Mycobionts of <Emphasis Type="Italic">Salix herbacea</Emphasis> on a glacier forefront in the Austrian Alps

Dwarf willows (e.g. Salix herbacea) are among the earliest ectomycorrhizal (EM) plants colonising primary successional sites such as glacier forefronts in the Tyrolean Alps. EM of S. herbacea were sampled at the Rotmoos glacier forefront (Otz Valley, Austria) three times a year during the growing season and once a year during winter when plants were covered with snow in 2005 and 2006. EM were investigated using morphological methods and by sequencing the rDNA ITS region. The degree of EM mycorrhization was high throughout both years (93%). We distinguished 21 EM morphotypes and identified 19 fungal species. Cenococcum geophilum, Sebacina spp., Tomentella spp. and Cortinarius spp. dominated the mycobiont community of S. herbacea. The observed species richness in this about 150-year-old soil was at least 59% of the estimated species richness. Fungal communities differed significantly between consecutive years, and spatial heterogeneity was high. These differences made it difficult to detect seasonal impacts. Abundances of C. geophilum EM increased throughout the 2-year sampling period. Sebacina incrustans EM were very abundant in 2005, but nearly disappeared in 2006, whilst its fruitbodies were still frequent in the sampling area. This suggests that the mycorrhizae were displaced from the roots by an outcompeting species, whereas the mycelium was still present in the soil.     

Tomentella alpina and other tomentelloid taxa fruiting in a glacier valley

Tomentella is a genus of resupinate basidiomycetes usually fruiting on rotten wood. Ecological studies based on molecular methods have reported many Tomentella species as mycobionts of alpine ectomycorrhizal plants, thus highlighting their importance for plant establishment and development under extreme conditions. For the first time, we report fruiting of eight tomentelloid species in an alpine site, and describe Tomentella alpina as a new species. In the rDNA ITS phylogeny, Tomentella alpina forms a distinct clade in the T. stuposa complex, from which it can be clearly separated based on spore size and shape. Closely related taxa are briefly described, and synonymy of Tomentella fungicola with T. stuposa is rejected. Tomentella alpina was found to be one of the most important mycorrhizal partners of Kobresia myosuroides, Bistorta vivipara and Salix herbacea at this alpine site. The mutualistic association with plants is a very successful life strategy for Tomentella spp. growing in primary successional habitats, where the lack of organic matter is generally a growth-limiting factor.     

<Emphasis Type="Italic">Polygonum viviparum</Emphasis> mycobionts on an alpine primary successional glacier forefront

Polygonum viviparum is one of the first ectomycorrhizal (EM) plant species colonising primary successional sites at the Rotmoos glacier forefront (Tyrolean Alps, Austria). On a site with soil development of about 150 years (2,400 m above sea level), mycobionts of P. viviparum were identified by morphotyping and fungal ribosomal deoxyribonucleic acid internal transcribed spacer sequencing. For studying seasonal dynamics and spatial heterogeneity, ectomycorrhizae were sampled on five plots during all seasons. P. viviparum root tips were always EM. In total, 18 mycobiont taxa of the following genera were identified: Cenococcum (1), Cortinarius (2), Helvella (1), Inocybe (3), Russula (1), Sebacina (2), Thelephora (2) and Tomentella (6). All were non-specific EM partners of EM plants. As early as 2 weeks after spring snow melt, EM were well developed, vital and showed high mycobiont diversity. The relative abundance of senescent root tips was lowest in spring and increased throughout the year, with a maximum in winter (frozen soil). Thus, mycobiont growth and physiological activity obviously start when soil is still under snow cover: We speculate that water availability is one important initiation factor for mycorrhizal development under snow cover, when temperatures still range around the freezing point. Irrespectively of the season, most abundant mycobionts at this primary successional site belonged to the genera Tomentella, Sebacina and Cenococcum, also in frozen soil. Spatial heterogeneity was high when considering species composition and diversity indices. Overall mycobionts species richness was restricted at this site, probably because of the limited availability of fungal partners. We regard the presence/absence of fungal partner and limiting abiotic impacts of the environment as key factors for the symbiotic status of P. viviparum. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

白皮松天然林外生菌根土壤繁殖体库

为更好地恢复和保存白皮松天然林,在陕西省白皮松残存林地采集根际土壤,采用幼苗检测法获取土壤外生菌根真菌繁殖体,用形态观察分类与ITS-PCR-sequencing相结合的方法进行菌根鉴定,研究白皮松林地外生菌根真菌土壤繁殖体库的组成.结果表明: 在白皮松幼苗菌根中共获得73个特异性序列;按照97%的相似度阈值,将序列划分为12个可操作分类单元(OTUs);稀疏曲线分析表明,本研究基本获得了白皮松土壤外生菌根繁殖体库的多样性.常见种有土生空团菌、Tomentella sp.、Tuber sp.等.出现频率最高的一个OTU(80%)与已知种类相似度较低(75%),说明其可能是一个新的菌根菌种类.白皮松残存天然林地的外生菌根繁殖体库中具有其他松科植物土壤繁殖体库常见的种类,但是频率最高的种类未能鉴定到已知属或科,说明白皮松菌根繁殖体库具有其宿主特异性.这种群落特异构成也说明研究和利用白皮松土壤外生菌根真菌繁殖体库具有特殊性和重要性.     

Diversity and abundance of resupinate thelephoroid fungi as ectomycorrhizal symbionts in Swedish boreal forests

Resupinate thelephoroid fungi (hereafter called tomentelloid fungi) have a world-wide distribution and comprise approximately 70 basidiomycete species with inconspicuous, resupinate sporocarps. It is only recently that their ability to form ectomycorrhizas (EM) has been realized, so their distribution, abundance and significance as mycobionts in forest ecosystems is still largely unexplored. In order to provide baseline data for future ecological studies of tomentelloid fungi, we explored their presence and abundance in nine Swedish boreal forests in which the EM communities had been analysed. Phylogenetic analyses were used to compare the internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) sequence data obtained from mycobionts on single ectomycorrhizal tips with that obtained from sporocarps of identified tomentelloid fungi. Five species of Tomentella and one species of Pseudotomentella were identified as ectomycorrhizal fungi. The symbiotic nature of Tomentella bryophila, T. stuposa, T. badia and T. atramentaria is demonstrated for the first time. T. stuposa and Pseudotomentella tristis were the most commonly encountered tomentelloid fungi, with the other species, including T. sublilacina, only being recorded from single stands. Overall, tomentelloid fungi were found in five of the studies, colonizing between 1 and 8% of the mycorrhizal root tips. Two of the five sites supported several tomentelloid species. Tomentelloid fungi appear to be relatively common ectomycorrhizal symbionts with a wide distribution in Swedish coniferous forests. The results are in accordance with accumulating data that fungal species which lack conspicuous sporocarps may be of considerable importance in EM communities.     

The potential for mycobiont sharing between shrubs and seedlings to facilitate tree establishment after wildfire at Alaska arctic treeline

Root‐associated fungi, particularly ectomycorrhizal fungi (EMF), are critical symbionts of all boreal tree species. Although climatically driven increases in wildfire frequency and extent have been hypothesized to increase vegetation transitions from tundra to boreal forest, fire reduces mycorrhizal inoculum. Therefore, changes in mycobiont inoculum may potentially limit tree‐seedling establishment beyond current treeline. We investigated whether ectomycorrhizal shrubs that resprout after fire support similar fungal taxa to those that associate with tree seedlings that establish naturally after fire. We then assessed whether mycobiont identity correlates with the biomass or nutrient status of these tree seedlings. The majority of fungal taxa observed on shrub and seedling root systems were EMF, with some dark septate endophytes and ericoid mycorrhizal taxa. Seedlings and adjacent shrubs associated with similar arrays of fungal taxa, and there were strong correlations between the structure of seedling and shrub fungal communities. These results show that resprouting postfire shrubs support fungal taxa compatible with tree seedlings that establish after wildfire. Shrub taxon, distance to the nearest shrub and fire severity influenced the similarity between seedling and shrub fungal communities. Fungal composition was correlated with both foliar C:N ratio and seedling biomass and was one of the strongest explanatory variables predicting seedling biomass. While correlative, these results suggest that mycobionts are important to nutrient acquisition and biomass accrual of naturally establishing tree seedlings at treeline and that mycobiont taxa shared by resprouting postfire vegetation may be a significant source of inoculum for tree‐seedling establishment beyond current treeline.     

Seasonal dynamics of ectomycorrhizal fungus assemblages on oak seedlings in the southeastern Appalachian Mountains

The potential for seasonal dynamics in ectomycorrhizal (EM) fungal assemblages has important implications for the ecology of both the host trees and the fungal associates. We compared EM fungus distributions on root systems of out-planted oak seedlings at two sites in mixed southeastern Appalachian Mountain forests at the Coweeta Hydrologic Laboratory in North Carolina, from samples taken in mid-July and early September. Species level EM fungus type specificity, and identification in some cases, was enabled by direct sequencing of the mycobionts from the seedling roots. Seventy-four EM fungal ITS types were documented, most of which occurred only in the midsummer or early-fall samples, respectively. Cenococcum geophilum (morphotyped) was ubiquitously present and accounted for the majority of root tips sampled. Abundance and relative frequency of types other than C. geophilum were significantly higher in the July samples, while C. geophilum was significantly more frequent and abundant in September. Several generalistic dominants were found fairly equally at both sites and on both sample dates. Other taxa with relatively high frequency were recovered from both sites and tree seedling species, but were reliable indicators occurring primarily in the July sample (e.g., Laccaria cf laccata). Notable shifts in mycobiont dominance were apparent in relation to sample date, including increases in Cortinarius spp. richness, decreases in Thelephoraceae richness, and the disappearance of Amanita spp. types in the early fall compared to midsummer samples. However, diversity and rarity were high and differences in overall community composition (other than C. geophilum) by season were not significant based on multi-response permutation procedures. Although these results based on a single growing season are preliminary, changes in abundance and frequency, detection of significant indicator species, and the apparent systematic affinities of shifting EM types support the potential for seasonal variability in EM associations in this system.     

Ectomycorrhizal fungal communities in late-successional Swedish boreal forests, and their composition following wildfire

This study was conducted to evaluate the effects of wildfires on ectomycorrhizal (EM) fungal communities in Scots pine ( Pinus sylvestris ) stands. Below- and above-ground communities were analysed in terms of species richness and evenness by examining mycorrhizas and sporocarps in a chronosequence of burned stands in comparison with adjacent unburned late-successional stands. The internal transcribed spacer (ITS)-region (rDNA) of mycobionts from single mycorrhizas was digested with three restriction enzymes and compared with an ITS–restriction fragment length polymorphism (RFLP) reference database of EM sporocarps. Spatial variation seemed to be more prominent than the effects of fire on the EM fungal species composition. Most of the common species tended to be found in all sites, suggesting that EM fungal communities show a high degree of continuity following low-intensity wildfires. Species richness was not affected by fire, whereas the evenness of species distributions of mycorrhizas was lower in the burned stands. The diversity of EM fungi was relatively high considering that there were only three EM tree species present in the stands. In total, 135 EM taxa were identified on the basis of their RFLP patterns; 66 species were recorded as sporocarps, but only 11 of these were also recorded as mycorrhizas. The species composition of the below-ground community of EM fungi did not reflect that of the sporocarps produced. EM fungal species present in our ITS–RFLP reference database accounted for 54–99% of the total sporocarp production in the stands, but only 0–32% of the mycorrhizal abundance.     

Fungal colonization of shrub willow roots at the forefront of a receding glacier

Shrub willows (Salix spp.) form associations with arbuscular mycorrhizal (AM), ectomycorrhizal (EM) and dark septate endophytic (DSE) fungi. Willow root colonization by these three types of fungi was studied on a deglaciated forefront of Lyman Glacier, Washington, USA. Root colonization was low; less than 1% of the root length was colonized by AM and 25.6% by DSE. EM colonized 25% of the root tips and 19.4% of the root length. AM and DSE colonization were not related to distance from the present glacier terminus or to canopy cover. EM colonization increased with distance from the glacier terminus based on gridline intercept data but not on root tip frequency data. Availability of propagules in the substrate was low, but numbers of propagules increased with distance from the glacier terminus. The EM communities were dominated by three ascomycetes showing affinity to Sordariaceae in BLAST analyses. Other frequent taxa on the glacier forefront included species of Cortinariaceae, Pezizaceae, Russulaceae, Thelephoraceae and Tricholomataceae. When occurrence of individual taxa was used as a response variable to canopy cover, distance from the glacier terminus, and their interaction, four different fungal guilds were identified: 1) fungi that did not respond to these environmental variables; 2) fungi that occurred mainly in intercanopy areas and decreased with distance from the glacier terminus; 3) fungi that were insensitive to canopy cover but increased with distance from the glacier terminus; 4) fungi that occurred mainly under willow canopies and increased with distance from the glacier terminus. We suggest that fungal colonization is mainly limited by fungal propagule availability. Environmental conditions may also limit successful establishment of plant-fungus associations. We propose that the four EM guilds partly explain successional dynamics. The initial EM community comprises fungi that tolerate low organic matter and nitrogen environment (first and second guilds above). During later community development, these fungi are replaced by those that benefit from an increased organic matter and nitrogen environment (third and fourth guilds above).     

Hyperdiversity of ectomycorrhizal fungus assemblages on oak seedlings in mixed forests in the southern Appalachian Mountains

Diversity of ectotrophic mycobionts on outplanted seedlings of two oak species (Quercus rubra and Quercus prinus) was estimated at two sites in mature mixed forests in the southern Appalachian Mountains by sequencing nuclear 5.8S rRNA genes and the flanking internal transcribed spacer regions I and II (ITS). The seedlings captured a high diversity of mycorrhizal ITS-types and late-stage fungi were well represented. Total richness was 75 types, with 42 types having a frequency of only one. The first and second order jackknife estimates were 116 and 143 types, respectively. Among Basidiomycetes, tomentelloid/thelephoroid, russuloid, and cortinarioid groups were the richest. The ascomycete Cenococcum geophilum was ubiquitously present. Dominant fungi included a putative Tuber sp. (Ascomycetes), and Basidiomycetes including a putative Craterellus sp., and Laccaria cf. laccata. Diversity was lower at a drier high elevation oak forest site compared to a low elevation mesic cove--hardwood forest site. Fungal specificity for red oak vs. white oak seedlings was unresolved. The high degree of rarity in this system imposes limitations on the power of community analyses at finer scales. The high mycobiont diversity highlights the potential for seedlings to acquire carbon from mycelial networks and confirms the utility of using outplanted seedlings to estimate ectomycorrhizal diversity.     

Rangewide analysis of fungal associations in the fully mycoheterotrophic Corallorhiza striata complex (Orchidaceae) reveals extreme specificity on ectomycorrhizal Tomentella (Thelephoraceae) across North America

Fully mycoheterotrophic plants offer a fascinating system for studying phylogenetic associations and dynamics of symbiotic specificity between hosts and parasites. These plants frequently parasitize mutualistic mycorrhizal symbioses between fungi and trees. Corallorhiza striata is a fully mycoheterotrophic, North American orchid distributed from Mexico to Canada, but the full extent of its fungal associations and specificity is unknown. Plastid DNA (orchids) and ITS (fungi) were sequenced for 107 individuals from 42 populations across North America to identify C. striata mycobionts and test hypotheses on fungal host specificity. Four largely allopatric orchid plastid clades were recovered, and all fungal sequences were most similar to ectomycorrhizal Tomentella (Thelephoraceae), nearly all to T. fuscocinerea. Orchid-fungal gene trees were incongruent but nonindependent; orchid clades associated with divergent sets of fungi, with a clade of Californian orchids subspecialized toward a narrow Tomentella fuscocinerea clade. Both geography and orchid clades were important determinants of fungal association, following a geographic mosaic model of specificity on Tomentella fungi. These findings corroborate patterns described in other fully mycoheterotrophic orchids and monotropes, represent one of the most extensive plant-fungal genetic investigations of fully mycoheterotrophic plants, and have conservation implications for the >400 plant species engaging in this trophic strategy worldwide.     

Significant difference in mycorrhizal specificity between an autotrophic and its sister mycoheterotrophic plant species of Petrosaviaceae

Petrosaviaceae is a monocotyledonous plant family that comprises two genera: the autotrophic Japonolirion and the mycoheterotrophic Petrosavia. Accordingly, this plant family provides an excellent system to examine specificity differences in mycobionts between autotrophic and closely related mycoheterotrophic plant species. We investigated mycobionts of Japonolirion osense, the sole species of the monotypic genus, from all known habitats of this species by molecular identification and detected 22 arbuscular mycorrhizal (AM) fungal phylotypes in Archaesporales, Diversisporales, and Glomerales. In contrast, only one AM fungal phylotype in Glomerales was predominantly detected from the mycoheterotrophic Petrosavia sakuraii in a previous study. The high mycobiont diversity in J. osense and in an outgroup plant, Miscanthus sinensis (Poaceae), indicates that fungal specificity increased during the evolution of mycohetrotrophy in Petrosaviaceae. Furthermore, some AM fungal sequences of J. osense showed >99 % sequence similarity to the dominant fungal phylotype of P. sakuraii, and one of them was nested within a clade of P. sakuraii mycobionts. These results indicate that fungal partners are not necessarily shifted, but rather selected for in the course of the evolution of mycoheterotrophy. We also confirmed the Paris-type mycorrhiza in J. osense.     

Lichen responses to nitrogen and phosphorus additions can be explained by the different symbiont responses

? Responses to simulated nitrogen (N) deposition with or without added phosphorus (P) were investigated for three contrasting lichen species - the N-sensitive Alectoria sarmentosa, the more N-tolerant Platismatia glauca and the N(2) -fixing Lobaria pulmonaria- in a field experiment. ? To examine whether nutrient limitation differed between the photobiont and the mycobiont within the lichen, the biomass responses of the respective bionts were estimated. ? The lichenized algal cells were generally N-limited, because N-stimulated algal growth in all three species. The mycobiont was P-limited in one species (A. sarmentosa), but the growth response of the mycobionts was complex, as fungal growth is also dependent on a reliable carbon export from the photobiont, which may have been the reason for the decrease of the mycobiont with N addition in P. glauca. ? Our findings showed that P availability was an important factor when studying effects of N deposition, as P supply can both mitigate and intensify the negative effects of N on epiphytic lichens.     

Ectomycorrhizal transfer of amino acid-nitrogen to the alpine sedge Kobresia myosuroides

Previous work in the Colorado alpine ecosystem has shown that amino acids are a potentially important N source for the sedge, Kobresia myosuroides . This plant is the only known sedge to harbour associations with ectomycorrhizal fungi. The aim of the present work was to test the hypothesis that these ectomycorrhizas transfer N from amino acids in the soil solution to the host plant, and thereby have an important role in the N nutrition of this species. We used a two-chamber system (rhizoboxes) in which K. myosuroides plants were separated from a soil chamber by nylon mesh that allowed fungal hyphae, but not plant roots, to cross it. Injections of [¹⁵N, 2-¹³C]glycine were made into the soil chamber. The hyphal crossings on half of the rhizoboxes were regularly disrupted to control for leakage of label across the barrier. Plants in the intact rhizoboxes showed significantly higher ¹⁵N enrichment than those in controls, and mycorrhizal root tips were significantly more enriched than bulk roots. The mycorrhizas transferred an average of 1.3% of the added ¹⁵N label to plants, a figure comparable to those obtained in previous studies in which plant roots were directly exposed to label. We conclude that fungal associations have an important role in the N nutrition of K. myosuroides by transferring N from amino acids to their hosts.     

Ectomycorrhizal fungus communities of Quercus liaotungensis Koidz of different ages in a northern China temperate forest

Ectomycorrhizal (ECM) fungal communities of Quercus liaotungensis of different ages (seedlings, young trees and mature trees) in the growing seasons (June and September) between 2007 and 2009 were studied in a temperate forest of northern China. A total of 66 ECM fungal taxa were identified based on ECM morphotyping, PCR-RFLP, and DNA sequence data. Of these fungal taxa, 51 were Basidiomycetes (77.3%) and 15 were Ascomycetes (22.7%). Cenococcum geophilum was the dominant species. Thelephoraceae (16 taxa), Sebacinaceae (12 taxa) and Russulaceae (seven taxa) were the most species-rich and abundant ECM fungi, accounting for 19.5%, 17.6% and 8.3% of the total ECM root tips, respectively. Results of multiple response permutation procedure (MRPP) analysis indicated that there were marginally significant effects of tree ages (A?=?0.01801, P?=?0.054) and growing seasons (A?=?0.01908, P?=?0.064) on the ECM fungal species composition of Q. liaotungensis in a temperate forest.     

嵩草属地理分布的研究

嵩草属Kobresia Willd.隶属于莎草科,全世界有64种5变种,中国有49种4变种,属下分为4个组。该属主要分布于北半球温带至寒带,亚洲种类最多,主要集中分布于喜马拉雅山地区和横断山地区。上述两地共有总数的90％以上的种类。因此,喜马拉雅-横断山地区为嵩草属的分布中心。与嵩草属最近缘的属Schoenoxiphium只分布在马达加斯加和非洲东南部山地。两个属可能有共同的祖先,发生于冈瓦纳古陆。随着印度板块与非洲大陆分离并向北方漂移,嵩草属的祖先被带到欧亚大陆,在两个板块相遇处——喜马拉雅-横断山地区产生了现在的嵩草属。其后,喜马拉雅山脉进一步抬升,气候与环境发生巨变,嵩草属也进一步分化形成现在的规模。印度板块在早第三纪与欧亚大陆相连接,嵩草属可能就是此时起源于喜马拉雅山地区,并开始分化,且沿北半球的山系向北扩散到欧洲和西伯利亚,又从欧洲到格陵兰再到加拿大东部,从西伯利亚通过白令海峡到阿拉斯加并沿落基山脉南下达到美国的科罗拉多,形成了嵩草属现今的分布格局。     

Significant diversity and potential problems associated with inferring population structure within the Cenococcum geophilum species complex

Cenococcum geophilum is perhaps the most widely distributed and most recognized ectomycorrhizal fungus with a host range of more than 200 tree species from 40 genera of both angiosperms and gymnosperms. We conducted a phylogenetic analysis on a large collection of isolates (n=74) from North America and Europe based on glyceraldehyde 3-phosphate dehydrogenase (gpd). A subset of isolates (n=22) also was analyzed with the more conservative LSU-rDNA locus. Significant nucleotide diversity was detected (approximately 20%) in the gpd region and the LSU-rDNA analysis supported that the C. geophilum isolates studied were monophyletic but distinct from two isolates, Am5-1 and N2-10, which previously were used in population genetic studies of this species. These results suggest that Am5-1 and N2-10 are likely two undescribed species or even genera. Our results suggest that C. geophilum sensu lato is a species complex and support previous molecular, physiological and morphological studies that have shown significant diversity in C. geophilum. This study also revealed that caution is advised when conducting population genetic studies in C. geophilum due to the possibility of pooling unrelated isolates. This potential problem also has implications for other fungal taxa because cryptic species routinely have been found in recent years based on molecular data.     

Symbiotic seed germination and evidence for <Emphasis Type="Italic">in vitro</Emphasis> mycobiont specificity in <Emphasis Type="Italic">Spiranthes brevilabris</Emphasis> (Orchidaceae) and its implications for species-level conservation

Orchid–mycobiont specificity in the Orchidaceae was considered controversial and not well understood for many years. Differences in mycobiont specificity during germination in vitro vs in situ have lead some to consider orchid–mycobiont specificity as being generally low; however, others have suggested that specificity, especially in vitro, is surprisingly high. Mycobiont specificity may be genus or species specific. An in vitro symbiotic seed germination experiment was designed to examine mycobiont specificity of the endangered Florida terrestrial orchid Spiranthes brevilabris using mycobionts isolated from both the study species and the endemic congener Spiranthes floridana. In a screen of mycobionts, isolates Sflo-305 (99.5%), Sflo-306 (99.5%), and Sflo-308 (89.9%) (originating from S. floridana) supported higher initial (stage 1) seed germination than isolate Sbrev-266 (32.4%) (originating from S. brevilabris) after 3 wk culture. However, only isolate Sbrev-266 supported advanced germination and protocorm development to stage 5 (53.1%) after 12 wk culture. These findings suggest that S. brevilabris maintains a high degree of mycobiont specificity under in vitro symbiotic seed germination conditions. High orchid–mycobiont specificity in S. brevilabris may be indicative of the rare status of this orchid in Florida.     

Characterization of surface n-alkanes and fatty acids of the epiphytic lichen Xanthoria parietina, its photobiont a green alga Trebouxia sp., and its mycobiont, from the Jerusalem hills.

Surface alkanes and fatty acids from the thalli of the lichen Xanthoria parietina, its photobiont Trebouxia sp., and its mycobiont were analysed by GC-MS. The green alga Trebouxia sp. synthesized mainly unsaturated fatty acids such as (Z,Z,Z)-9,12,15-18 : 3 (Z,Z)-9,12-18 : 2 and (Z)-9-18 : 1, and light alkanes C8-C15 (up to 83% of total n-alkanes). However, the mycobiont contained mainly saturated fatty acids such as hexadecanoic (16 : 0) and octadecanoic acid (18 : 0), and also very long-chain n-alkanes C22-C34. Dehydroabietic acid was found in both lichen and mycobiont. The occurrence of different amounts of n-alkanes and fatty acids in the photobionts and mycobionts of X. parietina was shown for the first time. Lichens collected from different locations in the Jerusalem hills contained n-alkanes ranging in concentration from 187 to 211 mg x (g dry wt)-1; n-alkane concentrations in the photobiont and mycobiont were 17-24 and 215-262 mg x (g dry wt)-1, respectively.     

Symbiotic seed germination of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid

The rapid loss of native orchid habitat throughout ecologically important areas (e.g., Florida) has prompted researchers to develop appropriate plans for the propagation and reintroduction of many native orchid species. Ideally, symbiotic orchid seed germination methods are utilized in the production of orchid seedlings to be used in plant reintroduction programs. In the current study we (1) describe an efficient symbiotic seed germination protocol to germinate seeds of the rare sub-tropical terrestrial orchid Habenaria macroceratitis; (2) discuss the in vitro fungal specificity demonstrated by this species; and (3) describe the effects of three photoperiods (0/24 h, 16/8 h, 24/0 h L/D) on in vitro symbiotic seed germination of H. macroceratitis. Six fungal mycobionts were isolated from both vegetative and flowering plants of H. macroceratitis from two geographically distinct sites. Symbiotic seed germination percent was highest (65.7%) and protocorm development was most advanced (Stage 2) when seeds were cultured with fungal mycobiont Hmac-310. Seeds of H. macroceratitis demonstrated a degree of specificity toward fungal mycobionts isolated from plants originating from the same site where seed was collected. Continual darkness (0/24 h L/D) inhibited initial seed germination (Stage 1; 17.1%), but stimulated subsequent protocorm development (Stage 2; 53.5%). These findings will aid in developing an efficient symbiotic seed germination protocol for the conservation of this rare Florida terrestrial orchid, and may prove useful in the conservation of other sub-tropical terrestrial orchid species.