Research Article |
Corresponding author: Celeste Santos-Silva ( css@uevora.pt ) Academic editor: Francesco Dal Grande
© 2021 Celeste Santos-Silva, Rogério Louro, Bruno Natário, Tânia Nobre.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Santos-Silva C, Louro R, Natário B, Nobre T (2021) Lack of knowledge on ecological determinants and cryptic lifestyles hinder our understanding of Terfezia diversity. MycoKeys 84: 1-14. https://doi.org/10.3897/mycokeys.84.71372
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Developing below the soil surface desert, truffles are hard to find. Within Terfezia genus, at least 18 species are described and many are endemic to the Mediterranean basin. Ecological and geographic information are key factors for species diagnosis, and so far Terfezia species are believed to be linked to either acidic or basic soils or to specific plant hosts. Thus, we have looked at Terfezia diversity within a relatively homogeneous geographical area in Portugal that is suitable for these species and that covered different soils and different dominant host species. We analyzed the observed intraspecific variability within the context of species ecological preferences (e. g. edaphic and putative host). One of our major findings was the discovery of T. grisea in acid soils in association with Tuberaria guttata, a puzzling information since, until now, this species was only found in alkaline soils. We also report on the linkage of different Terfezia lineages within species and ecologic parameters such as soil texture, soil pH and plant host. Additionally, by placing the collected specimens on the most recent genus phylogeny based on the ITS region, we also updated the number of known Terfezia species occurring in Portugal from three to ten. Terfezia dunensis is here reported for the first time for Portugal. Overall, our results show that the exploration of undersampled sites reveals itself as a good strategy to disclose unknown aspects of desert truffle diversity and ecology. These aspects are of prime importance when considering the economic value of the desert truffles for rural populations in the Mediterranean basin.
Desert truffles, host plants, phylogeny, soil properties, taxonomy
Desert truffles produce macroscopic fruitbodies partially or completely embedded in soil. These hypogeous Ascomycota encompass several genera within the Pezizaceae family. Terfezia Tul. & Tul. is the most diverse genera of desert truffle with 18 species described, typically found in arid and semi-arid areas throughout the world (
The interest in understanding diversity and the molecular phylogeny of fungi, in particular of desert truffles, has increased in recent years following up from the increasing importance of biotechnology and plant nutrition. In addition, and for Terfezia, the interest is even higher as the demand for ascocarp availability/production increased. Terfezia products are continuously gaining in relevance as exquisite components of the Mediterranean diet.
Early attempts at Terfezia classification relied on morphological characteristics, such as spore and peridium morphology, gleba colour, and chemical features (
The first step in linking diversity to its geographic and ecological determinants is to know the diversity that we are dealing with. Considered as separated species in pre-molecular era, Terfezia leptoderma (Tul. & C. Tul.) Tul. & C. Tul. and T. fanfani Mattir. are now regarded as one taxa (T. fanfani) since phylogenetic studies show a clear nesting of these species sequences in a well-supported monophyletic group (
Despite all the above contributions, the genus Terfezia is still undergoing frequent taxonomic revaluations. It now seems clear that combined efforts are needed: classic taxonomy, molecular biology and ecology have to be worked synergistically. The lack of available sequences regarding the most cryptic species and the lack of a clear description of its ecological and geographic preferences are still obstacles hindering our understanding of the genus diversity.
As with all other truffles, Terfezia species are obligate symbionts of specific host plants, mainly members of the Cistaceae (
At this point it seems that only through a multidisciplinary approach encompassing molecular, morphological and ecological features will we be able to broaden our understanding of Terfezia diversity. This especially applies in undersampled regions where the probability of discovering new species is favored due to the cryptic lifestyle of Terfezia. Adhering to these these stipulations, we have developed a case study in Portugal, where until 2018 Terfezia richness was greatly overlooked, with only three species documented. Since then, five more Terfezia species have been recorded T. cistophila, T. extremadurensis, T. lusitanica, T. pini and T. solaris-libera (
The sampling took place between 2013 and 2020 from February to June, in the most favorable months for desert truffle growth. The surveys occurred within the framework of two projects (
DNA extraction from the analyzed specimens was performed by CTAB method, following the protocol described in
Based on the most recent published phylogenetic reconstruction using UNITE curate sequences (
An ITS amplified fragment with gaps of 721 bp was aligned, comprising 67 bp of the partial sequence of the 18S ribosomal RNA gene; 228 bp internal transcribed spacer 1; 156 bp of the 5.8S ribosomal RNA gene; 221 bp of the internal transcribed spacer 2; and 49 bp of the 28S ribosomal RNA gene. The reconstructed phylogeny ample supports the existence of 18 distinct clades representing well supported monophyletic groups (Fig.
a Phylogenetic relationship between Terfezia species. The reconstructed phylogeny corresponds to the majority rule consensus tree higher than 0.50 of trees sampled in a Bayesian analysis, and the posterior probability values are shown for main nodes b clades with new sequenced specimens collected within the present study.
Concerning species distribution and representativeness, T. arenaria and T. fanfani were the most widespread and commonly found Terfezia species, being in abundance at every sampling site. All other 7 species seemed to have narrower distribution ranges, however, their stochastic appearance throughout the sampling period made it impossible to confirm their distribution and fructification patterns.
Regarding soil texture, Terfezia species occupied areas dominated by loamy sand soils (lSs) (51%) or sandy loam soils (sLs) (42%), and less frequently pure sandy soils (Ss) (7%). As to the soil pH, values varied from 5.1 to 7.3, with 5.6 the most frequent value, and half of the areas sampled showed pH values between 5.6 and 6.0. In other words, the sampled Terfezia specimens occupy strongly acidic to neutral soils, ranging from sandy to loamy soils (Table
Terfezia preferences relating to host plant and soil (see more details in Suppl. material
Species | Host plant | Soil type | Soil pH |
---|---|---|---|
T. arenaria | Tg | Loamy sand, Sandy loam | 5.2–7.3 |
T. cistophila | Cs, Cl | Loamy sand | 5.5–5.6 |
T. dunensis | Cs, P | Loamy sand | 6.1 |
T. extremadurensis | Tg | Sandy loam | 5.3–6.0 |
T. fanfani | Tg | Loamy sand, Sandy loam, Sandy | 5.1–6.4 |
T. grisea | Tg | Loamy sand, Sandy | 5.7–6.1 |
T. lusitanica | Tg | Loamy sand, Sandy | 5.5–6.2 |
T. pini | Q, P | Sandy loam | 5.3–6.0 |
T. solaris-libera | Tg | Sandy loam | 6.0 |
Despite the observed spatial heterogeneity of the different sampling sites, and the multiple putative plant hosts available, which in some sites included annual plants, Cistus shrubs and either Quercus or Pinus trees, the most frequent putative plant host was Tuberaria guttata (91%) (Suppl. material
While checking for possible relations between the specimen’s position in the reconstructed phylogenetic tree and the recorded ecological parameters, we found that proximity of sampling locations was not an influencing factor to explain the multiple lineages (i.e. subgroups) seen within each clade, since specimens from different locations were often grouped together in almost all the subgroups of a given clade. For instance, T. pini intraspecific variability, as shown by well supported branches in the reconstructed phylogeny (Fig.
Phylogenetic reconstruction of intra-species diversity (Fig.
T. arenaria occupies strongly acid to neutral sandy or loamy soils and its putative host is only T. guttata. In T. arenaria intraspecific reconstructed phylogenetic variability (Fig.
T. fanfani showed a larger range of soil textures and narrow pH soil preferences (Table
This is the first report of T. grisea in this region. More interesting, T. grisea was considered exclusively an alkaline soil species until the present work. We have shown T. grisea presence in moderately to slightly acidic soils, mainly sandy soils and in association to T. guttata (Table
T. lusitanica occurs in strongly to slightly acidic sandy soils exclusively with T. guttata (Table
T. pini occurs in strongly to moderate acid loamy soils associated with Quercus spp. and Pinus spp (Table
The recently described T. solaris-libera occurs in moderate acid loamy soils associated with T. guttata (Table
The nine Terfezia species collected in the present work are illustrated in Fig.
The introduction of the newly collected Terfezia samples on the most recent published phylogenetic reconstruction of the genus (
The comprehensive sampling along eight consecutive years, allowed us to update the existing knowledge on Terfezia species diversity in the region, and expand the number of species occurring in the country to 10 species (i.e. T. alsheikhii, T. arenaria, T. cistophila, T. extremadurensis, T. fanfani, T. grisea, T. lusitanica, T. pini, T. olbiensis and T. solaris-libera sp. nov.). Though Terfezia alsheikhii was only registered once for Portugal (
More importantly, the present work examined the observed intraspecific variability within the context of soil and host preferences. The here achieved better understanding of the edaphic preference and host specificity of the analyzed Terfezia species is of the utmost importance in the framework of desert truffle cultivation. Although we found that the sampling area was not an influencing factor to explain the multiple lineages seen within each clade, we were able to identify some tendencies linking different Terfezia lineages within species to ecologic parameters such as soil texture, soil pH and host plant.
As such, the finding of T. grisea in acid soils is puzzling and contradicts the original species description. Our reconstructed phylogeny suggests a separation between two variants, one associated with alkaline soils and hosted by Pinus spp. and the other with acid soils and linked to T. guttata. Yet, this separation is not clear-cut, since the two existing Spanish sequences associated with Helianthemum spp. were represented in both sub-clades. Further sampling of this species is still needed in order to clarify if this clade represents a group of cryptic species, a single species that is undergoing speciation or a single species that has a wide edaphic tolerance and low host specificity.
The other two species with clear intra-species variability are Terfezia arenaria and T. fanfani, both associated with a higher number of samples. These two species seem to be much more abundant but are also much more conspicuous because of their size. Whether the observed intra-species diversity can be linked to clear ecological preferences remains unknown. For T. arenaria we could observe a grouping tendency based on pH and soil type tolerance. For T. fanfani, differential preferences were also observed on these variables, albeit less defined. In both cases, the intra-specific diversity found in these species calls for a more detailed study including a set of meaningful ecological variables, forest and land management options. Concerning the last, it is reported that macrofungal richness, particularly for mycorrhizal taxa, are shaped by tree canopy density (
Understanding Terfezia diversity and its ecological constraints is highly relevant when considering the economic value of the desert truffles for rural populations on the Mediterranean basin. Desert truffles are a potentially important food source that is highly valued in local markets. A shift from expert collector to cultivation would enhance the socio-economic development of rural and/or local populations. To efficiently mass produce Terfezia one needs to explore the best genotype-host species combination but also learn the growing determinants that lead to a more efficient growth and fruitbodies production. T. arenaria and T. fanfani are by their abundance and size the most promising for cultivation purposes. In fact, most of the other Terfezia species have small size, do not fructify every year and are even harder to find. The attempt to describe its ecology is thus of upmost importance to confirm their identity, distribution and fructification patterns.
The present work attempts, to the best of our knowledge for the first time, to systematically associate the diversity of Terfezia species with soil type, pH and with a putative host plant in a geographically limited sampling area. By doing so, it contributes to our knowledge of the species in the region, increasing the number of species to ten, opening the cultivation possibilities to other species, other host plants and to a wider range of soil types. To notice the first reference of T. grisea in acidic soils. No doubt T. arenaria and T. fanfani are the most found Terfezia species, either by their size, by their abundance or by a combination of both. We need to increase our knowledge on the crucial ecological determinants affecting desert truffles if we want to understand their diversity and cultivation potential.
The authors acknowledge National Funds through FCT - Foundation for Science and Technology under the Project UIDB/05183/2020. This work was supported by the projects PRODER, PA 46134 Produção de Túberas, and Alentejo 2020, ALT20-03-0145-FEDER-000006 Micorrização de Cistus spp. com Terfezia arenaria (Moris) Trappe e sua aplicação na produção de túberas. TN acknowledges the support by “Fundação para a Ciência e Tecnologia”, FCT Portugal (PTDC/ASP-PLA/30650/2017).
Table S1
Data type: Taxonomic, geographical and ecological information
Explanation note: This file discloses all Terfezia sequences falling within each clade on the phylogenetic analysis generated in this work (including the 45 sequences selected from the most recent genus phylogenetic reconstruction and the 216 newly generated sequences from this work) and their respective accession numbers and bibliographic references. A collection/sampling number is also provided for each one of the new sequences pertaining to the samples deposited at the Évora University Herbarium (UEVH-FUNGI).