Assessing Biodiversity Associated with Four Monumental Trees in Madrid Region (Spain)

Molecular and Data Analysis

Molecular biology tools were used to support or extend latent information about fungi (including yeasts and lichens) and bacteria from phyllosphere and endosphere. Two samples were taken from canopy leaves in autumn and spring and one more from branch wood obtained under sterilised conditions (three samples per tree). We used 50 to 100 mg of mixed fragments from 4 leaves with different orientations (N, S, W, E) or 50 to 100 mg of branch sawdust. The DNA was extracted using the ZymoBIOMICS™ DNA Miniprep Kit (Zymo Research Corporation, Irvine, CA, USA) which includes a physical rupture method with glass beads of 0.1 and 0.5 mm. PCR amplification was made from 16S rRNA with primers 27F and 1492R for bacteria and from ITS (internal transcribed spacer) rDNA with primers ITS1 and LR5 for fungi (Heuer et al. 1997; Lindow and Brandl 2003; Stielow et al. 2015). Purified product was ligated with the Native Barcoding Kit SQK-NBD114.96 and sequenced with Oxford MinION portable nanopore sequencing device (MinION FLOMIN114 R10.4.1 flow cells)(Oxford Nanopore Technologies plc., Oxford, United Kingdom)(cf. Lu et al. 2016; Jain et al. 2017; Pavlovic et al. 2021; Ni et al. 2023). Read sequences were filtered by size (1.300 to 1.800 bp for rRNA 16S gen, no size filter for ITS region) and selected when quality scores were at least 15 with Nanofilt (Nanopack tools)(De Coster et al. 2018). Chimeras were removed during amplification process with VSearch bioinformatic tool (Rognes et al. 2016). EMu software (Axiell, Lund, Sweden) was used with 16S RNA database for taxonomic assignment in bacteria while ITS sequences were mapped against the fungal reference genome within the UNITE database (Kõljalg et al. 2013; Schoch et al. 2020; Abarenkov et al. 2021; Curry et al. 2022). Species with less than 10 sequence reads were not reported in the analysis except when few taxa were identified in the samples.

Additional sequences were obtained from mycelium of wood cultures to check their identity. PCR amplification was made from ITS rDNA with primers ITS1 and ITS4 (Martin and Rygiewicz 2005; Schoch et al. 2012) with the following PCR conditions: 95 °C for 15 min followed by 40 cycles of 95 °C for 30 s + 54 °C for 20s + 72 °C for 45s and a final step of 72 °C for 8 min with the TaqGold polymerase (Applied Biosystems; Thermo Fisher Scientific Inc., Waltham, MA, USA). PCR product was purified (DNeasy Plant Mini Kit; Qiagen, Venlo, The Netherlands) and checked in agarose gel and then sequenced with the BigDye® Terminator v3.1 kit (Applied Biosystems; Thermo Fisher Scientific Inc.) and capillary electrophoresis in an ABI 3730xl sequencer (Thermo Fisher Scientific Inc.). Sequences obtained were checked by pairwise comparison with the NCBI-Genbank sequence database (BLAST tool)(Altschul et al. 1990) to identify species.

Scanning electron microscopy pictures of insects, bacteria, and fungi were made with a JEOL JSM-IT500 InTouchScope™ SEM microscope (Jeol Ltd., Akishima, Tokyo). Samples were previously treated with critical point technique (Bray 2000).

Fauna Survey

An inventory and diagnosis of the insect communities directly or indirectly associated to the tree as food or shelter depending on their ecological role was made (phytophagous, xylophagous, saproxylic, radicivorous, corticolous, mycetophagous, sap-sucking, gallicolous, anthophagous, carpophagous, nectarivorous, parasitoids, predator species, etc.). Special attention was paid to the Hymenoptera: Anthophila and Symphyta, Lepidoptera, Coleoptera, and Diptera groups. These groups are involved in wood decay, pollination, and upper trophic relationships in mature trees. Moreover, some of them are mature tree bioindicators (Dajoz 2001; Marcos García and Ricarte Sabater 2009; Aguado Martín 2018; Bouget et al. 2019; Falk 2021; Wermenliger 2021).

The sampling techniques used were active or passive. Active techniques included the use of entomological sweep net, gill harvest, and insect capture into humus. Also, wood fragments with insect larvae were collected for ex situ culture in a terrarium (50-L transparent plastic box). Passive techniques were carried out using pitfall traps (buried plastic cups 8 cm in diameter and 10 cm in depth, two-thirds bait filled), baited traps, or traps with attractant in the canopy and wood emergence cavities. The baits used were acetic acid (5%) in pitfall traps and vaporized trementine (100%) in emergence cavities. Canopy traps were baited with α and β-Pinene as well as Cerambyx pheromones with a few meters scope to avoid incoming pests outside the targeted tree. All baits were replaced and traps cleaned when trees were visited. Insect collection was completed for unidentified species which needed a detailed taxonomy study. Insect identification was performed under a Meiji EMZ-10 stereo microscope (Meiji Techno Co. Ltd., Tokyo, Japan).

We checked macrofauna diversity (mammals, birds, reptiles) during sample visits. Birds were checked by using binoculars during fieldwork at 80 to 100 m from the tree. Infrared photo-trapping cameras were installed on trees outside urban environments and indirect evidence was also checked (nests, feathers, clutches, droppings, etc.).

Plant Survey

The study of terrestrial bryophytes and algae was done on the surface of branches and bark. The procedure was similar to that used in mycology, since once collected and dried they can be rehydrated later for identification as they are reviviscent. Individual collection in plants was only done for unidentified species which needed detailed taxonomy study.

Macrofungi and Lichen Survey

The mycological and lichenological study was similar in its approach to the entomofauna study: we selected about 7 to 14 days after rain to sample macrofungi. Ecological relationships were also analysed, being direct or indirect and acting as saprobes, parasites, or mutualists that interact with the tree in their category (foliicolous, corticolous, lichenised, mycetophilus, xylophagous, phytopathogenic, mycorrhizal, etc.).

Samples were taken from different parts of the tree in order to study both surface fungi (bark and wood of trunk and main branches, fallen branches, twigs or dead leaves, and humus) and endophytes (living leaves for phyllosphere, and living twigs for endosphere). Particular attention was paid to the presence of cankers, wounds, and hollows, as well as to areas showing signs of functional weakening.

Cutting instruments (chainsaw, pruning shears, razor, hand saw) were disinfected with 96% ethyl alcohol between small cuts to avoid contamination between samples and in the tree. Samples intended for culture were waxed on the cuts and stored in self-sealing plastic envelopes and kept in a refrigerator until the laboratory study.

Material for microscopic identification was placed in paper envelopes, and, when the water content was high, as in the case of carpophores, they were cut and dehydrated (Stöckli dehydrator, 50 °C for 12 hours) (A. & J. Stöckli AG, Netstal, Switzerland). For hyphomycetes and microscopic fungi, we used fungi plate cultures with Potato Dextrose Agar medium (PDA 3.9% Merck, 9-cm diameter cultivation Petri plates) and wet chamber cultures (crystal tuppers 14 × 20 × 6 cm with 25-ml added sterile water on dead leaves/wood fragments, sample included on site). Petri and wet chamber cultures were stored at room temperature with ambient indirect light; we checked for fungal colonies and fructifications every 2 days under a Faster SafeFAST (FASTER S.r.l., Milan, Italy) vertical laminar flow hood to avoid indoor spore air contamination.

Sampling of macrofungi was only done for unidentified species which needed light microscopy techniques (Nikon Labophot-2 microscope and Nikon SMZ745 stereo microscope)(Nikon, Tokyo, Japan), mainly corticioid fungi, small ascomycetes, and hyphomycetes from cultures. Fresh field data and microscopy analysis details were described in Daniëls et al. (2016).

Microorganism Survey (Monera and Protists)

Like fungi, bacteria were analyzed by plate culture and through molecular biology techniques from 8 samples of leaves and 4 of wood, as previously explained. Samples were washed with distilled water to avoid surface residues and then cut with sterilised scissors and scalpels, then crushed in a mortar with 0.9% NaCl water solution and put into a 50-mL Falcon tube (tissues covered with distilled water) to homogenize in a Vortex mixer. Extract was diluted to 1:100,000 in distilled water and a sample of 200 μl of the dilution was inoculated in a culture plate (Agar with Luria Bertani culture medium)(MacWilliams and Liao 2006) and then incubated at 21 °C (J.P. Selecta bench incubator)(JP Selecta S.A., Barcelona, Spain), checking growing strands daily. Isolated strands were stored at 4 °C until sequenced (Sanger PCR of 16S rRNA). A total amount of 24 bacterial cultures were made from wood and tree leaves. We also include the identification of the myxomycetes (Protists) found on the studied trees.

Protected Organisms and Endemisms

The obtained data allowed us to identify some species within the fauna and flora Directive 92/43 CEE or included within different protection categories such as the IUCN red list (i.e., van Swaay et al. 2010; Bilz et al. 2011; Cálix et al. 2018; IUCN 2024) or the Spanish red lists (i.e., Verdú and Galante 2009; López Jaime et al. 2019). Insects could constitute valuable elements as bioindicators of environmental quality due to their endemicity, rarity, or threat. Nymphalis polychloros is a rare butterfly included as vulnerable in the IUCN Red List Category (EU27)(van Swaay et al. 2010); it feeds on leaves when larvae and later on poplar fluids when adult (Aguado Martín 2007a, 2007b). Some clues of the biodiversity value of monumental trees were revealed by phototrapping. For example, the bird Jynx torquilla is included in Spain’s bird Red Book as vulnerable (López-Jiménez 2021).

The holm oak is a source of biodiversity conservation in terms of entomofauna, since 68 species develop and live in the tree. Between leaf litter and fallen wood, we found the Iberian endemic beetles Carabus lusitanicus, Cetonia aurataeformis, and Akis granulifera.

Exotic Species

Some exotic birds were found on monumental trees like the invasive Myiopsitta monachus on London plane and, possibly escaped from a cage, Nymphicus hollandicus located in the holm oak. Another exotic species is the leafhopper assassin bug (Zelus renardii) collected on the leaves of the San Isidro poplar. It is a generalist predator and a common invader of urban, anthropogenic environments and garden plants in Europe, without host plant specificity (Weirauch et al. 2012).

Biogeography and Rare Species

Some new records were found in our study. Among bacteria, Corticibacterium populi was identified on the poplar by NGS. This is a species recently described from China (Li et al. 2016) with no references from Spain.

The old branches of the poplar have some galleries of the carpenter bee Xylocopa violacea, and this is accompanied by very rare parasitoid species such as the wasp Polochrum repandum or the fly Satyromoeba etrusca; these latter 2 species are new records to the Community of Madrid. Another rare parasitoid wasp was found on the holm oak, Ctenochares bicolorus, and it could be the first published record in Madrid province (Selfa and Bordera 1995). The leafhopper assassin bug (Zelus renardii) is the third record for the province of Madrid (Tanco 2024). Pogonocherus sturanii has only 5 previous records in Madrid province (González Peña et al. 2007). Another rare bug is Acanthocinus aedilis, with declining populations in the Iberian Peninsula.

Acrogenospora sphaerocephala is a saprophitic dematiaceous hyphomycete, new to Madrid province, which grows in small twigs and branches of London plane (cf. Hernández Restrepo 2013). The basidiomycete Hohenbuehelia unguicularis has been mentioned once in Madrid (Daniëls and Borrajo Millán 2021). The ascomycete Thyronectria quercicola is a recently described species. This is the fourth world record and a second record to Madrid province (Hirooka et al. 2012; Tello Mora 2015). More macrofungi, Oedohysterium insidens and Gymnopus vernus found growing under holm oak, as well as Myrmaecium rubricosum found growing on pine needles, were first recorded to the Comunity of Madrid. Irpicodon pendulus and Byssomerulius albostramineus, growing on pine wood, are the first records in Spain, since no publication about these species were found.

Habitat and Ecological Relationships

Dead wood, cavities, and exposed heartwood of the San Isidro poplar had the highest score in biodiversity potential with a value of 17/20. However, this poplar is confined in a tree pit and fallen leaves or dead wood are often removed, so effective diversity decreases. The high diversity in the holm oak is justified by the high number of niches (15/19) present in its structure, the amount of substrate accumulated under its dense canopy (with higher humidity), and by the greater abundance of insects, which are typically favoured from an agricultural environment. The “Carretero” pine is located next to a forest track and is relatively isolated, receiving enough sunlight to dry out the ground surface quickly, especially after losing much of the canopy due to the snowstorm “Filomena”. The presence of a clear open canopy with many broken and rotting branches allows the presence of insects and birds. The value of potential diversity in this pine is 13/19. The London plane city tree is slowly declining, with regresive dead branches due to the presence of some insects and fungi with a low diversity (123 macroorganisms) and niche diversity (10/19). However, when microbiome is included in the study, diversity changes. Most taxa found on the London plane tree belonged to bacteria, reaching the maximum record with 232 different OTUs. The increase in bacteria diversity disagrees with the low rank of its niche diversity, and this could be due to contamination of urban environments (Laforest-Lapointe et al. 2017).

Some ecological relationships and niche preferences were recorded on the monumental trees. Higher numbers of microhabitats favoured insect and vertebrate diversity, especially on water filled tree holes (Yanovlak 2001; Paradise 2004; Nishadh and Anoop Das 2014; Patel et al. 2021). Syrphidae is a group of Diptera whose species are often considered bioindicators of old forests and mature trees (Marcos García and Ricarte Sabater 2009). Eristalis tenax and E. arbustorum larvae were found in the trunk or branch holes filled with water in poplar and London plane, respectively. However, green algae can also be found in this microhabitat. Oedogonium alga grew around a water filled hole in the canopy of the “Carretero” pine tree. Another green alga, Desmococcus olivaceus, covered the smooth bark of young holm oak branches. Green algae are of great ecological importance, as photosynthetic products of algae are used directly by bacteria and some animals as food material (Arora and Sahoo 2015).

Lichens and mosses are mainly found on the “Carretero” pine located in a forest environment (Evernia, Parmelina, Lecanora, Physcia, Ramalina, Rinodina, Xanthoria). There were enough foliose and fruticulose lichens to allow Marchandiomyces corallinus, a lichenicolous pathogen, to grow on Parmelina tiliacea.

Temporal distribution of ectomycorrhizal macrofungi is due to the host associations and microenvironmental factors, so many species do not fruit every year (Vogt et al. 1992; Straatsma et al. 2001). Three mycorrhizal fungi were found fruiting under the Holm oak: Lactarius zugazae, Russula amoenolens, and the earthstar Geastrum lageniforme. The pavement around the poplar and the London plane interfere with natural ectomycorrhization. No mycorrhizal fungi were found on the pine, mainly due to the short sampling period, climate conditions, scarce humus found in soil, and compaction by the path close to the tree.

Some species of bacteria were lichenicolous bacteria (Edaphobacter, Lichenibacterium, Lichenihabitans, Tundrisphaera), but many other act as decomposers on trees (Lasa et al. 2019). Bacteria from endosphere were mostly Alphaproteobacteria (73% in holm oak) with genera such as Caulobacter, Devosia, Endobacter, Methylobacterium, or Roseomonas, as well as other wood endophytes of Actinobacteria such as Amnibacterium or Jatrophihabitans (Gottel et al. 2011). On the urban London plane, wood endophytes such as Methylobacterium goesingense (Ni-tolerant), Flaviflagellibacter deserti (drought tolerant), and Variovorax paradoxus (contaminated environments) were frequent on small branches. Bacteria occupy the majority of the plant leaf surface, ranging from 10⁶ to 10⁷ bacteria/cm². Interest in phyllosphere microbiology has been driven by the need to better understand the behavior and control of the plant pathogens. Some bacteria incite frost injury, whereas others produce phytohormones. The numerous nonpathogenic microbes that inhabit the phyllosphere apparently play important roles in modulating population sizes of deleterious microbes. (Lindow and Leveau 2002).

However, a low number of genera were identified on phyllosphere (Crinalium, Gluconacetobacter, Lactobacillus, Massilia, Methylobacterium) because of plant chloroplast 16S rRNA contamination (Tian et al. 2017). Fungal phyllosphere have both epi- and endophytic yeasts of the genera Buckleyzyma, Filobasidium, Symmetrospora, Udeniomyces, and Vishniacozyma.

Most vertebrates on trees were birds. Tree cavities are often used for nesting, such as the rock pigeon (Columba livia), the scops owl (Otus scops), and the Western jackdaw (Corvus monedula) in the London plane. Storm “Filomena” created the presence of dead wood in the pine canopy which attracted insects and then many birds, such as the great spotted woodpecker (Dendrocopos major), the nuthatch (Sitta europea), or the short-toed treecreeper (Certhia brachydactyla). Most of the birds found in this tree (9/15) were insectivores. The common wall gecko (Tarentola mauretanica) was found in the poplar trunk; the presence of spaces between wood and bark to nest, and a spotlight close to the trunk to attract moths at night, improves their living conditions (Salvador 2015).

In autumn, some branches and acorns release molasses which feed some bees and wasps like Vespula germanica. It is the main food resource in this season, favouring the production of honeydew, which is essential for the survival and hibernation of queen bees in the Iberian Peninsula.

Wood Relationships

The decay of the poplar tree revealed 65 decomposers, saprophytes, or xylophages, and 13 phytopathogens. The fungi Inocutis levis and Rigidoporus ulmarius were the main responsible for tree decay. Rotting trunk with soft wood (white rot) and cavities inside led to reduced canopy height because of the structure loss and the increased risk of failure on pedestrians. Subsequent fungal saprobes (Cyclocybe cylindracea, Volvariella bombycina, Protofenestella ulmi) and secondary parasites (Cytospora chrysosperma, Nectria cinnabarina, Schizophyllum amplum) developed on branches and dead heartwood (Daniëls and Borrajo Millán 2021). Many xylophagous Coleoptera benefit from the poplar decay as they breed, feed, and take shelter in the rotten wood: Oryctes nasicornis, Dorcus parallelipipedus, Phyllognathus excavatus, Cetonia aurataeformis, Potosia cupraea, Oxythyrea funesta, Tropinota squalida, and Saperda carcharias. The high amount of rotten wood also benefits mycetophagous Coleoptera like Xyleborinus saxesenii. Two rare saproxylic Diptera emerged from cavities of ex-situ decayed wood in terrarium: Ferdinandea cuprea and Myathropa florea. The butterfly Paranthrene tabaniformis placed their eggs directly on the pruning cut areas, attracted by the volatile components of the fresh wood. Similarly, the wasp Tremex fuscicornis also lay eggs on wood, inoculating at the same time saproxylic fungi with their mycangia to improve larvae feeding through a symbiotic association (Marčiulynas et al. 2024).

In the “Carretero” pine, thick branches were colonised by saproxylic fungi like Stereum sanguinolentum, Leucogyrophana mollusca, Lentinellus micheneri, Peniophorella pallida, or Tubulicrinis glebulosus. Xilophagous beetles such as Chalcophora massiliesis, Pogonocherus sturanii, Buprestis haemorrhoidalis, Ergates faber, Spondylis buprestoides, and Monochamus galloprovincialis were also found. The latter species usually act as spreading vectors of the pinewood nematode pest Bursaphelenchus xylophilus and also of many filamentose fungi (Jankowiak and Rossa 2007; Naves et al. 2016). Great spotted woodpeckers (Dendrocopos major) nested on this tree, with some old holes in the rotting wood.

The abundance of dead small branches on the holm oak favours the presence of several endophytic saproxylic fungi such as Peniophora quercina, Eichleriella leucophaea, Byssomerulius corium, Stereum hirsutum, Merismodes anomalus, or Vuilleminia comedens. Twenty-five phytopathogen species were recorded in this healthy tree. Phytoparasites on trees are mostly detected by DNA analyses as latent endophytes in asymptomatic samples, so their richness does not necessarily imply an effective disease on the tree.

London plane was previously pruned and its wood removed before field work, so niche was reduced for dead saproxylic organisms. Among saproxylic fungi, we recorded Auricularia auricula-judae, Coprinellus truncorum, Pluteus cinereofuscus, and Schizophyllum commune. Some saproxylic and cortical insects were i.e., Bostrychus capucinus, Valgus hemipterus, or Uleiota planata. This latter species is used as a bioindicator of mature trees.