Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57696, enero-diciembre 2024 (Publicado Nov. 05, 2024)

Ectomycorrhizal fungi associated with Coccoloba uvifera

(Polygonaceae) in coastal ecosystems of Eastern Cuba

Mijail Mijares Bullaín-Galardis1*; https://orcid.org/0000-0001-8123-4906

Ludovic Pruneau2; https://orcid.org/0000-0002-7529-4387

Bettina Eichler- Löbermann6; https://orcid.org/0000-0001-8306-0452

Fatoumata Fall3; https://orcid.org/0000-0002-8533-7010

Raúl Carlos López-Sánchez1; https://orcid.org/0000-0003-0477-3572

Amadou Mustapha Bâ3,4,5; https://orcid.org/0009-0008-7398-4107

1. Centro de Estudios de Biotecnología Vegetal, Facultad de Ciencias Agropecuarias, Universidad de Granma, 85100,

Bayamo, Provincia Granma, Cuba; mbullaing@gmail.com (*Correspondencia), raulcarloslopez2015@gmail.com

2. Institut de Systématique, Évolution, Biodiversité, Unité Mixte de Recherche7205, Université des Antilles, Guadeloupe,

France; ludovic.pruneau@univ-antilles.fr

3. Laboratoire de Biologie et Physiologie Végétales, Université des Antilles, Fouillole, Pointe-à-Pitre, 97157 Guadeloupe,

France; fal1481@yahoo.fr, amadou.ba@univ-antilles.fr

4. Laboratoire des Symbioses Tropicales et Méditerranéennes, Unité Mixte de Recherche113/Université Montpellier2/

Centre de coopération internationale en recherche agronomique pour le développement/ Institut de Recherche pour

le Développement/Sup-Agro, Montpellier, France; amadou.ba@ird.fr

5. Académie Nationale des Sciences et Techniques du Sénégal, Dakar, Sénégal

6. Department of Plant Sciences/Faculty of Agriculture/University of Rostock, Rostock, Germany;

bettina.eichler@uni-rostock.de

Received 20-XI-2023. Corrected 18-VII-2024. Accepted 30-X-2024.

ABSTRACT

Introduction: Coccoloba uvifera, named also seagrape, establishes symbiotic relationships with many ectomycor-

rhizal fungi. However, in Cuba, these fungi have been little studied.

Objective: To characterize the diversity of sporocarps and ectomycorrhizae of ectomycorrhizal fungi associated

with C. uvifera in three coastal ecosystems of Eastern Cuba.

Methods: Four samplings of sporocarps and ectomycorrhizae were carried out at three-week intervals during the

rainy season, from June to September, in 2018 and 2019. Ectomycorrhizae were collected from three mature trees

and 30 young individuals per tree. The samples were transferred to the Abiotic Stress Laboratory of the Center

for Plant Biotechnology Studies of the University of Granma and the Laboratory of Plant Biology and Physiology

of the University of French West Indies for processing and subsequent identification.

Results: Five species of ectomycorrhizal fungi were identified from sporocarps collected under C. uvifera in the

three sampling sites (Scleroderma bermudense, Russula sp., Cantharellus sp., Inocybe sp., and Amanita sp.). Using

internal transcribed spacer sequencing, six taxa of ectomycorrhizal fungi were identified from ectomycorrhizas

of mature trees and seedlings (S. bermudense, two Tuber spp., Tomentella sp., Inocybe sp., and Thelephora sp.).

Only S. bermudense coincided (similarity 99-100 %) with sporocarps. S. bermudense was the most frequent

ectomycorrhizal fungus in the ectomycorrhizas and sporocarps collected. Mature trees and C. uvifera seedlings

shared between 75 and 100 % of the ectomycorrhizal fungal communities, being able to form potential common

ectomycorrhizal networks.

https://doi.org/10.15517/rev.biol.trop..v72i1.57696

BOTANY AND MYCOLOGY

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57696, enero-diciembre 2024 (Publicado Nov. 05, 2024)

INTRODUCTION

Many trees form ectomycorrhizal (ECM)

symbioses with diverse Basidiomycota and

Ascomycota, which play a key role in many

tropical forests (Bâ et al., 2012; Corrales et

al., 2018), affecting tree growth and nutri-

ent absorption as well as protection against

pathogens (Smith & Read, 2008). Large areas of

tropical and neotropical forests are dominated

by ECM trees (Bâ et al., 2012; Corrales et al.,

2018), suggesting a key role of these symbio-

ses in the functioning of some tropical forest

ecosystems. ECM surveys from tropical forests

show that Africa has the highest number of

confirmed ECM plant species, followed by

Neotropics, including Central and South Amer-

ica and the Caribbean (Corrales et al., 2018).

ECM host plants in the neotropical lowlands

are from predominantly tropical lineages in the

Fabaceae, Cistaceae, Dipterocarpaceae, Polygo-

naceae, and Nyctaginaceae families (Corrales

et al., 2018).

In Guadeloupe and Martinique (Lesser

Antilles), the ECM fungal diversity associ-

ated with the Polygonaceae Coccoloba uvifera

L. (seagrape) was quite poor (Bâ et al., 2014;

Séne et al., 2015; Séne et al., 2018) as compared

with the ECM fungal diversity of the Fabaceae

Afzelia africana Sm. from West Africa (Bâ et

al., 2012). This low diversity does not result

from insufficient sampling, because species

accumulation curves of sporocarps and ECM

fungal taxa reached an asymptote at all sites

in Guadeloupe (Séne et al., 2015). However,

other Caribbean and South American sites

Conclusions: At the three collection sites, sporocarp weakly reflected the belowground ectomycorrhizal fungal

community, ectomycorrhizal fungal diversity is quite limited, and S. bermudense was the only ectomycorrhizal

fungus that overlapped in sporocarps and ectomycorrhizae.

Key words: seagrape; fungal diversity; fruiting body; ectomycorrhizae; ITS sequencing.

RESUMEN

Hongos ectomicorrícicos asociados con Coccoloba uvifera (Polygonaceae)

en ecosistemas costeros del oriente cubano

Introducción: Coccoloba uvifera, también llamada uva de mar, establece relaciones simbióticas con varios hongos

ectomicorrícicos, sin embargo, en Cuba, estos hongos han sido poco estudiados.

Objetivo: Caracterizar la diversidad de esporocarpos y ectomicorrizas de hongos ectomicorrízicos asociados a C.

uvifera en tres ecosistemas costeros del oriente de Cuba.

Métodos: Se realizaron cuatro muestreos de esporocarpos y ectomicorrizas a intervalos de tres semanas durante

la temporada de lluvias, de junio a septiembre, entre 2018 y 2019. Las ectomicorrizas se recolectaron en tres árbo-

les maduros y en 30 individuos jóvenes por árbol. Las muestras se trasladaron al Laboratorio de Estrés Abiótico

del Centro de Estudios de Biotecnología Vegetal de la Universidad de Granma y al Laboratorio de Biología y

Fisiología Vegetal de la Universidad de las Antillas, para su procesamiento y posterior identificación.

Resultados: A partir de esporocarpos recolectados bajo C. uvifera en los tres sitios de muestreo se identificaron

cinco especies de hongos ectomicorrícicos (Scleroderma bermudense, Russula sp., Cantharellus sp., Inocybe sp. y

Amanita sp.). Utilizando la secuenciación espaciador transcrito interno se identificaron seis taxones de hongos

ectomicorrícicos a partir de ectomicorrizas de árboles maduros y plántulas (S. bermudense, dos Tuber spp.,

Tomentella sp., Inocybe sp., y Thelephora sp.). Solo S. bermudense coincidió (similitud 99-100 %) con esporocar-

pos. S. bermudense fue el hongo ectomicorrícico más frecuente en las ectomicorrizas y los esporocarpos recolec-

tados. Los árboles maduros y las plántulas de C. uvifera compartieron entre el 75 y el 100 % de las comunidades

fúngicas ectomicorrícicas, pudiendo formar redes ectomicorrícicas comunes.

Conclusiones: En los tres sitios de recolección, los esporocarpos reflejaron débilmente la comunidad de hongos

ectomicorrízicos subterránea, la diversidad de hongos ectomicorrízicos fue bastante limitada y S. bermudense fue

el único hongo ectomicorrícico que se superpuso en esporocarpos y ectomicorrizas.

Palabras clave: uva de mar; diversidad fúngica; cuerpo fructífero; micorrizas; secuenciación ITS.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57696, enero-diciembre 2024 (Publicado Nov. 05, 2024)

have to be investigated before this feature can

be generalized to the whole distribution area

of seagrape (Bâ et al., 2014; Põlme et al., 2017).

There is evidence that the number of host tree

species drives diversity (Ishida et al., 2007),

and indeed, seagrape is the only ECM host of

the investigated beach sites (Séne et al., 2015).

Study of sporocarps fruiting near seagrape

weakly reflected the belowground ECM fungal

community, although five fruiting species were

found in ECM roots. Seagrape seedlings and

mature trees had very similar communities of

ECM fungi and could share potential common

mycorrhizal networks that could play a major

role in nutrient transfers (e.g., C, P, and N)

through hyphae from mature trees to seedlings

of Coccoloba (Karst et al., 2022; Séne et al.,

2015). This tree is associated with ECM fungi

in the genera Amanita, Inocybe, Cantharellus,

Melanogaster, Cenococcum, Lactarius, Russula,

Thelephoraceae, Xerocomus, and Scleroderma

(Álvarez, 2012; Bâ et al., 2014; Guzmán et al.,

2004; Kreisel, 1971; Miller et al., 2000; Pegler,

1983; Séne et al., 2015; Séne et al., 2018). The

ECM fungal species Scleroderma bermudense

plays a major role in mitigating salt stress for

seagrape (Bandou et al., 2006; Bullaín-Galardis

et al., 2023; Bullaín et al., 2022).

In the flora of Cuba, 34 species of Coc-

coloba including seagrape are listed and 25 are

endemic, suggesting that Cuba is an important

center of specific diversity of the genus Coccolo-

ba in the Greater Antilles (Castañeda, 2014).

As host tree species drive diversity (Ishida et

al., 2007), higher ECM fungal diversity should

be expected in Cuba. However, the ECM fungi

associated with seagrape in Cuba are poorly

studied. Therefore, the objective of this research

was to characterize the diversity of sporocarps

and ectomycorrhizae (EMe) of ECM fungi

associated with seagrape in three coastal eco-

systems in Eastern Cuba.

MATERIALS AND METHODS

Study area: The ECM roots of seagrape

and sporocarps were collected at Las Coloradas

beach (19º55’31.9’’ N & 77º41’14.1’’ W), Cabo

Cruz (19º50’23.5’’ N & 77º43’14.4’’ W), and

Punta de Tomate (21º16’16.6’’ N & 76º31’ 18.5’’

W) in Cuba (Fig. 1).

These sites are representative of sand-

growing littoral seagrape forests with abun-

dant seedling recruitment in the crowns of the

mature trees, except on Cabo Cruz beach. Due

to their geographical proximity, barely 10 km,

Las Coloradas and Cabo Cruz both have an

average annual rainfall and temperature of 942

mm and 27.0 °C. The precipitation and aver-

age annual temperature of Punta de Tomate is

Fig. 1. Location of the three coastal collection sites (Punta de Tomate, Las Coloradas, and Cabo Cruz) in Cuba.

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57696, enero-diciembre 2024 (Publicado Nov. 05, 2024)

920 mm and 31 °C. Seagrape is the dominant

tree in the three sampling sites. In Las Colora-

das, the presence of trees of Thespesia populnea

(Majaguilla), Cocos nucifera, Terminalia cata-

ppa (Indian almond tree), Rhizophora mangle

(Red mangrove), and creeping plants such as

Ipomoea pes-caprae (beach sweet potato) were

observed. In Punta de Tomate trees such as

Casuarina equisetifolia, Rhizophora mangle, and

creeping plants such as Canavalia rosea (mate

of the coast) were present. In Cabo Cruz, only

creeping plants such as Ipomoea pes-caprae,

Canavalia rosea, and Sessuvium maritium (sea

purslane) shared the same habitat.

The soils of the three collection sites are

formed on limestone, especially Cabo Cruz,

where calcareous rocks predominate, mainly

limestone. Unlike Cabo Cruz and Punta de

Tomate, Las Coloradas is a beach protected

from strong winds and waves due to its location

in the Gulf of Guacanayabo, which has mostly

muddy bottoms with little contaminated sedi-

ment (Arencibia et al., 2014).

Sampling of sporocarps and EMe: The

study area was divided into two permanent

plots in each forest site. The dimensions of

the plots were 750 m2 (150 x 5). The first plot

extended from the shoreline to five meters

and the other from five to ten meters from the

shoreline, both with a length of 150 meters.

The sporocarps and ECM roots were col-

lected from both plots. Each plot was sampled

four times a year, with an interval of three

weeks between each sampling during the rainy

season, from June to September, in 2018 and

2019. With the help of a garden spade, the

sporocarps were collected, grouped according

to their morphology, photographed, and placed

separately in a woven wooden basket with

handle to be transported to the laboratory. A

fragment of fresh tissue from each sporocarp

was placed in 50 ml centrifuge tubes containing

silica gel, labeled with the sample number and

date and place of collection until DNA extrac-

tion. The voucher specimens were deposited

in the herbarium of the Laboratory of Abiotic

Stress of the Center for Plant Biotechnology

Studies of the University of Granma (Cuba) and

the herbarium of the Laboratory of Plant Biol-

ogy and Physiology of the University of French

West Indies (Guadeloupe). The identification

of the sporocarps was carried out in accor-

dance with Coker (1939), Courtecuisse (2006),

Courtecuisse (2009), Guzmán et al. (2004),

Miller et al. (2000), and Pegler (1983).

Natural regeneration of seedlings was

observed under the crown of all mature sea-

grape trees except at the Cabo Cruz site. Each

mature tree and its seedlings covered approxi-

mately 4 m2 (2 × 2 m) in area and were distant

by at least 2 m from the other sampled trees.

Three mature trees and 30 young individuals

(1-2 months old) with two cotyledons and two

leaves, < 20 cm in height, and < 2 mm diameter

at ground level were randomly chosen and

collected during the rainy season under each

mature tree, carefully avoiding mature tree

roots that sometimes intermingled with seed-

ling roots. To sample the ECM roots of mature

trees, we carefully sampled ten soil cores (15

cm diameter and 20 cm depth, approximately

250 g of soil) under the crown where seedlings

were absent, avoiding mixing the root sizes and

reducing the damage to the seedlings. The ECM

roots of mature trees and seedlings were gently

washed separately with tap water to remove the

excess substrate. Subsequently, they were placed

in a Petri dish with water using sleeved needles

and a scalpel under a stereoscopic microscope

with 4X magnification. Special attention was

paid to the presence of any type of swelling,

color change, or defined growth pattern rec-

ognized as a fungal morphotype, based on

macroscopic characteristics, such as the color

and texture of the mantle, and microscopic

characteristics, such as the presence or absence

of hyphae, mycelial filaments, and sclerotia,

according to Thoen and Bâ (1989) and Agerer

(1991). The ECM colonization was confirmed

by observing root apex sections of each fungal

morphotype to verify the presence of the fungal

mantle and the Hartig network. A fragment

of each fresh ECM morphotype was placed in

50 ml centrifuge tubes containing silica gel,

labeled with the sample number and date and

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57696, enero-diciembre 2024 (Publicado Nov. 05, 2024)

place of collection for later identification using

molecular techniques.

DNA extraction and amplification of the

ITS region: DNA was extracted from spo-

rocarps (100 mg dry weight) and from each

ECM root tip using the DNeasy Plant Kit

(Qiagen, USA) according to the manufacturer’s

instructions. Sporocarp and EMe were sepa-

rately placed in extraction tubes containing 400

μL of extraction buffer (AP1) and 4 μL of RNase

A. The samples were incubated for 30 min at 65

°C. Thereafter, 50 μL of buffer (AE) was added

to each tube, and the extracted DNA was stored

at 4 °C. The internal transcribed spacer (ITS)

region of nuclear rDNA was amplified in a mix

containing 10 μL of Sigma Taq 5X Ready To

Go beads (buffer, dNTPs, MgCl2), 1 μL of each

primer ITS1-F (5’-CTTGGTCATTTAGAG-

GAAGTAA-3’) and ITS4 (5’-TCCTCCGCT-

TATTGATAT GC-3’) at 10 μM each, 6 μL of

water, and 2 μL of the total DNA extract. The

PCR conditions were programmed as follows:

1 cycle of 4 min at 95 °C followed by 35 cycles

at 95 °C for 30 s, 53 °C for 30 s, 72 °C for 1 min

30 s, and extension at 72 °C for 7 min. Negative

controls (no DNA template) were included to

test for the presence of DNA contamination.

The PCR products were separated by electro-

phoresis in 1 % agarose gel (Promega, Spain)

in 1x TAE buffer (0.04 M Tris-acetate, 1 mM

EDTA, pH 8.0) with 10 μL of GelRed Nucleic

Acid Stain per 100 ml of gel. DNA bands were

visualized by fluorescence under ultraviolet

light. The amplification products were sent to

Genoscreen (Lille, France) for sequencing.

Sequencing of the ITS region: PCR prod-

ucts were sequenced for both strands. Forward

and reverse DNA sequences were edited and

manually corrected to obtain the consensus

sequence using the software BioEdit (version

7.0.8). For taxonomic affiliation of EMe and

sporocarps, consensus sequences were com-

pared with the GenBank database using the

BLASTN algorithm to identify the most simi-

lar sequences. ECM fungal taxa names were

defined following the BLAST score. Species

were considered to have been identified when a

sequence presented more than 97 % full-length

similarity to sequences derived from sporo-

carps or from well-identified sequences in Gen-

Bank. Alternatively, sequences with less than 97

% identity were identified at the genus or family

level. The phylogenetic tree was built with the

MEGA 6.06 software using the maximum like-

lihood method. Representative sequences for

each ITS OTU sequence were queried against

GenBank using BLAST to define fungal taxa.

The frequency of each fungal taxon was calcu-

lated as the ratio of the number of each fungal

taxon over the total number of fungal taxa.

RESULTS

Poor diversity of ECM fungi: Sampling

revealed limited sporocarp diversity at the three

study sites (Fig. 2, Fig. 3). In all, 111 sporocarps

were collected and five ECM fungal taxa were

identified from sporocarps, including Amanita

sp., Russula sp., Inocybe sp., Scleroderma ber-

mudense, and Cantharellus sp. (Fig. 2, Fig. 3). Of

these five taxa, only S. bermudense was subhy-

pogeous, the remaining four were epigeous. All

collected sporocarps belong to basidiomycetes.

Sporocarps of the five ECM fungal species were

found in Las Coloradas (Fig. 2B), two (S. ber-

mudense and Russula sp.) were found in Punta

de Tomate (Fig. 2C), and one (S. bermudense) in

Cabo Cruz (Fig. 2D). The ECM fungus S. ber-

mudense was the most common ECM fungus

found in the three sites and represented 50 % of

the sporocarps collected (Fig. 2A).

We morphotyped 169 ECM roots and

sorted them into six EMe (Table 1, Table 2, Fig.

4). All ECM roots were sampled for subsequent

DNA analysis and were identified within six

fungal taxa, of which four were identified as

Basidiomycota (66.7) and two as Ascomycota

(33.3 %). The frequencies of ECM fungal taxa

on roots were determined and S. bermudense

appeared the most representative on root tips

of seagrape (Fig. 5). Overall, S. bermudense was

the only ECM fungus detected on sporomes

and on EMe in all sites.

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57696, enero-diciembre 2024 (Publicado Nov. 05, 2024)

A low similarity between aboveground

and belowground ECM fungi: A comparison

of the aboveground and belowground ECM

fungi revealed that S. bermudense was identi-

fied from sporocarps and ECM roots (99-100 %

homology). Cantharellus sp., Russula sp., and

Amanita sp. were not found on roots. We were

not able to sequence ITS from Inocybe sp. Some

ECM fungi found on roots, such as Tuber spp.,

Inocybe sp., and Thelephoraceae (Tomentella

Fig. 2. Frequencies of sporocarps collected under adult seagrape trees and seedlings. A. In the three sites. B. Las Coloradas.

C. Punta de Tomate. D. Cabo Cruz.

Fig. 3. Sporocarps fruiting from under seagrape. A. Russula sp. SUA06. B. S. bermudense SUA09. C. Cantharellus sp. SUA07.

D. Amanita sp. SUA01. E. Inocybe sp. SUA13.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57696, enero-diciembre 2024 (Publicado Nov. 05, 2024)

Table 1

ECM fungi associated with C. uvifera in the three coastal zones of Cuba.

Herbarium

number Fungal taxa Origin Seagrape Fungal form Size ITS

(pb)

GenBank

accession numbers

Reference sequence in

NCBI and origin

Similarity

(%)

EUA01 Inocybe sp.Cuba

(Las Coloradas)

Seedling Ectomycorrhizae 647 OQ351717 Inocybe xerophytica

(Guadeloupe)

SUA13 Inocybe sp. Cuba

(Las Coloradas)

Seedling and

mature tree

Sporocarp nd nd nd nd

EUA02 Scleroderma

bermudense

Cuba

(Las Coloradas)

Seedling and

mature tree

Ectomycorrhizae 611 OQ351718 Scleroderma bermudense Ecu446

(Guadeloupe)

100

EUA03 Tomentella sp. Cuba

(Las Coloradas)

Seedling and

mature tree

Ectomycorrhizae 635 OQ351719 Tomentella sp. Ecu210

(Guadeloupe)

EUA04 Tuber sp. Cuba

(Las Coloradas)

Seedling and

mature tree

Ectomycorrhizae 564 OQ351720 Tuber beyerlei

(Colombia)

SUA01 Amanita sp. Cuba

(Las Coloradas)

Seedling and

mature tree

Sporocarp 475 OQ351727 Amanita arenicola BA 03.12.26

(Guadeloupe)

SUA02 Russula sp. Cuba

(Las Coloradas)

Seedling and

mature tree

Sporocarp 682 OQ351728 Russula aeruginea

(North West America)

SUA03 Scleroderma

bermudense

Cuba

(Las Coloradas)

Seedling and

mature tree

Sporocarp 685 OQ351729 Scleroderma bermudense Scu14

(Guadeloupe)

SUA07 Cantharellus sp. Cuba

(Las Coloradas)

Seedling and

mature tree

Sporocarp 538 OQ351733 Cantharellus cinnabarinus

(Guadeloupe)

82

EUA05 Scleroderma

bermudense

Cuba

(Punta de Tomate)

Seedling and

mature tree

Ectomycorrhizae 592 OQ351721 Scleroderma bermudense Scu14

(Guadeloupe)

100

EUA06 Thelephora sp. Cuba

(Punta de Tomate)

Seedling and

mature tree

Ectomycorrhizae 625 OQ351722 Thelephora sp. Cu P16

(Guadeloupe)

Nd = not determined; NCBI = National Center for Biotechnology Information.

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Fig. 4. EMe collected on seagrape. A. Bright white EUA02 of S. bermudense. B. Brown EUA01 of Inocybe sp. C. Yellow EUA04

of Tuber sp. D. Brown EUA03 of Tomentella sp. E. Brown light EUA06 of Thelephora sp. F. White EUA07 of Tuber sp.

Table 2

Number of sampled individuals, putative EMe, and ECM fungal taxa, and percentage of ECM fungal taxa fruiting and found

on roots of seagrape trees and seedlings in Las Coloradas, Punta de Tomate, and Cabo Cruz.

Las Coloradas Punta de Tomate Cabo Cruz

Tree Seedling Tree Seedling Tree Seedling

Number of sampled individuals 3 30 3 30 3 0

Number of putative ECM roots 36 28 54 36 15 0

Number of putative ECM sampled for DNA extraction 36 28 54 36 15 0

Number of putative ECM sampled for Sanger sequencing 26 22 31 21 7 0

Number of sequences 24 21 22 18 3 0

Number of ECM taxa 444320

Shared ECM fungal taxa (%) (1) 75 75 100 100 0 0

Similarity (%) of ECM fungal taxa found in sporocarps and EMe (2) 11 22 16 16 33 0

(1) % of ECM fungal taxa occurring on mature trees and seedlings at each site. / (2) % of ECM fungal taxa found aboveground

and belowground at each site.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57696, enero-diciembre 2024 (Publicado Nov. 05, 2024)

sp. and Thelephora sp.), were not found as spo-

rocarps, but were present in the roots. In each

site the ECM fungal taxa on roots that matched

with sporocarps were 11 % in Las Coloradas,

16 % in Punta de Tomate and 33 % in Cabo

Cruz (Table 2).

Sequence analysis and phylogenetic group-

ing of the ITS region was further used to

identify the unknown fungal symbionts by

comparing to a previously published database

of sequences from that region in GenBank (Fig.

6). Sequences from sporocarps and ECM roots

were compared to the GenBank database using

the algorithm BLASTN to identify the most

similar ITS sequences. In all cases, sequence

similarity was 97-100 % between unknown

EMe and their closest known genera within the

family or subfamily of Basidiomycota or Asco-

mycota sequences in the GenBank database.

Morphological identification of sporocarps

were all confirmed by phylogenetic analysis

except Inocybe sp. (Fig. 6). Two EMe were close-

ly related to the Thelephoroid taxa (Tomen-

tella and Thelephora), two to Tuber, and one to

Scleroderma (Fig. 5). Phylogenetic analysis as

displayed by the high bootstrap values shown

in the neighbor-joining-based tree in Fig. 6

also demonstrates that the different species fall

into the seven ECM fungal lineages proposed

by Tedersoo et al., (2010), including six from

Basidiomycota (/russula-lactarius-lactifluus, /

amanita, /cantharellus, /thelephora-tomentella,

/pisolithus-scleroderma, /inocybe) and one

from Ascomycota (/tuber-helvella) (Fig. 4).

Based on phylogenetic analysis and % of simi-

larity (99-100 %), only three common ECM

fungi compared to what was found using Blast,

one sporocarp S. bermudense and two EMe of

Inocybe sp. EUA01 and Thelephora sp. EUA06

of Cuba matching with Inocybe xerophytica and

Thelephora sp. Cu P16 of Guadeloupe, respec-

tively (Fig. 6).

Many ECM fungi are shared by mature

seagrape trees and seedlings: Of the six ECM

fungal taxa identified on roots, four were shared

between mature trees and seedlings (Table

2, Fig. 5). The other ECM fungi, Inocybe sp.

EUA01 and Thelephora sp. EUA06, were found

only on seedlings in Las Coloradas and mature

trees in Punta de Tomate, respectively. Two

fungal taxa belonging to Ascomycota, Tuber

sp. EUA04 and EUA07, were found on roots

of mature trees and seedlings in Las Coloradas

and Punta de Tomate, respectively (Table 2). In

all, mature trees and seedlings share between 75

Fig. 5. Relative frequencies of ECM fungal taxa on seagrape at Las Coloradas, Punta de Tomate, and Cabo Cruz.

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57696, enero-diciembre 2024 (Publicado Nov. 05, 2024)

and 100 % of the fungal taxa in Las Coloradas

and Punta de Tomate respectively (Table 2).

DISCUSSION

Poor diversity of ECM fungi: The lim-

ited diversity and uneven distribution and fre-

quency of ECM fungi in the three sampling

sites, based on sporocarp sampling, coincides

with what was observed by Séne et al. (2015)

at four sampling sites on the island of Guade-

loupe (France). In that study of 546 collected

sporocarps only seven species of ECM fungi

were identified. The distribution of the sporo-

carps of the different species was different in

the four sampling sites and S. bermudense and

R. cremeolilacina were the most abundantly

fruiting genera at the four sites. However, S.

bermudense was the most abundantly fruiting

species in the littoral forests of the Greater

Fig. 6. Phylogenetic tree showing the relationships between ITS sequences of ECM fungi of seagrape from Cuba (in bold)

compared to reference sequences in GenBank. The geographical locations of ECM fungi are indicated in square brackets.

GenBank accession numbers are indicated in brackets. Host plants are underlined. The herbarium numbers “EUA” represent

EMe and “SUA” sporocarps. Three common ECM fungi compared to what was found using Blast, one sporocarp S.

bermudense and two EMe of Inocybe sp. EUA01 and Thelephora sp. EUA06 of Cuba matching with Inocybe xerophytica and

Thelephora sp. Cu P16 of Guadeloupe, respectively. The phylogenetic tree was rooted with strains of Funneliformis mosseae

and Glomus mosseae.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57696, enero-diciembre 2024 (Publicado Nov. 05, 2024)

Antilles (Cuba) and the Lesser Antilles (Marti-

nique and Guadeloupe) (Bâ et al., 2014; Séne et

al., 2015). This result coincides with the report

that S. bermudense is an ECM fungus associated

with seagrape and follows its distribution in the

tropics (Guzmán et al., 2004).

Given the few sites investigated in Cuba,

it is difficult to identify the causes of the poor

diversity of ECM fungi and the differences

between the three collection sites in terms of

the presence of sporocarps of ECM fungi, but

this behavior could be related to the properties

of the soil, particularly salinity, and the average

annual rainfall at the three sites. Other authors

also suggest that the distribution of ECM fungi,

like many vascular and nonvascular plants, is

largely defined by adaptation and competi-

tion for niches related to stress tolerance (e.g.,

drought, soil acidity) and resource availability

(especially organic N, NH4+, and NO3−) in soils

(Dickie et al., 2002; Koide et al., 2005).

Precipitation could also structure the diver-

sity, distribution, and fruiting of ECM fungal

species in the littoral forest where soil water

potential partly depends on the levels of annual

rainfall and salinity, which may be two impor-

tant factors structuring the ECM fungal com-

munity (Aučina et al., 2011; Séne et al., 2015).

Alternatively, the low diversity of ECM

fungi from sporocarps and ECM roots observed

in Cuba and Guadeloupe may be the result of

the adverse environmental conditions in which

seagrape grows, in sandy, calcareous, and rocky

soils with a low percentage of organic matter

and high levels of salinity (Bullaín et al., 2022;

Bullaín-Galardis et al., 2023; Parrotta, 2000;

Séne et al., 2015). Additionally, Bâ et al. (2014)

observed that the number of mycorrhizal roots

was almost three times greater in seagrape trees

that grew in soils with a lower level of salin-

ity (0-2 %) than those that grew in soils with

a higher level of salinity (2-15 %). They also

observed that the amount of ECM fungi col-

lected under seagrape was lower than under

mature trees of C. swartzii and C. pubescens that

grew at a greater distance from the coastline

with a lower level of salinity.

Another possible cause may be the fact that

seagrape is the only ECM host of the investi-

gated sites. Ishida et al. (2007) suggest there is

evidence that the number of host tree species

drives fungal diversity. On the other hand, it is

well known that there are various levels of spec-

ificity on the fungal side of the ECM symbiosis

(Borowicz & Juliano, 1991; Molina et al., 1992).

A low similarity between aboveground

and belowground ECM fungi: The poor simi-

larity between aboveground and belowground

ECM fungi in this research could be the result

of insufficient sampling, since Arnolds (1991)

states that the type and number of ECM spo-

rocarps reflect the belowground abundance of

mycorrhizas to a large extent, but exceptions

occur. Alternatively, Lang and Polle (2008)

report that belowground mycorrhizal diversity

drives aboveground diversity. In this sense, it

is well known that the Thelephoraceae fam-

ily rarely produces sporocarps but they com-

monly form EMe on roots of trees (Kõljalg

et al., 2000). The family Thelephoraceae is

very common worldwide, but rare and often

inconspicuous in terms of sporocarps. Its fungi

are frequent in species-poor ECM fungal com-

munities of tropical rainforests (Bâ et al., 2012;

Diédhiou et al., 2010; Furtado et al., 2023) and

dominate on Nyctaginaceae spp. from South-

ern Ecuador (Henkel et al., 2005). According

to Furtado et al. (2023), the members of this

family establish ectomycorrhizal associations

with low fungal diversity in the Neotropics, but

in a study carried out by Alvarez et al. (2018),

Nyctaginaceae was the family with the highest

number of ECM host species. Our study has

once again confirmed that belowground ECM

fungal diversity from ECM roots is dissimilar

from that of aboveground sporocarps (Ebenye

et al., 2017; Séne et al., 2015; van der Heijden

et al., 1999). Therefore, neither the diversity

nor abundance of aboveground ECM fungi can

be used to assess belowground ECM diversity

or abundance (van der Heijden et al., 1999),

underlining the importance of molecular analy-

ses for the assessment of ECM fungal diversity.

However, the absence of Amanita sp., Russula

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57696, enero-diciembre 2024 (Publicado Nov. 05, 2024)

sp., Inocybe sp., and Cantharellus sp. on roots

suggests that a better view of the ECM fungal

diversity is achieved by combining sporocarps

and ECM roots surveys.

The two ECM fungal taxa belonging to the

genus Tuber had not previously been reported

from seagrape forests in the Lesser Antilles and

the Greater Antilles (Bâ et al., 2014; Séne et

al., 2015). This may be related to the criterion

that host diversity contributes to ECM fungal

diversity (Ishida et al., 2007), as the greatest

speciation and radiation of the genus Coc-

coloba in the Antilles occurred in Cuba, and

Eastern Cuba, with 26 species, of which 15

are endemic, is considered the main center of

diversification on the largest island of the Antil-

les (Castañeda, 2017).

The low number of species of ascomycetes

detected is likely due to their specific ecological

requirements more than a methodological issue

(Tedersoo & Smith, 2013). Previous studies in

tropical ecosystems also found low diversity of

ECM ascomycetes (Diédhiou et al., 2010; Ebe-

nye et al., 2017; Henry et al., 2015). The ECM

fungus Scleroderma bermudense was the most

common fungal taxa on roots of mature trees

and seedlings whatever the studied site (Fig.

5). This ECM taxon may form potential com-

mon mycorrhizal networks (CMNs) between

these two cohorts. Similarity of ECM fungal

taxa composition between mature trees and

seedlings has often been reported in temperate

(Aučina et al., 2011) and tropical forests (Diéd-

hiou et al., 2010; Ebenye et al., 2017; Séne et al.,

2015). For instance, mature trees and seedlings

of dominant seagrape forests shared three ECM

fungal taxa representing 80% of the ECM colo-

nization (Séne et al., 2015). In a mixed tropical

rainforest in Guinea, ECM fungi shared by

mature trees and seedlings represented 79 % of

the ECM colonization (Diédhiou et al., 2010).

The CMNs and their impact on the nutrition,

growth, and fitness of regenerating seedlings

should be further investigated experimentally

in seagrape coastal forests.

As in Guadeloupe, sampling at the three

coastal collection sites in Cuba showed that

sporocarps of ECM fungi associated with sea-

grape only weakly reflected the belowground

ECM fungal community, although some fruit-

ing species were also found on roots. The diver-

sity of ECM fungi associated with seagrape was

rather limited, which is also true for other ECM

plants of the Polygonaceae family. In seed-

lings and mature trees, the same representa-

tives of the ectomycorrhizal fungal community

coincide with S. bermudense, Thelephoraceae

(Tomentella and Thelephora), and Tuber spp.

predominating in the roots of both cohorts,

which may allow seedlings to join the poten-

tial CMN existing under mature trees, where

S. bermudense forms the main potential CMN

between mature trees and seedlings. The role

of CMNs in the regeneration of seagrape seed-

lings remains to be clarified. Given the few sites

sampled and investigated in Cuba, it is difficult

to make broader comments on the differences

in sporocarps and EMe in the whole territory

of the largest island of the Antilles. Thus, more

exploration and sampling work should be car-

ried out to contrast the results obtained.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENTS

We thank Editor and both reviewers for

correcting the manuscript. We are grateful for

the support of the French Embassy in Cuba,

the Laboratory of Plant Biology and Physiol-

ogy of the University of the Antilles and the

Laboratory of Abiotic Stress of the Center for

Plant Biotechnology Studies of the University

of Granma.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57696, enero-diciembre 2024 (Publicado Nov. 05, 2024)

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