Morchelle delle dune sabbiose della zona costiera dell’Emilia-Romagna (Mar Adriatico nord-occidentale, Italia)
10.6092/issn.2531-7342/9374
Le spugnole (Morchella spp.) sono funghi commestibili apprezzati per le loro ottime caratteristiche culinarie e proprietà medicinali. I loro ambienti di sviluppo si trovano nelle regioni temperate dell'emisfero settentrionale. La tassonomia dei funghi del genere Morchella è sempre stata controversa a causa della loro elevata plasticità fenotipica. La maggior parte delle spugnole è considerata saprotrofa, ma è noto che alcune specie interagiscono con le radici di molte specie vegetali formando diversi tipi di associazioni. In Emilia-Romagna (Italia), le spugnole sono entrate a far parte della tradizione culinaria, specialmente per le popolazioni delle aree costiere. In questo lavoro gli ascomi raccolti lungo le dune costiere dell’Emilia-Romagna sono stati caratterizzati morfologicamente e molecolarmente e sono state verificate le relazioni instaurate con le specie vegetali presenti nella stessa area. Tutti gli ascomi, raccolti dal 2001 ad oggi, condividevano caratteristiche morfologiche simili e la loro variabilità morfologica era dovuta principalmente al periodo di raccolta. Le analisi delle sequenze delle regioni ITS dell’ rDNA, effettuate sugli esemplari raccolti nel 2017, hanno permesso di raggruppare tutti gli ascomi esaminati all'interno del clade Mes-17, del gruppo Esculenta. Cordoni miceliari incrostati di sabbia sono stati trovati connettere la base degli ascomi e le radici di nove diverse specie di piante. La quantità degli ascomi raccolti sulle dune sabbiose costiere dimostra che l'area di studio è particolarmente adatta per la fruttificazione delle spugnole Da questo studio si evince la necessità di proteggere il sistema dunale al fine di preservare la diversità genetica si questi funghi.
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Morels on the sand dunes of the Emilia-Romagna coast (Northwestern Adriatic Sea, Italy)
Text.Serial.Journal
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2019-04-26
2019-03-01
2020-01-20
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Ital J Mycol
Alessandra Zambonelli
2019/05/03
10.6092/issn.2531-7342/9374
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Alma Mater Studiorum - University of Bologna
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White TJ, Bruns TD, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications (Innis MA, Gelfand DH, Sninsky J, White TJ, eds). Academic Press, San Diego, pp 315–322.
Morels on the sand dunes of the Emilia-Romagna coast (Northwestern Adriatic Sea, Italy)
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48
Snabl et al.                                                               Italian Journal of Mycology 48:16-25 (2019)

DOI: https://doi.org/10.6092/issn.2531-7342/9374

Original paper

Morels on the sand dunes of the Emilia-Romagna coast
(Northwestern Adriatic Sea, Italy)
    Martin Snabl1, Urbano Guidori1, Carmelo Gianchino2, Mirco Iotti2, Alessandra Zambonelli3

1
 Italian Mycological Society (UMI), viale Fanin 44, 40127 Bologna, Italy
2
 Department of Health, Life and Environmental Sciences, University of L’Aquila, via Vetoio, Coppito 1, 67100 L’Aquila,
Italy
3
 Department of Agricultural and Food Sciences, University of Bologna, via Fanin 44, 40127 Bologna, Italy

Corresponding author e-mail: mirco.iotti@univaq.it
Received 4/02/2019; accepted 18/04/2019.


Abstract
Morchella species are known as famous and prized edible fungi due to their culinary flavor and medicinal properties. The
asomata are collected throughout the temperate regions of the northern hemisphere. Morchella spp. taxonomy has long
been debated as a result of the high phenotypic plasticity charaterizing the genus. Most morels are considered saprobic
but some species has been reported to interact with roots of many plant species forming different types of associations.
In Emilia-Romagna (Italy), morels became a part of the culinary tradition, especially for the populations of the coastal
areas. This work aimed to describe and identify the ascomata collected on the white dune habitat as well as to verify the
extent of the interaction with plant species growing in the same area. All ascomata collected since 2001 shared a similar
morphology with a range of variability mainly due to the harvesting period. Ascomata collected in 2017 were grouped
within the Mes-17 clade, in the Esculenta group, based on their ITS rDNA sequences. Nine different plant species were
found to be connected with morel ascomata through characteristic sand-mycelial structures never described before. The
amount of the ascomata collected on the sand dunes demonstrates that the study area is particularly suitable for morel
fruiting. Therefore, the protection of the dune system is imperative for preserving Morchella genetic diversity within the
local population.

Key words: Morchella vulgaris; Morchella esculenta; Morchella dunensis; white dunes; host plant


Introduction

True morels (Morchella, Ascomycetes) are among the most valuable edible mushrooms, which also
have medicinal properties (Ajmal et al., 2015). Most of the morels in the market are harvested from
the wild but recent attempts to cultivate these mushrooms were successful (Du et al., 2017). Before
this success, other cultivation strategies were developed around the world but the low reproducibility
and the high costs prevented large-scale cultivation (Peng et al., 2015). Species in the genus
Morchella are collected throughout the temperate regions of the northern hemisphere where they
produce ascomata during a few weeks’ time period in the springtime (Du et al., 2015).
    Taxonomy of morels has long been debated among mycologists due to the high phenotypic
plasticity of their ascomata, which often do not reflect their phylogeny (Kellner et al., 2005).
Moreover, these fungi exhibit a variety of trophic habits as well as a broad range of growth habitats.
Most morels are considered saprobic because they can complete their life-cycle without host plants,
but a number of species have been reported to interact with roots of many plant species forming
different types of associations (Pilz et al., 2007). Morels can be found in many forest types, meadows,
burned sites, coastal dunes and a broad range of disturbed soils and man-made environments (Pilz et
al., 2007).
    In Italy, morels are locally appreciated but have not yet gained a significant market share among
edible mushrooms. Most of the morels commercialized in Italy come from Hungary, Turkey,
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Snabl et al.                                                                    Italian Journal of Mycology 48 (2019)




Romania and Bulgaria and their wholesale price is 20-28 € per kg whereas the retail price is 30-40 €
per kg (Luigi Dattilo, Appennino Food Group SpA, personal communication). Unlike other high-
priced edible mushrooms such as truffles (Tuber spp.) and porcini (Boletus edulis s.l.), the ecology
and biology of morels in Italy have been poorly investigated. These mushrooms are particularly
sought-after in the littoral area of the Emilia-Romagna region where their ascomata are collected
either from the coast or from the inland pine and hardwood forests. In this work, we aimed i) to
barcode the morels collected on the sand dunes of the Emilia-Romagna coast and ii) to investigate
the type and extent of their interactions with the local plants.

Materials and Methods

The area under investigation consists of a series of sand dunes extended along the coastline from
Punta Marina and Marina di Ravenna (Ravenna, northeastern Italy). The collection sites are
approximately 100 m from the sea at an elevation of 2 to 5 m asl, within the EU habitat 2120
[Echinophoro spinosae-Ammophiletum australis (Br.-Bl. 1933) Géhu, Rivas-Martinez & R. Tx. 1972
(Géhu et al., 1984)]. In this area, the dune system is extremely fragmented and threatened by touristic
facilities (Sytnik and Stecchi, 2015). The dune vegetation of the study area is dominated by
Calamagrostis arenaria (L.) Roth [=Ammophila arenaria (L.) Link subsp. australis (Mabille) Laínz],
Echinophora spinosa L., Eryngium maritimum L., Euphorbia paralias L., Medicago marina L.,
Cyperus capitatus Vand., Pancratium maritimum L., Stachys maritima Gouan and Spartina juncea
auct., non (Michx.) Willd., often associated to allochthonous species such as Cenchrus incertus
M.A.Curtis (=Cenchrus spinifex Cav.), Ambrosia psilostachya DC (=Ambrosia cronopifolia Torr. &
A. Gray), Yucca gloriosa L. and Oenothera stucchii Soldano (Merloni et al., 2015). A coastal Pinus
spp. forest extends in the inland area behind the dune system.
   Ascoma production was assessed every week during the fruiting season (from mid-March to mid-
April) from 2001 to 2017. Voucher specimens were deposited in the private herbarium of the first to
authors. The range of interactions between morels and host plant species was investigated by
following the mycelial structures connecting the base of the stipe and the host roots. A trench was
dug in proximity of each ascoma by using a trowel and the mycelial connecting structure was carefully
unearthed with a paint brush. A total of 68 trenches were opened throughout the study period, most
of them on the sand dunes but some also in the immediate surrounding anthropized areas (Table 1).

               Table 1 - List of the surveys in which sand-mycelial structures were unearthed. Host plant
               and number of ascomata collected in the same day of excavation are reported
                Survey date        Locality              Number of Host plant
                                                         trenches
                8 April 2001       Marina di Ravenna     3              Cyperus capitatus
                                                         1              Hypochaeris radicata
                14 April 2001      Punta Marina          2              Yucca gloriosa
                8 April 2004       Punta Marina          2              Reseda alba
                7 April 2006       Punta Marina          6              Yucca gloriosa
                                                         8              Tamarix gallica
                                                         4              Elaeagnus angustifolia
                30 March 2008      Punta Marina          1              Elaeagnus angustifolia
                3 April 2008       Punta Marina          2              Pinus pinaster
                5 April 2008       Punta Marina          5              Yucca gloriosa
                11 April 2008      Punta Marina          4              Yucca gloriosa
                                                         3              Hypochaeris radicata
                19 April 2010      Punta Marina          3              Yucca gloriosa
                                                         2              Sedum sp.
                10 April 2015      Punta Marina          7              Hypochaeris radicata
                                                         2              Cyperus capitatus
                1 April 2016       Punta Marina          4              Ambrosia cronopifolia
                                                         6              Hypochaeris radicata
                24 March 2017      Marina di Ravenna     3              Cyperus capitatus

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Snabl et al.                                                              Italian Journal of Mycology 48 (2019)




    Macro and micro-morphology of the specimens were assessed through the analysis of fresh
ascomata. Micro-morphological analyses were carried out under a Laborlux K microscope (Leitz) on
sections of the hymenia and the sterile ridges mounted either in water or Congo red. Measurements
were made with an ocular micrometer at × 400 magnification for asci, paraphyses and elements of
sterile ridges and × 1000 magnification for spores. One hundred measures were taken for each
taxonomic character in two out of five ascomata collected in 2017.
    Five ascomata collected on March 2017 were barcoded by sequencing the internal transcribed
spacer (ITS) region of the nuclear ribosomal DNA (rDNA). Few sterile hyphae were used directly as
target material for DNA amplification through polymerase chain reaction (PCR), bypassing the need
for DNA isolation (Iotti and Zambonelli, 2006). ITS region was amplified in a 50-μl volume reaction
using the primer pair ITS1F-ITS4 (White et al., 1990; Gardes and Bruns, 1993) and a SimpliAmp
thermal cycler (ThermoFisher). PCR reactions contained BioMix mastermix (Bioline), 400 nM for
each primer and 30 μg of bovine serum albumin. Thermal cycler conditions were the following: 30
cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with an initial denaturation at 94 °C for
8 min and a final extension at 72 °C for 10 min. Amplicons were purified with the NucleoSpin Extract
II kit (Macherey-Nagel) and sequencing was performed with ITS5 and ITS4 as forward and reverse
primers at GATC Biotech AG (Constance, Germany). ITS sequences were aligned and edited
manually with BioEdit 7.0.5.3 (Hall, 1999) and consensus sequences were compared to those
deposited       in      GenBank         (www.ncbi.nlm.nih.gov)           and     Morchella       MLST
(http://www.cbs.knaw.nl/morchella/) databases. Genotyped specimens were deposited in Herbarium
Mycologicum Aquilanum (AQUI9839 to AQUI9841) and their ITS sequences submitted to GenBank
(accession numbers MK388505 to MK388509).
    The phylogenetic analysis of the genotyped ascomata was inferred by using the maximum
likelihood (ML) estimation implemented in Raxml (version 8.2; Stamatakis, 2014) and MrBayes.
Sequences generated in this study were analyzed together with other ITS sequences from GenBank.
Alignment was performed by CLUSTAL W (Thompson et al., 1994) and gaps and ambiguous sites
were excluded prior to phylogenetic analyses. Hasegawa-Kishino-Yano (HKY) model was identified
as the best fit-model of nucleotide substitution by using JModelTest v2.1.4 (Darriba et al., 2012).
Branch support of ML tree was assessed by bootstrap analysis of 1,000 replicates. Bayesian analyses
were conducted using the software package MrBayes v3.2.2 (Ronquist et al., 2012). To ensure
convergence, two independent runs were conducted, each with four chains and for 5,000,000
generations, sampling every 100 generations. A 50% majority-rule consensus tree was generated after
the exclusion of the first 250 trees and the posterior probabilities of clades were computed.

Results

During the period of survey, about 200 ascomata were collected on sand dunes and their immediate
surroundings. All the ascomata collected during the period of investigation shared the same micro
and macro-morphology. The main source of morphological diversity was due to the collection period
(Fig. 1). In general, the earlier fruiting strains (first half of March) generated little and sterile ascomata
with darker pits while the ascomata collected in April were yellowish with a larger size and mature
ascospores.

Macro-morphological description
Ascomata 50-130 mm high. Hymenophore 30-80 mm high and 20-60 mm wide at the widest point;
irregularly spherical up to conical with a rounded apex; pitted and ridged; adnate at the point of
attachment. Ridges glabrous; light gray when young, becoming ochre-yellowish, ochre-grayish or
light brown with maturity; rusty-brown or orange brown when exposed to the sun or sea air; widely
rounded to nearly flat when young, becoming thinned in age. Pits glabrous; usually darker than ridges.
Stipe 20-50 mm high, almost entirely buried in the soil; 15-30 mm wide; more or less equal,
occasionally lacunose and basally subclavate; glabrous or slightly pruinose; whitish to beige,
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Snabl et al.                                                                   Italian Journal of Mycology 48 (2019)




sometimes with brown spots; 1.5-2 mm thick, becoming thickened near the base. Context whitish;
sterile inner surface whitish and finely warty. Flesh firm, elastic, with slight spermatic typical odour.




Fig. 1 - Ascomata collected in the study area during the period of investigation. a) immature specimen (Mvu10) hosted
by Hypochaeris radicata, collected on March 24, 2017; b) specimen (Mvu37) hosted by Cyperus capitatus, collected on
March 24, 2017: the darker side of the hymenophore faced the sea with < 10% of spore-containing asci; c) mature
specimen collected on April first, 2016; d) mature specimen collected on April 17, 2010


Micromorphological description
Ascospores pale yellowish in deposit, (13.2) 16.7-20.4 (23.2) µm × (8.2) 10.2-12.3 (13.9) µm; Q (1.1)
1.5-1.8 (2.3); elliptical; smooth; thin-walled; hyaline; contents homogeneous, sometime
microguttulate at poles. Asci 275-325 x 15-20 µm; eight-spored; cylindrical to subclavate, sinuose at
the base; thin-walled; operculate; hyaline; uniseriate; inamyloid. Paraphyses 200-250 x 5-9 µm;
cylindrical; hyaline; apices rounded to subcapitate; thick-walled; 2-3-septate; sometimes branched in
the lower half. Elements on sterile ridges 140-150 x 12-25 µm; septate; terminal cell clavate to
capitate; thick-walled; hyaline or with granular content.

Molecular analyses
PCR amplification of the five specimens collected in 2017 generated ITS1f/ITS4 amplicons of 1225
bp. Two ITS1-5.8S-ITS4 types with 99.64% similarity (4 variable positions out of 1099 bp) were
obtained after sequencing. Blastn search against Morchella MLST database yielded the best hit to a
M. esculenta sequence (JQ723102), having > 99.5% similarity with both ITS types. According to the
clustering criteria formulated by Kõljalg et al. (2013), ITS sequences generated in this study fallen
into the M. spongiola species hypothesis (SH181478.07FU) within the UNITE database.
Phylogenetic analyses placed all the sequences generated in this work within the clade Mes-17
identified by Du et al. (2012a). Phylogenetic tree from this work (Fig. 2) was generated by using the
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Snabl et al.                                                                    Italian Journal of Mycology 48 (2019)




full-length ITS1-5.8S-ITS2 sequences of Mes-17 clade deposited in Genbank and a sequence of the
closely related clade Mes-6 (JQ322060) as outgroup.




Fig. 2 - Maximum Likelihood (ML) tree generated from full ITS1-5.8S-ITS2 sequences (1015 bp) of Mes-17 clade after
Du et al. (2012a). Tree topologies from ML and Bayesian analyses were congruent at all supported nodes. Bootstrap
values (1000 replications) and Bayesian posterior probabilities are reported on each branch. Ascomata genotyped in this
work are labelled by their isolation code while the specimens from GenBank are identified by their accession number and
the associated species epithet. The tree was edited using FigTree v1.4.3 (http://tree.bio.ed.ac.uk/)


Host plant connections
Characteristic mycelial structures (Fig. 3) were found to connect the base of the morel stalks to the
roots of nine different plant species (Table 1). They appeared as a sand-mycelium cord, simple or
ramified, brittle to the touch, 5 to 30 mm in diameter and 20 to 100 cm long, composed of entangled
hyphae encrusted by sand grains. Fine roots were completely embedded within these mycelial
structures whereas the surface of the rhizomes and conductive roots (> 0.5 cm in diameter) was only
partially colonized. During the field surveys, any other type of fungal structure (e.g. sclerotia) was
found on the roots of host plants or in the soil.

Discussion

In this work, morels from white dunes of the Italian coast were genotyped for the first time. A number
of Morchella species have been reported to grow on sand dunes throughout Atlantic (Rotheroe, 1993;
Castañera and Moreno, 1996; Guinberteau, 2012) and Mediterranean coasts (Loizides, 2016; Melis
et al., 2017). However, the phylogeny and ecology of morels growing in this habitat have been poorly
investigated. The morels of the coastline from Punta Marina and Marina di Ravenna (Ravenna,
northeastern Italy) were previously described by Snabl and Guidori (2015) and identified as M.
esculenta based on morphological traits of their ascomata. Morphology of these morels is shared by
different species in the Morchella genus which can be found on coastal sand dunes [M. dunensis
(Castañera, J.L. Alonso & G. Moreno) Clowez] but also in other habitats [M. vulgaris (Pers.) Gray
and M. esculenta (L.) Pers.]. The growth habitat has often been considered as a key character in morel
taxonomy.

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Snabl et al.                                                             Italian Journal of Mycology 48 (2019)




                       Fig. 3 - Sand-mycelium cord of an ascoma associated to Cyperus
                       capitatus

    In a recent work, M. dunensis was described as a xerophilous and sand-inhabiting species, with
rufescent ascomata, ochraceous or ochraceous-brown in color, misshapen stipe and coarse ridges
(Loizides et al., 2016). As main differences with M. dunensis, M. vulgaris shows gray tinges and
contrasting dark pits whereas M. esculenta displays a stipe rather smooth, cylindrical and weakly
inflated at the base, regularly polygonal pits with more or less smooth ridges. Long-term monitoring
of ascomata collected in the study site highlighted a substantial range of morphological variation
attributable to seasonal climate dynamics and maturity stage. In this context, intraspecific variability
of ascoma morphology masked the differences among closely related morel species.
    In recent years, significant efforts have been made to improve the molecular-based taxonomy of
true morels by genotyping specimens from North America and Asia (O’Donnell et al., 2011; Du et
al., 2012b) and later from Europe (Taşkin et al., 2012; Richard et al., 2015; Loizides et al., 2016).
Despite the increasing interest in phylogeny of the Morchella genus, the genetic diversity of the Italian
specimens remains almost unexplored. To our knowledge, only three ascomata from alpine and sub-
alpine habitats were barcoded before December 2017: M. conica Pers., M. elata Fr. (Osmundson et
al., 2013) and M. semilibera DC. (Richard et al., 2015). No genetic data are available on morels
growing in the rest of the peninsula and, in particular, in the littoral areas where our investigation was
conducted.
    All the ascomata genotyped in this work are nested within the clade Mes-17 identified by Du et al.
(2012a). Phylogeny of the specimens within this clade is controversial and the high number of
misidentified GenBank records within the Esculenta group (Du et al., 2012a) does not facilitate the
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Snabl et al.                                                          Italian Journal of Mycology 48 (2019)




identification to species. Buscot et al. (1996) molecularly identified this species as Morchella
spongiola Boud. and, later, a number of authors accepted this epithet (Kellner et al., 2005; Kanwal et
al., 2011; Barseghyan et al., 2012). Based on priority criteria, Richard et al. (2015) applied the name
Morchella vulgaris (Pers.) Gray to the collections nested within Mes-17 clade and indicated a number
of synonyms: M. dunensis (Castañera & G. Moreno) Clowez, M. acerina Clowez & C. Boulanger,
M. andalusiae Clowez & L. Romero, M. anthracina Clowez & Vanhille, M. lepida Clowez & Franç.
Petit, M. robiniae Clowez and M. spongiola. In the same work, two members belonging to this clade
were described from coastal areas of northern France, including the withe dune habitat with A.
arenaria. Later, M. dunensis was elevated to species rank by Loizides et al. (2016) because most of
the Mediterranean and coastal collections nested in a subclade within M. vulgaris.
    Morels in the Esculenta group seem to feed on the roots of a broad range of plant species forming
different types of structures such as mycorrhiza-like structure (Pilz et al., 2007) or mycelial muffs
(Buscot and Roux, 1987). In the study site, plant/ascoma association through sand-mycelium cords
seem to be the main nutritional strategy to support the fruiting phase. These structures were found to
finish their growth on a variety of underground plant organs differing in size, type (roots or rhizomes)
and species. Similar structures, called radiscisclerotia, were found by Stefani et al. (2010) under
ascomata of Morchella tomentosa M. Kuo a year after a forest fire in a black spruce forest in Alaska.
Radiscisclerotia were described as whitish, ramified and compact hypogeous structures, 5-15 mm in
diameter, which represent time-resistant nutrient reservoir that could last a few years and support
ascoma production. On the contrary, the fungus-host connective structures described in this work
were fragile and ephemeral sand aggregates, with a likely seasonal lifetime. However, all the
assumptions made on nutritional strategies of this morel group need to be confirmed and explored
more in depth by molecular methods such as stable isotopes (Hobbie et al., 2016).
    On white dunes, the ascomata were mostly associated with the rhizomatous plants C. capitatus, A.
cronopifolia and H. radicata while a heterogeneous range of hosts, both herbaceous and woody
plants, were found in the surroundings anthropized areas. Sand dunes represent an optimal condition
to study the morel-plant interaction in the wild because the sandy soil allows to follow the growth of
the sand-mycelium cords in the soil without damage these mycelial structures.
    Considering the limited extension of the habitat under investigation and the amount of ascoma
production, the study area seems to be particularly suitable for morel production. Coastal dune
systems in Emilia-Romagna have an important role as ecosystem service to the coast. They provide
a number of services such as coastal defense, groundwater and carbon storage, water purification and
preserving biodiversity (Drius et al., 2016). Unfortunately, these systems are highly threatened by a
number of factors such as urbanization and tourism (Sytnik and Stecchi, 2015). In Spain, Moreno et
al. (2013) listed M. dunensis among the fungal species threatened by the loss of coastal ecosystems
due to climate changes. Although these fungi are also able to colonize heavily anthropized
environments, the protection of the dune system is imperative for preserving genetic diversity within
the local population of wild morels.

References

Ajmal M, Akram A, Ara A, Akhund S, Nayyar BG (2015) Morchella esculenta: an edible and health
     beneficial mushroom. Pakistan Journal of Food science 25(2):71-78.
Barseghyan GS, Kosakyan A, Isikhuemhe OS, Didukh M, Wasser SP (2012) Phylogenetic analysis
     within genera Morchella (Ascomycota, Pezizales) and Macrolepiota (Basidiomycota,
     Agaricales) inferred from nrDNA ITS and EF-1a sequences. In: Systematics and evolution of
     fungi (Misra JK, Tewari JP, Deshmukh SK, eds). CRC Press, Boca Raton, pp. 159-205.
Buscot F, Roux J (1987) Association between living roots and ascocarps of Morchella rotunda.
     Transactions of the British Mycological Society 89(2):249-252.



                                                                                                         22
Snabl et al.                                                         Italian Journal of Mycology 48 (2019)




Buscot F, Wipf D, Di Battista C, Munch JC, Botton B, Martin F (1996) DNA polymorphism in
       morels: PCR/RFLP analysis of the ribosomal DNA spacers and microsatellite-primed PCR.
       Mycological Research 100(1):63-71.
       https://doi.org/10.1016/S0953-7562(96)80101-8
Castañera V, Moreno G (1996) Una Morchella (Morchella esculenta forma dunensis f. nov.)
       frecuente en las dunas de Cantabria. Yesca. Revista Sociedad Micólogica de Cántabria 8: 27.
Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and
       parallel computing. Nature Methods 9:772.
       https://doi.org/10.1038/nmeth.2109
Drius M, Carranza ML, Stanisci A, Jones L (2016) The role of Italian coastal dunes as carbon sinks
       and diversity sources. A multi-service perspective. Applied Geography 75:127-136.
       https://doi.org/10.1016/j.apgeog.2016.08.007
Du XH, Zhao Q, Yang ZL, Hansen K, Taşkin H, Büyükalaca S, Dewsbury D, Moncalvo JM, Douhan
       GW, Robert VARG, Crous PW, Rehner SA, Rooney AP, Sink S, O’Donnell K (2012a). How
       well do ITS rDNA sequences differentiate species of true morels (Morchella)? Mycologia
       104(6):1351-1368.
       https://doi.org/10.3852/12-056
Du XH, Zhao Q, O’Donnell K, Rooney AP, Yang ZL (2012b). Multigene molecular phylogenetics
       reveals true morels (Morchella) are especially species-rich in China. Fungal Genetics and
       Biology 49(6):455-469.
       https://doi.org/10.1016/j.fgb.2012.03.006
Du XH, Zhao Q, Yang ZL (2015). A review on research advances, issues, and perspectives of morels,
       Mycology 6(2):78-85.
       https://doi.org/10.1080/21501203.2015.1016561
Du XH, Zhao Q, Xia EH, Gao LZ, Richard F, Yang ZL (2017) Mixed-reproductive strategies,
       competitive mating-type distribution and life cycle of fourteen black morel species. Scientific
       Reports 7: 1493.
       https://doi.org/10.1038/s41598-017-01682-8
Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes – application
       to the identification of mycorrhizae and rusts. Molecular Ecology 2(2):113–118.
       https://doi.org/10.1111/j.1365-294X.1993.tb00005.x
Géhu JM, Costa M, Scoppola A, Biondi E, Marchiori S, Peris JB, Frank J, Caniglia G, Veri L (1984)
       Essai synsystématique et synchorologique sul les vegetation littorals italiennes dans un but
       conservatoire. I. Dunes et vases salees. Documents Phytosociologique 8:394-474.
Guinberteau J (2012) Les Champignons des dunes non boisées du littoral aquitain: un univers
       méconnu! Mycologia Montenegrina 15:53-77.
Hall TA (1999) Bioedit: a user-friendly biological sequence alignment editor and analysis program
       for Windows 95/98/NT. Nucleic Acids Symposium Series 41:95-98.
Hobbie EA, Rice SF, Weber NS, Smith JE (2016) Isotopic evidence indicates saprotrophy in post-
       fire Morchella in Oregon and Alaska. Mycologia 108(4):638-645.
       https://doi.org/10.3852/15-281
Iotti M, Zambonelli A (2006) A quick and precise technique for identifying ectomycorrhizas by PCR.
       Mycological Research 110(1):60-65.
       https://doi.org/10.1016/j.mycres.2005.09.010
Kanwal HK, Acharya K, Ramesh G, Reddy MS (2011) Molecular characterization of Morchella
       species from the western Himalayan region of India. Current Microbiology 62(4):1245-1252.
       https://doi.org/10.1007/s00284-010-9849-1
Kellner H, Renker C, Buscot F (2005) Species diversity within the Morchella esculenta group
       (Ascomycota: Morchellaceae) in Germnay and France. Organisms Diversity and Evolution
       5(2):101-107.
       https://doi.org/0.1016/j.ode.2004.07.001

                                                                                                        23
Snabl et al.                                                          Italian Journal of Mycology 48 (2019)




Kõljalg U, Nilsson RH, Abarenkov K, Tedersoo L, Taylor AFS, Bahram M, Bates ST, Bruns TD,
      Bengtsson‐Palme J, Callaghan TM, Douglas B, Drenkhan T, Eberhardt U, Dueñas M, Grebenc
      T, Griffith GW, Hartmann M, Kirk PM, Kohout P, Larsson E, Lindahl BD, Lücking R, Martín
      MP, Matheny PB, Nguyen NH, Niskanen T, Oja J, Peay KG, Peintner U, Peterson M, Põldmaa
      K, Saag L, Saar I, Schüßler A, Scott JA, Senés C, Smith ME, Suija A, Taylor DL, Telleria MT,
      Weiss M, Larsson KH (2013) Towards an unified paradigm for sequence-based identification
      of fungi. Molecular Ecology 22(21):5271-5277.
      https://doi.org/10.1111/mec.12481
Loizides M (2016) Morels: the story so far. Field Mycology 18(2):42-53.
      https://doi.org/10.1016/j.fldmyc.2017.04.004
Loizides M, Bellanger JM, Clowez P, Richard F, Moreau PA (2016) Combined phylogenetic and
      morphological studies of true morels (Pezizales, Ascomycota) in Cyprus reveal significant
      diversity, including Morchella arbutiphila and M. disparilis spp. Nov. Mycological Progress 15:39.
      https://doi.org/10.1007/s11557
Melis JLL, Mir G, Prats MC (2017) Aportación al catálogo micológico de las illes Balears. Menorca
      II. Micobotánica-Jaén 12(1):1-32.
Merloni N, Rigoni P, Zanni F (2015) La vegetazione delle dune litoranee nella riserva di foce Bevano.
      In: Spiagge e dune dell’Alto Adriatico (Nobili G, Gabbianelli G, eds). Corpo Forestale dello
      Stato, Ufficio territoriale per la Biodiversità, Punta Marina Terme, Ravenna, pp. 79-98.
Moreno G, Manjón JL, Álvarez-Jiménez J (2013) Los hongos y el cambio climático. In: Desert truffle
      (Kagan-Zur V., Sitrit Y, Roth-Bejerano, NA Morte, eds). Springer Verlag, Berlín, pp. 129-135.
O’Donnell K, Rooney AP, Mills GL, Kuo M, Weber NS, Rehner SA (2011) Phylogeny and historical
      biogeography of true morels (Morchella) reveals an early Cretaceous origin and high
      continental endemism and provincialism in the Holarctic. Fungal Genetics and Biology
      48(3):252-265.
      https://doi.org/10.1016/j.fgb.2010.09.006
Osmundson TW, Robert VA, Schoch CL, Baker LJ, Smith A, Robich G, Mizzan L, Garbelotto MM
      (2013) Filling gaps in biodiversity knowledge for macrofungi: contributions assessment of an
      herbarium collection DNA barcode sequencing project. PlosOne 8(4):e62419.
      https://doi.org/10.1371/journal.pone.0062419
Peng W, Chen Y, Tan H, Tang J, Gan B (2015) Artificial cultivation of morels is blooming in Sichuan.
      Bulletin of the world society for mushroom biology and mushroom products 13.
      www.wsmbmp.org/Bol13/5.html
Pilz D, McLain R, Alexander S, Villarreal-Ruiz L, Berch S, Wurtz TL, Parks CG, McFarlane E,
      Baker B, Molina R, Smith JE (2007) Ecology and management of morels harvested from the
      forests of western North America. General Technical Report PNW-GTR-710, USDA, Forest
      Service, Pacific Northwest Research Station, Portland.
Richard F, Bellanger JM, Clowez P, Hansen K, O’Donnell K, Urban A, Sauve M, Courtecuisse R,
      Moreau PA (2015) True morels (Morchella, Pezizales) of Europe and North America:
      evolutionary relationships inferred from multilocus data and a unified taxonomy. Mycologia
      107(2):359-382.
      https://doi.org/10.3852/14-166
Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard
      MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient bayesian phylogenetic inference and model
      choice across a large model space. Systematic Biology 61(3):539–542.
      https://doi.org/10.1093/sysbio/sys029
Rotheroe M (1993) The larger fungi of welsh sand dunes. CCWR report 1993.
Snabl M, Guidori U (2015) Il genere Morchella – 1° contributo. Biologia di M. esculenta in natura.
      Rivista di Micologia 58(3):195-213.
Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large
      phylogenies. Bioinformatics 30(9):1312–1313.
      https://doi.org/10.1093/bioinformatics/btu033
                                                                                                         24
Snabl et al.                                                     Italian Journal of Mycology 48 (2019)




Stefani FOP, Sokolski S, Wurtz TL, Piché Y, Hamelin RC, Fortin JA, Bérubé JA (2010) Morchella
      tomentosa: a unique belowground structure and a new clade of morels. Mycologia 102(5):1082-
      1088.
      https://doi.org/10.3852/09-294
Sytnik O, Stecchi F (2015) Disappearing coastal dunes: tourism development and future challenges,
      a case of study from Ravenna, Italy. Journal of Coast Conservation 19(5):715-727.
      https://doi.org/10.1007/s11852-014-0353-9
Taşkin H, Büyükalaca S, Hansen K, O’Donnell K (2012) Multilocus phylogenetic analysis of true
      morels (Morchella) reveals high levels of endemics in Turkey relative to other regions of
      Europe. Mycologia 104(2):446-461.
      https://doi.org/10.3852/11-180
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of
      progressive multiple sequence alignment through sequence weighting, positions-specific gap
      penalties and weight matrix choice. Nucleic Acids Research 22(22):4673–4680.
      https://doi.org/10.1093/nar/22.22.4673
White TJ, Bruns TD, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal
      ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and
      Applications (Innis MA, Gelfand DH, Sninsky J, White TJ, eds). Academic Press, San Diego,
      pp 315–322.




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