizas whereas invading trees form ectomycorrhizas. Therefore, tree encroachment into heathlands can be regarded as the replacement of a resident mycorrhizal community by an invading one.
2 . This study examined how fungi form mycorrhizas with Betula and Pinus in lowland heathlands. We addressed the question of whether there are mycorrhizal fungi that mediate invasion using a molecular ecology approach to compare the mycorrhizal inoculum potential of soil at three levels of invasion (uninvaded heathland, invaded heathland and woodland) and the fungi forming mycorrhizas on tree seedlings and trees across diverse sites.
3.  We show that in lowland heathlands: (i) seedlings have severely limited access to ectomycorrhizal fungi relative to woodlands, (ii) there are few keystone spore-dispersed ectomycorrhizal fungi that can mediate tree invasion, (iii) tree seedlings can remain non-mycorrhizal for at least one year when no inoculum is present, even near saplings, and (iv) mycorrhizal seedlings achieve greater biomass than non-mycorrhizal seedlings. Within uninvaded heathland we detected only Rhizopogon luteolus , Suillus variegatus , S. bovinus ( Pinus symbionts) and Laccaria proxima (primarily a Betula symbiont).
4. Synthesis . Overall, ectomycorrhizal inoculum in lowland heathlands is rare; most tree seedlings growing in heathland soil are not mycorrhizal due to limited spore dispersal, poorly developed spore banks and weak common mycorrhizal networks. These seedlings can persist awaiting mycorrhization to boost their growth.     

Nitrous oxide production by the ectomycorrhizal fungi Paxillus involutus and Tylospora fibrillosa

Nitrous oxide (N(2)O) production by filamentous fungi has been demonstrated in pure culture and has been estimated indirectly in soils. However, it is unknown whether ectomycorrhizal fungi can also produce N(2)O. We demonstrate for the first time the ability of nitrogen (N)-tolerant ectomycorrhizal fungi (Paxillus involutus and Tylospora fibrillosa), found in forest soils under moderate to high rates of N deposition, to produce N(2)O from nitrate reduction. The N(2)O concentrations from the ectomycorrhizal fungal treatments after a 10-day pure culture experiment were 0.0117±0.00015 (P. involutus) and 0.0114±0.0003 (T. fibrillosa), and 0.0114±0.00043 μmol N(2)O L(-1) from a known fungal denitrifier (Fusarium lichenicola). No N(2)O was detected in the control treatment. Our results indicate the potential for these two N-tolerant ectomycorrhizal fungi to contribute to N(2)O production. Given that these species are abundant in many forest soils, the strength and regulation of fungal N(2)O production should now be verified in situ.     

The fungal sheath of ectomycorrhizal pine roots: an apoplastic barrier for the entry of calcium,magnesium, and potassium into the root cortex?

The apoplastic permeability of the fungal sheath of two different ectomycorrhizal associations of Pinus sylvestris L. was analysed by laser microprobe mass analysis (LAMMA) and energy-dispersive X-ray spectroscopy (EDXS) after stable isotope labelling with 25Mg, 41K and 44Ca. Entry of 25Mg and 44Ca into the outer cortical apoplast of non-mycorrhizal roots was detected after 4 min of labelling. After a longer exposure time the endodermis with its Casparian band acted as an efficient apoplastic diffusion barrier for the radial movement of 25Mg and 44Ca into the stele. A fraction of approximately one-third of the apoplastic cations of the root cortex could not be exchanged against the external label even after longer exposure times. The ectomycorrhizal sheath of the two fungal species used, Pisolithus tinctorius (Pers.) Coker & Couch and Suillus bovinus (L. ex Fr.) Kuntze, does not completely inhibit the apoplastic movement of ions into the mycorrhizal root cortex, but retarded the penetration of isotopes into the cortical apoplast. In roots inoculated with S. bovinus, a clear labelling of the cortical apoplast could first be detected after 24 h of exposure to the stable isotope solution. At this time the labelling of the cortical apoplast in these mycorrhizal roots was higher than those of non-mycorrhizal roots and, with EDXS, changes in the element composition of the apoplast were detected. The results indicated that possibly hydrophobins localized in the fungal cell wall might be involved in the increased hydrophobicity of mycorrhizal roots and the lower permeability of the ectomycorrhizal sheath.     

The effects of Laccaria proxima and fibrous pulp waste on the growth of nine container-grown conifer seedling species

 Japanese larch (Larix kaempferi), white spruce (Picea glauca), black spruce (Picea mariana), red spruce (Picea rubens), jack pine (Pinus banksiana), mugo pine (Pinus mugo), red pine (Pinus resinosa), Japanese black pine (Pinus thunbergii) and Douglas-fir (Pseudotsuga menziesii var. menziesii), were inoculated to test the effective host range of the ectomycorrhizal fungus Laccaria proxima and the possibility of utilizing pulp waste as a potting medium for containerized seedling production. Laccaria proxima tended to improve the container growth of Japanese black pine and white spruce, and significantly improved that of jack pine, mugo pine, black spruce, red spruce and Douglasfir. The growth of red pine and Japanese larch were only slightly improved with L. proxima. Pulp waste (33% by volume) had negative effects on tree seedling growth, except for Douglasfir (no significant effect). The interactions of Laccaria proxima and pulp waste varied; the hosts were significantly positive (P<0.01) in the case of jack pine and black spruce, but there was no significant effect for the rest. Negative effects were found with Japanese black pine. Use of pulp waste in seedling production of jack pine, black spruce, mugo pine, red spruce and Douglasfir inoculated with L. proxima and of Japanese black pine both with and without L. proxima is feasible, but further research is necessary to determine the optimal percentage of pulp waste that can be utilized in seedling production of tree species and the field performance of these seedlings. Accepted: 30 August 1995     

Ectomycorrhizal development on Scots pine (Pinus sylvestris L.) seedlings in different soils

In the present study ectomycorrhizal development of Laccaria bicolor, Rhizopogon luteolus and Suillus bovinus associated with Scots pine (Pinus sylvestris) seedings was studied as affected by primary stand humus, secondary stand humus, podsolic sandy soil or peat in perspex growth chambers. After 9 weeks, ectomycorrhizal development with S. bovinus was significantly greater in peat and primary stand humus than in secondary stand humus or podsolic sandy soil. Ectomycorrhizal development with R. luteolus in secondary stand humus was higher than in primary stand humus. Degree of ectomycorrhizal development of L. bicolor, R. lutuelus and S. bovinus on Scots pine was related to potassium concentration, organic matter content and pH of the soils suggesting that chemical composition of the soils affects ectomycorrhizal development.     

Compatible and Incompatible Interactions Between Callus Tissue and Mycorrhizal or Pathogenic Fungi

Tissue cultures of Nicotiana tabacum were utilized to investigate the mechanisms associated with host specificity and non-host incompatibility in mycorrhizal and pathogenic fungi. They were tested for expression of resistance to different species of mycorrhizal fungi and to a fungal pathogen of tobacco, Thielaviopsis basicola , by monitoring the production of callose, phenolic compounds and peroxidases in dual cultures. Tobacco cells reacted to the presence of all the mycorrhizal fungi with callose deposits, whereas callose was nearly always absent in tobacco cells inoculated with their pathogen T. basicola. The broad-host range ectomycorrhizal fungi Hebeloma crustuliniforme, Lac-caria laccata and Suilhis granulatus elicited less intense responses than did Hymenoscyphus ericae. The results obtained for phenolic production and peroxidase activity were consistently similar to those obtained for callose deposition. They showed that H. ericae , an endomycorrhizal symbiont of Ericaceae, was highly incompatible with tobacco cells and that the tobacco pathogen T. basicola did not elicit strong reactions in the cells of its host. In this paper, the possibility of utilizing callus cultures as a simple model system to study both the different degrees of compatibility and the early events of recognition between mycorrhizal fungi and their host or non-host plants is discussed.     

Effect of dual inoculation of Douglas fir with the ectomycorrhizal fungus Laccaria laccata and mycorrhization helper bacteria (MHB) in two bare-root forest nurseries

Douglas fir (Pseudotsuga menziesii) seedlings in two bare-root forest nurseries were inoculated with the ectomycorrhizal fungus Laccaria laccata, together or not with one of five mycorrhization helper bacteria isolated from L. laccata sporocarps or mycorrhizas and previously selected by in vitro and glasshouse screenings. With the most efficient MHB isolates, when compared to the control with no bacteria, the percent of mycorrhizal short roots was increased from 60 to 90 or from 80 to 100, depending on the nursery, with inoculation doses as low as 10⁶ living cells per m². A dual inoculum made of calcium alginate beads containing the two microorganisms appears to be a valuable technique for increasing the efficiency of ectomycorrhizal inoculation of planting stocks in forest nurseries.     

The evolution of reproductive isolation in the ectomycorrhizal Hebeloma crustuliniforme aggregate (Basidiomycetes) in northwestern Europe: a phylogenetic approach

Abstract. To reconstruct the evolution of reproductive isolation in the ectomycorrhizal Hebeloma crustuliniforme aggregate (Basidiomycetes), phylogenetic relationships were determined between strains that belong to a clade consisting of nine intercompatibility groups (ICGs, biological species). Four of these nine ICGs are partially compatible and belong to the H. crustuliniforme aggregate. Different levels of partial compatibility have been found between these four ICGs. Between ICGs 3 and 4, 15% of the combinations were compatible. One strain was compatible with all isolates of both ICGs 3 and 4 and also with one isolate of ICG 2. Both a nuclear phylogeny, based on ribosomal IGS sequence data, and a mitochondrial phylogeny, based on a group-I intron located in the large subunit ribosomal RNA gene (LrRNA), were reconstructed. The level of incompatibility was compared with the phylogenetic history of individuals belonging to this clade.
Different relationships were found between the level of compatibility and the relative age of the most recent common ancestor (MRCA) for different ICGs. On the one hand, the evolution of incompatibility between ICGs 2 and 3/4 is most consistent with the class of "divergence- first" models because a positive correlation was found between the relative age of the MRCA and the level of incompatibility for ICG 2 versus 3/4. On the other hand, the lack of such a correlation for ICGs 3 and 4 shows that (partial) incompatibility between these ICGs has arisen without strong divergence. The ecological (and to a lesser extent geographical) differences found between ICGs 3 and 4 suggest that selection for incompatibility, associated with host tree preference, has been important in the evolution of incompatibility between these two ICGs. The incongruence between the nuclear and mitochondrial trees for ICG 1 could be explained by a hybrid origin of this ICG, with different donors of the mitochondrial and nuclear sequences.