Halimium as an ectomycorrhizal symbiont: new records and an appreciation of known fungal diversity

Halimium is a genus of Cistaceae, containing a small group of shrub species found in open vegetation types and in degraded forest patches throughout the western and central Mediterranean region. We recently described the morpho-anatomical features of the ectomycorrhizae formed by Scleroderma meridionale on Halimium halimifolium, but the mycorrhizal biology of this host plant genus is still largely unexplored. Here, we report new data on the ectomycorrhizal fungal symbionts of Halimium, based on the collection of sporocarps and ectomycorrhizal root tips in pure stands occurring in Sardinia, Italy. To obtain a broader view of Halimium mycorrhizal and ecological potential, we compiled a comprehensive and up-to-date checklist of fungal species reported to establish ectomycorrhizae on Halimium spp. on the basis of field observations, molecular approaches, and mycorrhiza synthesis. Our list comprises 154 records, corresponding to 102 fungal species and 35 genera, revealing a significant diversity of the Halimium ectomycorrhizal mycobiota. Key ectomycorrhizal genera like Russula, Lactarius/Lactifluus, Amanita, Inocybe, and Cortinarius account for more than half of all mycobionts. A large proportion of Halimium fungal species are shared with other host plants in various ecological settings, suggesting a critical role of common mycorrhizal networks in the function played by this shrub in various Mediterranean ecosystems.


Introduction
Shrublands occupy a specific niche in the Mediterranean biome, with an increasingly appreciated ecological function. In particular, plants occurring in this peculiar environment improve water and light regime, protect soil from erosion, and desertification, and act as "nurse" species for tree seedlings, thus favoring the establishment of late-successional species (https://php.radford.edu/~swoodwar/biomes/?page_id=98). To perform such tasks, the shrubs and small trees that integrate this vegetation system developed adaptations to withstand adverse and stressful conditions such as drought and fire (Rundel and Cowling 2013). The presence of a large number of ectomycorrhizal fungi, mainly associated with Cistaceae Juss., is another common trait of Mediterranean shrublands. The role played by ectomycorrhizal fungi in optimizing plant fitness and increasing nutrients' availability in a wide range of terrestrial ecosystems, especially where cold and dry conditions limit decomposition, is largely appreciated (Smith and Read 1997). Cistus is the dominant ectomycorrhizal host plant in Mediterranean shrublands. Over 250 fungal species belonging to 40 genera have been reported to be associated with Cistus, with 35 host-specific species; members of the Cortinariaceae and Russulaceae make up the most of both generalist and Cistus-specific mycobionts (Comandini et al. 2006;Loizides 2016).
The genus Halimium (Dunal) Spach belongs to the Cistaceae (Page 2017), with 13 accepted species of evergreen  2000); however, both the status of this taxon and that of H. syriacum K. Koch, reported from subalpine levels in Lebanon and Syria, are still unresolved. Halimium is closely related to Cistus; some botanists in the past have considered Halimium species as belonging to Cistus (e.g., Demoly 2006), but the most recent molecular phylogenetic analyses have clearly shown the two genera as distinct Vargas 2005, 2009;Civeyrel et al. 2011). The two genera overlap largely in distribution within the Mediterranean basin although Halimium is restricted to the western part of the floristic region (Civeyrel et al. 2011) (Fig. 1). Halimium species occur usually in open vegetation types, like matorral shrublands and garrigues, but they can also been found in degraded forest patches, at the verges of woods, abandoned fields, pasturelands, and on coastal sandy areas and dry dunes (Zunzunegui et al. 2002(Zunzunegui et al. , 2009). The mycorrhizal biology of Halimium is poorly known. The genus may form ectomycorrhizae and possibly vesicular arbuscular mycorrhizae (Camprubi et al. 2011;Buscardo et al. 2012;Beddiar et al. 2015). To expand the current knowledge of mycorrhizal interactions of Halimium, we started a research program focusing on the isolation and full characterization of the ectomycorrhizae formed by the fungal symbionts associated with Halimium spp. We recently described, for the first time, the morphoanatomical features of an ectomycorrhiza on Halimium, formed by Scleroderma meridionale Demoulin & Malençon on Halimium halimifolium (L.) Willk. (Leonardi et al. 2018). In the current paper, we report new data on Halimium mycobionts, as observed in pure stands occurring in Sardinia, Italy, through both sporocarps and ectomycorrhizal root tip collections. Also, we provide a comprehensive and up-to-date checklist of fungal species reported to establish ectomycorrhizae on Halimium spp. on the basis of field observations, molecular approaches, and mycorrhiza synthesis, a type of information that is widely dispersed in the mycological literature, with no specific account existing on the topic. The data presented here reveal a high diversity of the Halimium ectomycorrhizal mycobiota.

Collecting site and fungal sampling
Sporocarps of ectomycorrhizal fungi were harvested in a costal sandy area (from 39°15′ 17.42″ N, 8°24′ 32.75″ E to 39°15′ 46.07″ N, 8°24′ 46.89″ E and from 58 to 123 m asl) close to Gonnesa, about 70 km west of Cagliari, Sardinia, Italy. Collection surveys were conducted weekly during growing season (October-January) and monthly during the rest of the year, from 2015 through 2019. Sporocarps were photographed in situ and identified on the basis of published descriptions of macroscopic and microscopic characters. Fungal species names retrieved from literature were verified for nomenclatural and taxonomic s y n o n y m s i n I n d e x F u n g o r u m ( h t t p : / / w w w . indexfungorum.org) and MycoBank (http://www. mycobank.org) and current names adopted. The collection site is characterized by extended stands H. halimifolium (Fig. 2) that here occurs practically in pure form. No other potential ECM host plants are present on the site, with the exception of a few scattered Cistus salviifolius L. shrubs. For ectomycorrhizae, 40 soil cores (about 20 × 20 × 20 cm) were excavated randomly in proximity of Halimium shrubs (not underneath sporocarps), at least 5 m apart from each other. Soil cores were immersed overnight in water, and ectomycorrhizal roots were carefully separated under a dissecting microscope. Ectomycorrhizae were classified into morphotypes following the methods and indications of Agerer (1991), and several tips for each type were immediately transferred into 90% EtOH and stored at − 20°C for subsequent DNA analysis or fixed in 2.5% (v/v) glutaraldehyde in 10 mM Na-phosphate buffer (pH 7.2) for morpho-anatomical description of characterizing features. Reference materials for sporocarps and ectomycorrhizae are deposited in CAG, at the collection of the Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy.

Molecular characterization of the fungi
Identification of sporocarps and ectomycorrhizae using a molecular approach was based on PCR amplification and sequencing of the complete internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (Gardes and Bruns 1993). Genomic DNAs of the sporocarps were isolated from 20 mg of each dried sample using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), and the ITS amplifications were performed following the protocol reported by Leonardi et al. (2005). A direct PCR approach was applied to identify ectomycorrhizal tips isolated from soil samples as described by Iotti and Zambonelli (2006). Three ectomycorrhizal tips were selected as PCR targets and directly amplified using ITS1F/ITS4 primer pairs (White et al. 1990;Gardes and Bruns 1993). Two microliters of 20 mg/ml BSA (bovine serum albumin) solution (Fermentas, Vilnius) was added to each reaction tube to prevent PCR inhibition (Leonardi et al. 2013). The amplified products were purified using the QIAquick PCR Purification Kit (Qiagen, Milan, Italy) and sequenced by Eurofins Genomics service (Ebersberg, Germany). Sequence-based fungal identification was performed following the indications and recommendations reported in Hofstetter et al. (2019). Sequences of sporocarps and ectomycorrhizae are deposited in GenBank (https://www. ncbi.nlm.nih.gov/genbank/) under accession numbers specified in Table 1. In selected cases, to confirm the identity of the host shrub roots, the plastid trnL region of ECM root tip DNA was amplified using primer pairs trnC/ trnD following Tedersoo et al. (2008). In these cases, the chloroplast trnL region obtained by PCR amplification of DNA extract from H. halimifolium leaves was used as positive control.

Compiling the list of records
Data on the association between Halimium spp. and ectomycorrhiza-forming fungi presented here are largely   based on reports of field observations of sporocarps associations with potential hosts. The dataset contains both personal collections and observations and information collated from a variety of published and web-based sources. Literature databases available to authors (e.g., Agricola, Scopus, PubMed, ISI Web of Science, ResearchGate) were searched for articles on Halimium and associated mycobionts. Sequences of Halimium ECM fungi were retrieved from either GenBank or UNITE. Fungal taxa belonging to genera for which the mycorrhizal status is currently uncertain were not listed (for a comprehensive list of ectomycorrhizal fungal genera and the criteria used to ascertain mycorrhizal status, see Rinaldi et al. 2008 andComandini et al. 2012). Only records clearly mentioning (potential) Halimium hosts were included in the data matrix (this includes records from mixed Cistus/Halimium stands). Evidence from studies on the morpho-anatomical and/or molecular characterization of ectomycorrhizae formed by fungal species on Halimium spp. were also inserted in the data set, excluding uncultured fungus sequences and fungal species not identified at least at genus level. In addition to studies concerning naturally occurring, field-collected mycorrhizae, data coming from synthesized mycorrhizae were also considered, although it must be stressed that associations induced in laboratory experiments may not occur under field conditions. Despite all efforts to cover as many bibliographic sources as possible, our literature survey might clearly be partial and incomplete. Reports of putative mycorrhizal relationships based solely on sporocarps associations rather than confirmed by direct inspection of ectomycorrhizae are obviously subject to an unquantifiable degree of error, especially when  Vidal et al. 1997 ECM description or report of naturally occurring ectomycorrhizae, and/or sequence isolated from ECM or roots. All other records are about sporocarp collections, if not specified otherwise; sECM description or report of synthesized ectomycorrhizae § For names of taxa and synonymy, we followed Index Fungorum (http://www.indexfungorum.org/) and MycoBank (http://www.mycobank.org). The higher classification system used in this paper is that outlined by Kirk et al. (2008) in the Dictionary of the Fungi *Usually reported as specific or typical Cistus mycobiont (see Comandini et al. 2006) # Previously reported as associated with Cistus (see Comandini et al. 2006;Gelardi et al. 2014 Moreau et al. 2007a more than one potential plant hosts are present (e.g., mixed Halimium stands with Cistus, Pinus, and/or Quercus). Finally, the identification of some fungi in the references that we have considered may not be correct.

Results
The Halimium ectomycorrhizal guild Our effort to gauge the diversity of ectomycorrhizal fungi linked to Halimium, through both direct field sampling and the compendium of literature records, resulted in 154 listed entries, corresponding to 102 species belonging to 35 genera from Ascomycota, Basidiomycota, and Zygomycota (Table 1). This tally excludes a few cases of possible synonymy, e.g., Inocybe halophila R. Heim = I. pruinosa R. Heim and Cantharellus cfr. pallens = C. subpruinosus Eyssart. & Buyck, and the dubious record of Lactarius subdulcis (Pers.) Gray, a known Fagus symbiont whose sequence was probably misbranded and it is likely to be L. hepaticus Plowr. Fiftyseven of the listed records, which refer to 41 different species, were provided by our field work in Halimium plots in southwestern Sardinia, Italy; of these, 29 species are reported here for the first time as linked to Halimium. Most of the ecologically key ectomycorrhizal fungal genera are represented in the list, with Russula (13 species), Amanita (12 spp.), Inocybe (10 spp.), Lactarius/Lactifluus (9 spp., including the synthesized ECMs of L. deliciosus (L.) Gray, see below), and Cortinarius (8 spp.), accounting for more than half of all species. As in other genera of Cistaceae (e.g., Cistus and Helianthemum), it is apparent from the entries in the list that hypogeous ascomycetes make a significant part of the Halimium ectomycorrhizal contingent (Table 1) Fig. 3e), molecularly confirming the identity of host plant (data not shown). Belonging to the Puberulum group, or the so-called whitish truffles, T. oligospermum has been reported previously as a Quercus and Cistus mycobiont (Comandini et al. 2006;Lancellotti et al. 2016), and it is the first Tuber species ever to be proven to form ECMs with Halimium.

Walking hand in hand with Halimium
Some of the mycorrhizal fungi we found associated with Halimium deserve a special mention and further notes. One of the most common ectomycorrhizal fungal species present in our H. halimifolium plots was Scleroderma meridionale (MycoBank MB#323250). Basidiomata of this species are large, globose, characterized by a smooth-to-finely furfuraceous peridium of an intense sulfur yellow color, which becomes brighter in the long pseudostipe, usually immersed deep in the soil (Fig. 4a). The habit is solitary, occasionally gregarious, found mostly in sandy soils and dunes. Originally described on the basis of collections from southern Portugal, continental France, Corsica, and Morocco, it occurs in all the Mediterranean basin, including Greece, Macedonia, and Turkey (Rusevska et al. 2014;Dimou et al. 2016). It is also reported from North America, from Florida to Arizona, and Oregon (Guzmán and Ovrebo 2000;Kuo 2004, http://www. svims.ca/council/Sclero.htm). However, the identity of these collections still awaits confirmation: indeed, preliminary molecular data seem to indicate that the North American "S. meridionale" belongs to a distinct, so far undescribed, taxon (D. Puddu, M. Leonardi, A.C. Rinaldi, unpublished data).
Based on field observations, S. meridionale has been reported as associated with both evergreen and deciduous species of Quercus, Pinus, and also Cistus (Comandini et al. 2006;Phosri et al. 2009;Dimou et al. 2016). We recently described the morpho-anatomical features of the ectomycorrhizae formed by S. meridionale on H. halimifolium, with the help of confocal laser scanning microscopy; the mycobiont and host plant identity from the ECM root tips were verified through molecular tools (Leonardi et al. 2018). This was the first description of an ectomycorrhiza on Halimium. The features of this ECM-felty mantle surface, whitish with silver patches, mantle surface characterized by a network of branched hyphae organized in hyphal bundles, small dimension of mycorrhizal system-are similar to those reported for the few described naturally occurring Scleroderma ECMs and to other ECMs formed by Cistaceae (Leonardi et al. 2018).
Another interesting species recorded in our collection site was Alessioporus ichnusanus (MB#808530). The taxon corresponds to a boletoid species recently segregated from Xerocomus Quél. to typify the new genus Alessioporus Gelardi, Vizzini & Simonini which currently accommodates two species. Its type and epitype were collected in Sardinia in 1980 (Galli 1981) and in 2003 (Gelardi et al. 2014), respectively, in Quercus spp. and Cistus spp. forests, in the province of Medio Campidano, 60 km from our Halimium plots. In addition to the type collections, the taxon has been recorded in different localities in Sardinia, as well as in Sicily, continental Italy (Brotzu 1988;Alessio 1991;Brotzu and Colomo 2009;Gelardi 2010;Illice et al. 2011) and other Mediterranean countries, as France (Eyssartier and Roux 2011), Greece (Polemis et al. 2012), and Spain (Muñoz 2005). Alessioporus ichnusanus can be recognized by the ochraceous-brown, dark olive-brown-to-copper-brown pileus with brownish-black fibrils (Fig. 4b). But one of the most important morphological character that define this species is a narrow pseudo-annulus in the middle part of the stipe formed by the remnants of the connection between the pileus margin and the stipe cortex during the primordial stage (Galli 2013). Currently, Alessioporus ichnusanus is considered an uncommon or rare species that has recently been included in the Red List of Italian macrofungi as an endangered species (Rossi et al. 2013) and in the IUCN red list as vulnerable (Persiani 2019;Angelini et al. 2020).
Keeping with the Boletales, Gyroporus pseudolacteus G. Moreno, Carlavilla, Heykoop, Manjón & Vizzini (MycoBank MB#356882) is an interesting finding. This species has been recently described from a material collected in Spain, on sandy soil under Pinus pinaster (Crous et al. 2016). To the best of our knowledge, this is the first confirmed record of this species not only for Sardinia but also for Italy. Gyroporus pseudolacteus can be distinguished from the common and closely related Gyroporus cyanescens (Bull.) Quél. by its larger habit, the longer stipe in relation to the pileus diameter, and the "deep and persistently indigo blue when handled or bruised" (Crous et al. 2016) (Fig. 4c). According to Vizzini and co-workers, G. cyanescens should be considered a complex of cryptic species (Vizzini et al. 2015) which is being unraveled with the help of molecular tools (see Crous et al. 2017), even though some of these taxa are still being treated as synonyms by some fungal names databases. While this is the first mention of Halimium as the probable host, the putative association of Gyroporus with cistaceous plants is not unprecedented. Although the six (not universally accepted) known European species of Gyroporus-G. ammophilus  (Vizzini et al. 2015;Crous et al. 2016Crous et al. , 2017, G. ammophilus was found along the Atlantic coast of the Iberian Peninsula "on fixed dunes in association with Pinus spp., or, less frequently with other trees and shrubs, such as Quercus suber L. and Cistus salviaefolius (sic!), on sandy soils" (Castro and Freire 1995).
Lactarius hepaticus (MycoBank MB#224000) was, by far, the most common milkcap in our Halimium stands (Fig. 4d). This was an unexpected finding, since this species belonging to the subgenus Russularia is commonly associated with Pinus and, more occasionally, other conifers such as Picea and Pseudotsuga (Heilmann-Clausen et al. 1998;Basso 1999). Uncommon to rare/absent in northern Europe is more frequent in Britain, the Netherlands, and, above all, in the Mediterranean area. This species is characterized by its convex to applanate with a depressed center cap, with liver-brown color (hence the epithet); the milk is white, turning yellow on a tissue (Pierotti 2005). Intriguingly, in central Portugal, L. hepaticus was part of shared ECM networks between understory shrubs and pine trees in a Pinus-dominated forest, being detected with molecular tools on the roots of both Pinus pinaster and Halimium lasianthum and H. ocymoides (Lam.) Willk. (Buscardo et al. 2012). Along the coastal area in Sardinia, where our Halimium plots are situated, Pinus stands are also frequent, originated from extensive reforestation plans carried out during the last century. We collected L. hepaticus in these stands as well, where H. halimifolium is frequent both in the understory and at the edges of the pine forest. Lactarius hepaticus was abundant as sporocarps among Halimium shrubs and on their roots in proximity of Pinus, confirming the existence of shared ECM networks, but it occurred also in pure Halimium plots distant several kilometers from pine trees stands, in areas where Pinus, at the best of our knowledge, has never been present. This suggests colonization of new ECM plant hosts (like Halimium) by L. hepaticus by means other than root networking, such as spore dispersal. More work is underway to investigate L. hepaticus ecological plasticity.
Lactifluus brunneoviolascens (Bon) Verbeken (MycoBank MB#564601) is another uncommon species, reported here for the second time in Sardinia (see Lalli and Pacioni 1992). It was previously named L. luteolus Peck, which is now known to be the correct name for a North American species (Verbeken et al. 2012;De Crop et al. 2017). Both species belong to the newly erected section Phlebonemi (R. Heim ex Verbeken) Verbeken (= Lactarius subsect. Luteoli Pacioni & Lalli) (Verbeken et al. 2012). Lactifluus brunneoviolascens is easily distinguished in the field by the whitish/whitish-cream color of the pileus, with velvety cuticle, dry even in very humid weather, finely crenulated at the edge, stained ocherbrown with age; the context is firm, whitish then darker ocherbrownish, with a sweet taste and an unpleasant fishy smell; the latex is fluid, abundant, opalescent white, immutable if isolated on glass, slowly but strongly browning in contact with the lamellae or on absorbent paper (Fig. 4e). We studied two different collections of this Lactifluus from almost pure H. halimifolium stands. Another collection was recently recorded from southeastern Sardinia, under Quercus (Alberto Mua, personal communication), a more usual habitat (sometimes it also occurs in mixed Quercus-Pinus forests) for this species with a prevalently Mediterranean distribution that prefers dry and sandy soils (Basso 1999;Pierotti 2002).
Among the various Cortinarius species encountered during this study and likely linked to Halimium, Cortinarius coeruleopallescens Contu (MycoBank MB#459976) deserves a mention (Fig. 4f). This taxon, not uncommon in our plots, was described in 1999, when Marco Contu raised to species level a fungus he encountered in Sardinia, and that had been observed by other researchers 2 years earlier in Spain and originally thought to be a variety of C. croceocoeruleus (Pers.) Fr., C. croceocoeruleus var. meridionalis Bidaud, A. Ortega & Mahiques (Ortega et al. 1997;Contu 1999). The collections from both Spain and Sardinia were associated with Cistus, while C. croceocoeruleus is typical of central European coniferous and broadleaved forests. Another species linked to Halimium and worthy of remark is Cortinarius cedretorum var. halimiorum Brotzu & Peintner (Mycobank MB#580057) (Table 1). This beautiful variety in the subgenus Phlegmacium was described (originally reported in a field guide as C. halimiorum; see Brotzu and Colomo 2009) on the basis of material collected on a dune system in the northeastern part of Sardinia (Brotzu and Peintner 2009). Despite the fact that the vegetation system in this case is more complex than the one present in our H. halimifolium stands, with psammophile coastal associations where H. halimifolium is present together with other floristic entities, such as Cistus spp., Pistacia lentiscus L., Juniperus phoenicea L., and Arbutus unedo L. (see Arrigoni 1996), the link between this specific Cortinarius and Halimium was apparent to the researchers (Brotzu and Peintner 2009 Buscardo and colleagues, it is showed that about 30% of the identified ECM fungal species were common to pine and Halimium spp., with shared ECM fungal species representing up to 80% of the total fungal abundance in some stands (Buscardo et al. 2012 (Taudiere et al. 2015).
The ability of Cistaceae to develop common mycelial networks by sharing ECM fungal partners with neighboring plants is a crucial ecological trait that has not been appropriately appreciated. As stressed by Randy Molina and Thomas Horton, "common mycelial networks (CMNs) of mycorrhizal fungi connecting neighboring host plants affect ecosystem processes and community dynamics including seedling establishment, plant succession, and ecosystem resiliency" (Molina and Horton 2015). Based on our current knowledge of Halimium and Cistus ECM communities, it is apparent that these are largely shaped by "ecological specificity" rather than host-specificity. Despite the fact that both genera have host preferential (or even exclusive) fungal partners, large part of their mycorrhizal associations seems rather driven by environmental (soil composition, for example) and biological factors, like the presence of other host plants in the same or neighboring areas, thus going beyond host-fungus genetic compatibility due to co-evolution. Either in pure shrublands, as in our Sardinian stands, or at the edges or in the understory of Quercus and Pinus forests, especially when growing on poor and/or degraded soils, Halimium might thus play a key ecological role, maintaining ECM fungal diversity, favoring vegetation succession and dynamics, and assisting ecosystem resilience following disturbance, thanks to common mycelial networks and possibly spore dispersal of ECM mycobionts. A similar function has been recognized for Cistus. In Spain, many of the fungal species associated with Cistus ladanifer L. were found to be shared with Pinus pinaster Aiton, suggesting a role in the regeneration of Pinus stands after wildfire (Martín-Pinto et al. 2006). In this context, it is relevant to note that Cistus and possibly Halimium are dual-mycorrhizal plants, capable of hosting both arbuscular mycorrhizal and ectomycorrhizal associations. Several early successional ECM hosts like Alnus, Populus, and Salix share this feature. In the Mediterranean ecosystem, Cistaceae definitely play a major role in secondary succession following major disturbances like fire (or even human activity). Benefits deriving from dual-mycorrhizal colonization thus extend from interested plants-endowed with greater survival, growth, and nutrient uptake-to ecosystems, favoring establishment and improving survival on adverse sites of connected ECM plants (Teste et al. 2020).

Conclusions
We are just starting to unveil the complexity of Halimium mycorrhizal biology and ecology, especially for what concerns host-specificity of associated mycobionts and patterns of shared mycorrhizal networks with neighboring host plants. The general poor knowledge of Halimium as an ectomycorrhizal host has led to relatively few records of potentially associated fungal species based on observations of sporocarp occurrence. Hopefully, this and other works will increase the awareness of researchers, providing us in the near future with fresh data coming from fungal forays. Also, wellplanned molecular studies examining mycorrhizal specificity at the root tip scale are bound to disclose many details of the structure and dynamics of Halimium-linked ectomycorrhizal communities in multiple ecological settings.
Funding Open access funding provided by Università degli Studi di Cagliari within the CRUI-CARE Agreement. ANMF was supported by Coordenação de Aperfeiçoamento Pessoal de Nível Superior-Brazil-Finance Code 001 (CAPES-DS and PDSE fellowship grants).
Data availability All associated data are deposited in public repositories.

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