Iry Andrianjara
email: iry.andrianjara@paris.fr

Marianne Bordenave-Jacquemin

Virginie Roy

Cécile Cabassa

Pierre Federici

David Carmignac

Yoan Marcangeli

Germinal Rouhan

Mathilde Renard

François Nold

Jean-Christophe Lata

Patricia Genet

Séverine Planchais

Urban tree management: Diversity of Tilia genus in streets and parks of Paris based on morphological and genetic characteristics

Keywords: green space management, microsatellite markers, morphological identification, urban ecology

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Author's contribution I.A. led the writing of the manuscript, acquired, analysed and interpreted data; M.B-J. analysed, determined morphological identification, wrote and revised manuscript; V.R. analysed and interpreted genetic data; G.R. contributed to morphological identification and revised manuscript; C.C. and J-C.L. designed sampling and revised manuscript; P.F. contributed to the acquisition and analysis of genetic data; D.C. contributed to harvest and prepare samples; Y.M. contributed to harvest and prepare samples and conceived graphical abstract; M.R. and F.N. revised manuscript; P.G. and S.P. conceived the ideas and designed methodology, harvested samples, analysed and interpreted data, contributed to the writing and supervised the research group. All authors gave final approval for publication.

Introduction

In a world of ever-expanding urbanization, urban trees contribute to ecosystem service bundles including the mitigation of urban heat islands or the improvement of air quality [START_REF] Livesley | The urban forest and ecosystem services: impacts on urban water, heat, and pollution cycles at the tree, street, and city scale[END_REF]. These services depend on morphological and physiological behaviors of urban trees and their adaptation to urban pressures such as space constraints, soil compaction, limitation in the access to water and air pollution [START_REF] Roman | Street tree survival rates: meta-analysis of previous studies and application to a field survey in Philadelphia, PA, USA[END_REF]. Tree species or genotypes do not respond in the same manner to these constraints and this response may change with age. This leads to potential and effective services [START_REF] Sieghardt | The abiotic urban environment: impact of urban growing conditions on urban vegetation[END_REF] or even disservices, e.g. spreading of allergenic pollens [START_REF] Zong | Assessing the composition, diversity, and allergenic risk of street trees in Qingyang District of Chengdu City[END_REF]. Moreover, for citizens and policymakers, historical or cultural/patrimonial aspects for some trees also matter [START_REF] Blicharska | Incorporating social and cultural significance of large old trees in conservation policy[END_REF]. All these aspects must be taken into consideration by managers when selecting the right tree species or genotype in order to successfully achieve, and monitor, city development plans.

Urban trees are often considered by citizens and managers as a "green patrimony". Green space managers would like to integrate criteria such as the resilience of species to future climate change and city conditions, and promote tree diversity in cities together with the use of indigenous species. Beyond media aspects, this issue is scientifically interesting. In Paris city (France), there are currently 205.311 trees (2020), originating from German and Dutch tree nurseries or from Paris municipal nurseries (DEVE -City of Paris, 2020; [START_REF] Laurian | Planning for street trees and human-nature relations: lessons from 600 years of street tree planting in Paris[END_REF]. During the 1980's and 1990's, concerns about the origin of trees were less prevalent and a large proportion came from the East of France, Italy and Germany (Personal communication, L. Chadirac, DEVE, 06/09/2020). A better control of supplies provided by tree nurseries, in particular the knowledge of tree genetics, and a clear traceability of the production methods are needed [START_REF] Wolff | Tilia cultivars in historic lime avenues and parks in the UK, Estonia and other European countries[END_REF]. There are both advantages and disadvantages of having genetically diverse trees. Diverse urban trees can facilitate the connectivity between natural or naturalized populations (birds, insects, trees) by providing diverse resources and habitats [START_REF] Dallimer | Contrasting patterns in species richness of birds, butterflies and plants along riparian corridors in an urban landscape[END_REF][START_REF] Rivkin | A roadmap for urban evolutionary ecology[END_REF]. However, this requires the planting and managing of heterogeneous batches of trees that may not meet all of the desired aesthetic standards. Planting clones also has some advantages. For example, retaining homogeneous tree alleys contributes to the prestige of famous avenues. Moreover, managers usually plant clones because, in addition to aesthetics, they are genetically tested and selected to resist urban pressures (pollution, soil compaction, etc). It also facilitates tree management, e.g. managers ensure how clones develop and when pruning is required [START_REF] Alphand | Les promenades de Paris : histoire, description des embellissements, dépenses de création et d'entretien des Bois de Boulogne et de Vincennes, Champs-Elysées, parcs, squares, boulevards, places plantées[END_REF][START_REF] Roloff | Urban tree management: for the sustainable development of green cities[END_REF]. They also have more control on the losses and financial costs of replacing clone trees. The question of the genetic diversity of trees in urban areas is therefore complex.

Among the most planted tree species in Europe, Linden are often present as ornamental trees in streets and parks [START_REF] Hansen | DNA markers provide insight about common lime in historical plantings -An example from the Royal Danish Gardens[END_REF]. In Paris city, after Plane trees and Horse-Chestnut trees they represent the third most planted species, representing more than 10% of planted trees in streets (APUR, 2010;DEVE -City of Paris, 2020). They are considered as resistant to urban stresses such as water scarcity and drought, pollution and pruning [START_REF] Sjöman | Selecting trees for urban paved sites in Scandinavia -A review of information on stress tolerance and its relation to the requirements of tree planners[END_REF]. All over Europe, different linden species are planted in cities, such as Tilia platyphyllos Scop. (large-leaved lime), Tilia cordata Mill. (small-leaved lime) and Tilia dasystyla Steven (Caucasian lime) [START_REF] Pigott | Lime-trees and basswoods: a biological monograph of the genus Tilia[END_REF]. Tilia cordata is also a parent in two different inter-specific hybrids named Tilia ×euchlora K.Koch (Crimean lime -a hybrid between T. cordata and T. dasystyla) and Tilia ×europaea L., known as common lime (a hybrid between T. cordata and T. platyphyllos), both described in [START_REF] Pigott | Lime-trees and basswoods: a biological monograph of the genus Tilia[END_REF]. Morphological identification of the different linden species is complex as leaves and fruits are not always visible when young trees are in nursery. Furthermore, when looking at adult street trees, it is often difficult to unequivocally identify species, especially for hybrids combining characteristics of both parents. This Tilia genus has even been viewed by some as 'taxonomic torture' [START_REF] Santamour | Checklist of cultivars of linden (Tilia) species[END_REF]. Knowledge of tree genetics for reliable affiliation to a particular linden species is therefore a crucial point. Different molecular biology tools are available to study Tilia species. Species boundaries amongst North American Tilia have been studied by phylogenetic analysis of plastid DNA sequences [START_REF] Mccarthy | Morphological variation in North American Tilia and its value in species delineation[END_REF]. Nuclear markers such as microsatellite repeated sequences were characterized in Tilia platyphyllos and then used to test for cross-amplication in 23 species in the Tilia genus [START_REF] Phuekvilai | Characterization of microsatellite loci in Tilia platyphyllos (Malvaceae) and cross-amplification in related species[END_REF]. These microsatellite loci were used to characterize the natural clonal propagation of Tilia americana L. in North America [START_REF] Evans | Isolated coastal populations of Tilia americana var. caroliniana persist long-term through vegetative growth[END_REF] and many other studies used microsatellite loci on planted taxa and clones of Tilia spp.

in cities over Europe. For example, in Denmark [START_REF] Hansen | DNA markers provide insight about common lime in historical plantings -An example from the Royal Danish Gardens[END_REF][START_REF] Wolff | Tilia cultivars in historic lime avenues and parks in the UK, Estonia and other European countries[END_REF], UK [START_REF] Pigott | The clones of common lime (Tilia ×vulgaris Hayne) planted in England during the seventeenth and eighteenth centuries[END_REF][START_REF] Pigott | Lime-trees and basswoods: a biological monograph of the genus Tilia[END_REF][START_REF] Wolff | Tilia cultivars in historic lime avenues and parks in the UK, Estonia and other European countries[END_REF], Belgium and the Netherlands [START_REF] Vanden Broeck | Genetic diversity loss and homogenization in urban trees: the case of Tilia ×europaea in Belgium and the Netherlands[END_REF], researchers studied the historical plantations of T. cordata and T. ×europaea. They found that, during the 17th & 18th centuries, a majority of T. ×europaea planted in avenues in England and Sweden were clones exported from Netherlands and Belgium nurseries. Their results revealed that most of T. ×europaea planted in Northwest Europe were highly uniform and belonged to few clones (Wolff et al., 2019). However, there is a paucity of information on the genetic diversity of Tilia species in Paris as compared to other European cities. Moreover, for city managers, this implies complex and costly laboratory work.

Inexpensive identification according to morphological criteria is still helpful for green space managers to determine the identity of planted Tilia sp. and for maintaining traceability in tree inventories. Organs that are most used for species determination are: leaves (shape, margin dentation, and color), hairs (presence/absence, and type), flower bracts, fruits (form, hairiness and ribs) and twigs (color and hairiness) [START_REF] Pigott | Lime-trees and basswoods: a biological monograph of the genus Tilia[END_REF]. These criteria have led to two morphological keys for Tilia species. The first morphological key took all organs into account [START_REF] Pigott | Lime-trees and basswoods: a biological monograph of the genus Tilia[END_REF] and the second morphological key for Tilia species cultivated in Western Europe was based on vegetative characteristics visible during a large part of the year [START_REF] De Langhe | Malvaceae) -Vegetative key to species cultivated in Western Europe[END_REF]. Although morphological methods are more easily applicable in the field and population genetics methods are more used in the laboratory, both provide information that will allow managers to better understand the history and origin of their linden trees in parks and streets and to adapt their management to the local context.

Moreover, morphology gives information on the plasticity due to environmental effects. The choice between morphological and/or molecular methods depends on the objectives of the study and access to laboratory techniques.

Here, we wished to increase the knowledge base on tree intra-interspecific diversity in urban areas and, as a result, improve management of these trees by taking into account their potential resilience. We also wanted to establish a clear correspondence between morphological and genetic analysis. First, the identification of different linden species was done by morphological criteria on leaves, twigs and fruits (both on fresh and dry materials). Then, by microsatellite loci analysis, different genetic groups were assigned to different species to check for intraspecific variations in each group.

Materials and methods

Study sites and sampling methods

In total, 80 linden trees from streets (named T01 to T40) and parks (named TP01 to TP40), plus 3 Tilia cordata reference trees (SU1, SU2, SU3) from Sorbonne University campus, were sampled in Paris (France) (48.8534°N; 2.3488°E) in September 2018 when leaves were fully mature. These trees were selected with city managers, across different districts, urban landscapes (streets and parks), and varying tree age.

To prepare herbarium specimens, two sun-exposed twigs, when possible with fruits, were collected from the lower part of the crown (2-5m above the ground). Three trees ("Léon Bollée" avenue) were higher and not accessible with a ladder, and were sampled at 6-7m above ground with a gondola. Best-preserved twigs with leaves and -when available -fruits were selected and dried by pressing samples within folded newsprints for several days. They were then mounted on white paper labelled with their inventory number, localization description and collection date.

For genetic analysis, leaves were sampled and pooled in a plastic bag labelled accordingly.

Leaves were washed, towel-dried and then frozen in liquid nitrogen and stored immediately at -20°C.

Age of trees was determined using dendrochronology by estimating the number and width of growth rings and abnormal wood rings for each tree. Tree wood cores were collected in 80 linden trees with a sterilized Pressler increment borer (length: 400 mm; diameter: 4.3 mm). Due to the tree health policy of Paris municipality, only one core was sampled per tree. The dating was conducted by Xylotree (www.xylotree.fr, Saintes, France).

Morphological analysis of collected specimens

Several studies have shown that if pruning has an effect on leaves, the effect is visible on leaf area rather than on leaf shape [START_REF] Pinkard | A physiological approach to pruning[END_REF][START_REF] Rajaona | Effect of pruning history on growth and dry mass partitioning of jatropha on a plantation site in Madagascar[END_REF][START_REF] Albarracín | Responses of shoot growth, return flowering, and fruit yield to post-pruning practices and growth regulator application in olive trees[END_REF]. No information was found on the impact of pruning on leaf margins. Parisian green space managers prune trees generally every seven years (Personal communication, B. Rizzo, DEVE, 22/01/2021). The pruning history is therefore not considered as a major impact in our morphological determination. As [START_REF] Bengtsson | Variation in common lime (Tilia ×europaea L.)[END_REF] said "[…] it is of great importance to study the samples when they are fresh. Otherwise some important characters like the sheen or lustre and the micromorphology of the upper lamina may soon be lost when the leaves are dried". Therefore, photographs of fresh samples were used to compare characters that can be lost when leaves are dried. Some of the collected specimens had no reproductive organs, and therefore lacked floral and fruit characters.

Each collected specimen (fresh and dry) was assigned to one species by using both De Langhe (2005) identification key that relies solely on vegetative character together with the authoritative reference, and Pigott's world monograph (2012), which describes the characters of all vegetative and reproductive organs of the twigs. The samples were also compared to the following references: i) type specimens of Tilia species (Supp. Table 1) using high definition images accessed through JStor (https://plants.jstor.org), ii) T. cordata well-identified specimens collected from the Sorbonne university campus (Paris) (named SU1, SU2 and SU3), and iii) herbarium specimens kept in the Herbarium of the National Museum of Natural History (MNHN) in Paris, France. Finally, each character and their description were added to a database in Xper3 biodiversity collaborative management platform which helped us discriminate available traits in our specimens [START_REF] Vignes-Lebbe | Xper3: new tools for collaborating, training and transmitting knowledge on botanical phenotypes, in: Botanists of the Twenty-First Century: Roles, Challenges and Opportunities[END_REF]). An interactive key was then easily generated using the Xper3 platform tools (Supp.

Table 2). The main distinction between species is based on the characteristics of the lower laminar surfaces. Morphological identification of a tree was based on the combination of several criteria.

Microsatellite marker analyses

Genetic diversity of our 80 trees was assessed by using 9 microsatellites (Tc915, Tc7, Tc6, Tc5, Tc11, Tc927, Tc31, Tc943, and Tc937) previously characterized in T. platyphyllos [START_REF] Phuekvilai | Characterization of microsatellite loci in Tilia platyphyllos (Malvaceae) and cross-amplification in related species[END_REF] and successfully used on trees of the Tilia genus [START_REF] Hansen | DNA markers provide insight about common lime in historical plantings -An example from the Royal Danish Gardens[END_REF][START_REF] Erichsen | Genetic and clonal structures of the tree species Tilia cordata mill. in remnants of ancient forests in Denmark[END_REF]. Sequence and labelling of primers are shown in Supp. Table 3. Our references T. cordata (SU1, SU2 and SU3) collected and determined through morphological analysis were included in the same run. One T. tomentosa tree (CLT01) from the arboretum of Versailles-Chèvreloup (Yvelines, France; Latitude: 48°50'1.02"N, Longitude: 2° 6'7.88"E) was added as a reference, and its allele sizes are shown in Supp. Table 4.

DNA extraction

Frozen leaves were ground in liquid nitrogen. Fifty mg of leaf powder was dissolved in 800 μl of CTAB (CetylTrimethylAmmonium Bromide) buffer and DNA extraction was done as described by [START_REF] Phuekvilai | Characterization of microsatellite loci in Tilia platyphyllos (Malvaceae) and cross-amplification in related species[END_REF]. Since our leaf samples contained mucilages, CTAB buffer was heated to 60°C at the first step of DNA extraction. After DNA precipitation, pellets were resuspended in 100 μl of water. DNA concentration was estimated at 260 nm and its purity at 280 nm (NanoPhotometer, Implen®).

PCR

Forward primers were 5' labeled with NED, PET, VIC or 6-FAM dyes (Life technologies; Supp. Table 3). PCR amplifications were performed in 10 μL reactions containing 10 ng of DNA, 1× reaction buffer, 0.2 mM of each dNTP, 0.4 µM of each primer, and 0.5 U of GoTaq G2 DNA polymerase (Promega). PCR program was 95°C for 5 min, followed by 15 cycles of 95°C for 15 s, 54°C for 15 s, and 72°C for 15 s, and 20 cycles of 89°C for 20 s, 52°C for 20 s, 72°C for 20 s, and a final extension step of 72°C for 30 min. DNA amplification was made in three multiplexes combining different primers and fluorochromes (M1: Tc915, Tc7, Tc6 and Tc5; M2: Tc11 and Tc927; M3: Tc31, Tc 943, and Tc937). Samples were diluted 1:10 in Hi-Di Formamide (Life Technologies SAS), a size standard was added (Genescan 600 LIZ size standard v2.0, Life Technologies SAS) and runs were made in a Genetic Analyzer from Applied Biosystems (AB3500). Length of alleles at each locus was determined by using GeneMapper® software (Applied Biosystems). Raw data were analysed with AutoBin v 0.9 to homogenize allele sizes [START_REF] Salin | AutoBin[END_REF].

Ploidy estimation

Because some Tilia species are known to be polyploid, determining allele copy number in partially heterozygous genotypes is challenging [START_REF] Dufresne | Recent progress and challenges in population genetics of polyploid organisms: an overview of current state-of-the-art molecular and statistical tools[END_REF]. We used the POLYSAT version 1.7 package in R to determine the maximum number of microsatellite alleles and to estimate ploidy for each individual [START_REF] Clark | POLYSAT : an R package for polyploid microsatellite analysis[END_REF]. The ploidy results were used for exporting the data (Supp. Table 4) for subsequent genetic analyses.

Cluster analysis

In an exploratory phase, microsatellite data were used for delimiting putative species, i.e. partitioning sampled individuals into putative species without a priori knowledge about the number of species [START_REF] Hausdorf | Species delimitation using dominant and codominant multilocus markers[END_REF]. For this, we used a Bayesian clustering approach implemented in the software STRUCTURE 2.2 [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF]. As STRUCTURE is searching for groups of individuals that are in approximate Hardy-Weinberg equilibrium, a condition that is not realized in planted trees, it was used in a first approach to evaluate the number of genetic groups and each individual probability of belonging to a genetic group. Genetic clusters proposed by STRUCTURE were seen as species hypotheses (i.e. groups whose members share similar patterns of variation) that were re-evaluated with morphological characters. Simulations were run with an admixture model, allele frequencies correlated, a burn-in of 50,000 and 100,000 Markov chain Monte Carlo (MCMC) repetitions. In order to discard all dosage information in the input file and perform the analysis correcting for the unknown dosage, the RECESSIVEALLELES flag was set to 1 [START_REF] Meirmans | The analysis of polyploid genetic data[END_REF]. Ten iterations were run for each assumed cluster (K= 1 to (n+2), with n the number of species found during morphological identification). Structure Harvester software version 0.6.94 [START_REF] Earl | STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method[END_REF] was used to determine the optimal K-value following Delta K method [START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study[END_REF]. Once the number of clusters was defined, all iterations were aligned using the Greedy algorithms implemented in cluMPP [START_REF] Jakobsson | CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure[END_REF] and the final plot was edited using Structure Plot V2.0 [START_REF] Ramasamy | STRUCTURE PLOT: a program for drawing elegant STRUCTURE bar plots in user friendly interface[END_REF].

Clone assignment

Clones were identified by sorting allele sizes for each locus in an Excel table. We considered that our individuals were clones if they did not differ by more than two alleles at one or two loci. For each genetic cluster, we evaluated clones and their proximities to other genotypes through trees based on a matrix of genetic dissimilarities (Lynch.distance function) obtained using the POLYSAT package. The trees were constructed by Unweighted Pair Group Method with Arithmetic mean (UPGMA) with the hclust function (plot in R version 3.4.2).

Results

Morphological determination of species

Tree ages determined by dendrochronology varied between 20 and 146 years old and are distributed across all districts and local environments (parks, streets) (Supp. Table 4.). The analysis of seven major characters on our specimens compared to our references (collected specimen, MNHN herbarium, jstor database, [START_REF] Pigott | Lime-trees and basswoods: a biological monograph of the genus Tilia[END_REF] and De Langhe ( 2005)) shows that, regardless of age, our specimens could be divided into 5 morphogroups that correspond to 5 species, including two described hybrids (Table 1; Fig. 1).

Tree identification, based on several morphological characters, was therefore possible even if certain characters were sometimes absent (e.g. fruits). Uncertainty persists for only 3 trees: T05, T35 and T36. The T05 and T36 individuals were assigned respectively to T. dasystyla and T. platyphyllos.

The individual T35 could not be determined because only a small amount of leaf sample was available and this was used for genetic analysis.

Initial identification was based mainly on the shape of leaf margins. Indeed, T. cordata, T. platyphyllos and T. dasystyla exhibit distinct leaf margin teeth shapes, very triangular with a very acute apex in T. dasystyla, convexly triangular with an acute apex in T. platyphyllos, and convexly triangular with a blunt apex in T. cordata. Interestingly, hybrids between T. cordata and the two other species showed intermediate leaf margin shapes as compared to their two parent species (Fig. 1,D). In order to further confirm the identification, a closer examination was performed on the leaf abaxial and/or adaxial side hairs, which persist when dry (Figs 1, E andF). It is important to note that the observed leaves were at the same stage of maturity because hair color changes according to leaf stage in this species. Both T. ×euchlora and T. dasystyla exhibited numerous club-shaped brownish hairs along veins, often on both sides of the leaves, a feature generally absent in the other species, where club-shaped hairs are less numerous, restricted to the abaxial side of the leaf, smaller, and lighter colored (Fig. 1,G). Distinction between T. ×euchlora and T. dasystyla could then be based on fruit shape (Fig. 1,A). The color of twig of the year turned out to be useful (Fig. 1,C), being usually green in T. ×euchlora, and brownish in T. dasystyla. Among the studied taxa, T. platyphyllos was the easiest species to determine, with leaf veins harbouring distinctive, numerous, perpendicular simple hairs.

The shape, hardness and size of its ribbed fruits were also unique among studied species. In contrast, T. ×europaea was more difficult to identify, as its leaves closely look like those of T. cordata (Fig. 1,B). However its fruits are distinctive, similar to both T. cordata regarding their size (although slightly larger), and to T. platyphyllos regarding their shape and the presence of marked ribs (Fig. 1,A). Moreover, its leaf abaxial fascicular hairs exhibited a duller color than T. cordata ones. Finally, T. cordata could easily be identified by its reddish tufts of fascicular hairs in the vein axils on the leaf abaxial side, and by its small, easily breakable mature fruits, with slightly asymmetric apiculus, and very faint ribs.

In our tree population, there were 36 T. ×euchlora, 19 T. cordata, 18 T. platyphyllos, 6 T. dasystyla and 3 T. ×europaea (Table 2). According to these identifications, a map of the location of our samples was drawn and showed the distribution of Tilia spp. trees across different Parisian districts (Fig. 2).

Genetic profiling by using microsatellite markers 2.2.1 Genetic group repartition of urban Tilia spp.

Structure Harvester analysis found the highest Delta K value at K=2 (ΔK=208.4), followed by K=3 (ΔK=175.6), which is also interesting for analysing substructuring of the data. References for T. cordata (SU1, SU2 and SU3) and T. tomentosa (CLT01) were first integrated in this analysis (Supp.

Table 4). Since none of the individuals matched to T. tomentosa genetic profile, CLT01 is not shown in these results. For K=2, the first cluster included individuals affiliated to T. cordata, T. dasystyla, T. ×europaea and T. platyphyllos, and the second cluster included individuals affiliated to T. ×euchlora (Supp. Fig. 1). For K=3, genetic group 1 showed a clear correspondence with T. cordata, genetic group 2 with T. ×euchlora and genetic group 3 with T. platyphyllos (Fig. 3). Four individuals morphologically identified as T. dasystyla (TP11, TP31, TP32 and TP37) showed an admixed profile, with a posterior probability of assignment to genetic group 2 of 0.56 (TP11), genetic group 1 of 0.55 (TP31), genetic group 3 of 0.61 (TP32), genetic group 3 of 0.72 (TP37). The TP04 and TP07 individuals, identified as T. ×europaea showed a mixture of group 1 (T. cordata) and group 3 (T. platyphyllos) corresponding to T. ×europaea parents. Seven individuals had their genetic group not corresponding to their morphological assignation (T05, T06, T12, T33, TP03, TP19 and TP33). For example, we observed that the T06 and T12 individuals were assigned to genetic group 3, while their morphological assignments were respectively T. cordata and T. ×europaea. For K=4, a cluster may correspond to T. dasystyla, but the ΔK was 0.563 which is too low for adequate interpretation of the results (Supp. Fig. 2).

Genetic similarity

Distance trees were constructed (Fig. 4) for each genetic group/species identified by STRUCTURE analysis (K=3). Conflicting genetic data were related specifically to 4 lindens (TP11, TP16, TP32, and TP37). Morphological identification of TP16 led to T. ×euchlora; TP11, TP32, and TP37 led to T. dasystyla, but they were indexed to genetic group 3 (T. platyphyllos). Indeed, these individuals had a low genetic distance with each other in genetic group 3 (Fig. 4,C), although TP16, TP32 and TP37 were a mixture of genotypes between the three groups (Fig. 3).

Concerning genetic groups, different clonal subgroups were found in group 1 (T. cordata) as in group 2 (T. ×euchlora), no clonal subgroups were found for T. platyphyllos (Table 3, Supp. Tables 5, 6 and7). These clonal groups come from tree nurseries that probably use propagation by cuttings or layering to produce new trees instead of planting seeds [START_REF] Bengtsson | Variation in common lime (Tilia ×europaea L.)[END_REF][START_REF] Pigott | Lime-trees and basswoods: a biological monograph of the genus Tilia[END_REF]. In group 1, 6 trees (T01, T05, T07, T16, TP10, TP13) corresponded to the same clonal subgroup, TP04 and TP07 to another clonal subgroup (Fig. 4, A, Supp. Table 5). SU3 and TP06 belong to a third clonal subgroup but this is a hypothesis because of missing data (Fig. 4, Supp. Table 5). In total, group 2 showed 20 individuals belonging to the same clonal subgroup (T03, T08, T17, T23, T25, T26, T27, T29, T31, T39, T40, TP08, TP09, TP19, TP24, TP25, TP28, TP29, TP35, TP36), 2 individuals to another subgroup (T20 and TP27) and 3 individuals to a third subgroup (T24, TP40, TP30) (Fig. 4,B, Supp.Table 6).

Geographical location of the genetic profiles of the 80 trees is random inside Paris city, without distinction between streets or parks. Moreover, the same clonal group could have been indifferently planted in different locations or in the same street for several decades (Supp. Tables 5,6 and 7). For example, a clone was planted in "Louis Vicat" street (15 th district, T08, T17, T26, T27, T29, T39, T40), in "Léon Bollée" avenue (13 rd district, T03) and in a park named "Square des Epinettes" (17 th district, TP28 and TP36). Dendrochronology revealed different ages among clonal individuals, indicating possible use of the same clone for more almost fifty years (Table 4).

Discussion

Morphological vs genetic identification

Genetic variability (genotypes) results in morphological variations (phenotypes). Local environment also plays a role and can have significant impact on urban tree growth [START_REF] Kjelgren | Growth and water relations of Liquidambar styraciflua L. in an urban park and plaza[END_REF]. Genetic variability between and within a species is important because each individual responds differently to environmental conditions. For example, twig color is one of the criteria used to differentiate T. ×euchlora and T. dasystyla but one should keep in mind that twigs well exposed to light often turn from green to red [START_REF] Pigott | Lime-trees and basswoods: a biological monograph of the genus Tilia[END_REF].

Our integrative study shows that there is a good correspondence (73 out of 80 trees) between morphological criteria and genetic analysis by microsatellites. Some individuals (TP04, TP07, T12) were difficult to differentiate from their T. cordata parent but were finally assigned to T. ×europaea.

For these hybrids, one parental genome could be expressed more than the other on its progeny genome, thus exacerbating some associated characters [START_REF] Rapp | Epigenetics and plant evolution[END_REF]. During our morphological analysis, concerns were raised that we could have mistakenly considered the cultivar 'Pallida' or 'Zwarte linde' of T. ×europaea as T. ×euchlora individuals. However, our T. ×euchlora individuals had all the characters described by [START_REF] Pigott | Lime-trees and basswoods: a biological monograph of the genus Tilia[END_REF] and did not correspond to the description of these cultivars. Tree identification therefore involves several criteria, some discriminating more than others, such as fruits, leaf margins and the position and nature of hairs on the abaxial side of leaves.

We defined 5 species based on morphological characters, microsatellite analysis allowed us to show that we have only 3 genetic groups among our trees (genetic groups 1, 2 and 3 showed a clear correspondence respectively with T. cordata, T. ×euchlora and T. platyphyllos). When morphology was difficult, genetic analysis (assignment of an individual to a genetic group) although complex, costly and time-consuming, is necessary to confirm species determination [START_REF] Wolff | Tilia cultivars in historic lime avenues and parks in the UK, Estonia and other European countries[END_REF].

However, some individuals identified as T. dasystyla and T. ×europaea and whose morphology was not obvious also proved difficult to identify through genetic analysis because of their mixed profile. In our study, the number of individuals identified as T. dasystyla and T. ×europaea was low compared to the other species, making it difficult to differentiate between species with a Bayesian clustering approach.

Genetic diversity within the genus Tilia in Parisian streets and parks

Twenty trees of T. ×euchlora belonged to the same clonal group and other individuals in genetic group 2 were closely related to this clone. Often they were located within the same street, illustrating the practice of selecting a homogeneous batch to create a landscape alignment, with a particular aesthetic goal. In contrast, when clones were present in several places, scattered across Paris, they probably correspond to a clone produced in nurseries over several decades. These clones are likely easy to reproduce and appreciated by the services of the city for their aesthetic characters (Personal communication, L. Chadirac, DEVE, 06/09/2020). In our study the most planted clones was T. ×euchlora, while other studies described T. ×europaea clones in different European cities [START_REF] Hansen | DNA markers provide insight about common lime in historical plantings -An example from the Royal Danish Gardens[END_REF][START_REF] Vanden Broeck | Genetic diversity loss and homogenization in urban trees: the case of Tilia ×europaea in Belgium and the Netherlands[END_REF][START_REF] Wolff | Tilia cultivars in historic lime avenues and parks in the UK, Estonia and other European countries[END_REF]. As our sampling includes only a small number of T. ×europaea, it is difficult to compare the clone planting policy between Paris and European cities.

The fact that we have a significant proportion of clonal individuals in urban areas is not very surprising when we consider that a small number of nurseries supply the municipality with linden trees (Personal communication, L. Chadirac, DEVE, 06/09/2020). Moreover, vegetative multiplication is favoured over sowing seeds in tree nurseries because germination is often a difficult and time consuming task [START_REF] Tylkowski | Effects of dormancy breaking in stored seeds on germinability and seedling emergence of Tilia platyphyllos[END_REF]. Nevertheless, tree production from seeds is possible for species with easier seed germination, leading to more genetic diversity such as for T. platyphyllos whose seeds show higher germination rates than T. cordata [START_REF] Magherini | Propagation of selected Tilia spp. by seed and semihardwood cuttings[END_REF]. The high degree of genetic similarity within urban tree species is not unique to Paris, nor to the urban environment. For example, in Fredensborg Slotshave (Denmark), old T. ×europaea were removed and replaced by trees of the same genetic profile found in commercial tree nurseries in order to restore an historical avenue [START_REF] Hansen | DNA markers provide insight about common lime in historical plantings -An example from the Royal Danish Gardens[END_REF].

Nurseries when possible should therefore develop programs dedicated to seed storage, viability and testing for clones. Indeed, promoting planting of different clonal groups of the same species would provide genetic diversity while maintaining positive aspects of clonality. Aesthetic aspects would remain the responsibility of managers.

Scientific and management perspectives on the genetic diversity of urban trees

In a context of global change, which tree species should be planted in our cities to conserve all the ecological services they provide? How can we ensure sufficient diversity? The low genetic diversity between trees of the same species probably reflects management practices that have been in place for decades. The aim was to plant, as much as possible, trees that are as similar to each other as possible, mainly for aesthetic purposes, but that are also resistant to urban challenges [START_REF] Laurian | Planning for street trees and human-nature relations: lessons from 600 years of street tree planting in Paris[END_REF]. However, poor genetic diversity could be a threat to urban trees in the case of emerging diseases, pest proliferation or an abrupt change in the environmental situation (e.g., air pollution, extreme temperatures, increased dust formation) [START_REF] Raupp | Street tree diversity in eastern North America and its potential for tree loss to exotic borers[END_REF][START_REF] Roloff | Urban tree management: for the sustainable development of green cities[END_REF]. In addition to the observance of Santamour's guidelines (1990), cities have many challenges to face in order to increase tree diversity in their streets. They need to identify new insights and progress in terms of production and purchasing practices. According to the location in the city, different possibilities of tree management can be considered. When the location is classified as an historical or cultural landscape, managers are required to maintain homogeneous alignments, taking great care of phytosanitary status of trees. In other places, plantations of diversified batches of trees (intra-and interspecific diversity) should be prioritized. By increasing tree diversity with a wise selection of species in one location, they will also support the diversity of tree-associated species (insects, herbaceous plants etc.) [START_REF] Omar | Colonization and extinction dynamics among the plant species at tree bases in Paris (France)[END_REF]. Other advantages, such as having a varied architecture for cooling and air circulation [START_REF] Vos | Improving local air quality in cities: to tree or not to tree?[END_REF] and reducing allergic risks [START_REF] Zong | Assessing the composition, diversity, and allergenic risk of street trees in Qingyang District of Chengdu City[END_REF] can also be anticipated.

Conclusion

This study showed that there is a strong correspondence between morphological identification, which is an important tool for green space managers to identify Tilia species, and genetic based determinations. Cities have developed tree inventories and open data to improve understanding of tree diversity in urban areas. Adding genetic profiles of trees to inventories could be an interesting tool for identifying where clones are planted in the same street and for evaluating potential disease risks. Only Tilia species were mentioned in this study but it is clear that similar investigations on other species is needed if we are to maintain and enhance tree diversity and improve ecosystem services in urban areas.

Figures Legends

  The existence of this clonality could lead to fruitful interactions between managers and scientists interested in comparisons of growth and resistance of trees isolated from different environments. For instance,[START_REF] Lobo | Local adaptation through genetic differentiation in highly fragmented Tilia cordata populations[END_REF] studied T. cordata scions of twelve populations originated from different geographical areas in Denmark (including Northern and Southern areas), and grown for eight years in the same orchard located in the South of Denmark. Scions from the Northern part of Denmark kept their original phenology and showed a delayed bud burst compared to those from the South. Phenotypic changes which were monitored in this study could be due to adaptation of trees to local conditions.

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Comparison of morphological characters of the five Tilia species, identified in the samples collected in streets and parks of Paris. (A): fruit; (B): general view of the second leaf from the apex of the twig of the year; (C): twig of the year; (D): leaf margin; (E): base of main veins on the abaxial leaf side; (F): minor veins on the abaxial leaf side; (G): club-shaped hairs along minor veins on the abaxial leaf side. Inset bars represent 2 mm in (A), 1 cm in (B), 5 mm in (C) and (D), 1 mm in (E), 500 µm in (F), or 200 µm in (G). In (G) plain arrows indicate club-shaped hairs, and 4-time magnified views of these hairs are included inside inset circles, corresponding to the 50 µm scale bars

Figure 4 .

 4 Figure 4. Genetic distance trees (UPGMA method) of 83 linden in Paris, separated in three genetic groups 1 (A), 2 (B) and 3 (C), determined by STRUCTURE with K=3. Individual trees in red showed an admixed profile. Name of trees are T01 to T40 (in streets) and TP01 to TP40 (in parks)

Acknowledgements I.A. research was supported by a CIFRE doctoral fellowship from Ville de Paris and A.N.R.T. We wish to acknowledge Béatrice Rizzo (City of Paris) for the access to Parisian Tilia sp, Régis Maldiney (CNRS) and Emilie Crilat (Sorbonne Université) for helping with DNA extractions and PCR expertise, Anthony Venon (INRAe) for technical help with microsatellite markers, Pauline Moretti, Valentin Puzcilewicz and Alexandre Abadie for technical and field help. The MNHN gives access to the collections in the framework of the RECOLNAT national Research Infrastructure.

Data Availability Statement

http://www.xper3.fr/xper3GeneratedFiles/publish/identification/-3498028717715930076/

Tables legends

Table 1. Character states of seven major morphological characters assigned the 83 linden trees to five morphogroups subsequently identified as the five mentioned species based on Pigott's monograph (2012)


M Pérouse

P Schultz

R Pérouse

C Debry

Laryngeal spasm and tracheostomy after hydroxyapatite injection laryngoplasty

Keywords: spasmodic dysphonia, presbyphonia, tracheostomy, laryngeal spasm, hydroxyapatite

Introduction: Adductor spasmodic dysphonia (Ad-SD) is usually treated by botulinum toxin injection into the thyroarytenoid muscles. In older patients, atrophy of these muscles is responsible for glottic leak, causing presbyphonia and is managed by vocal fold medialization using autologous fat or hydroxyapatite (HA). We report the first case of uncontrollable laryngeal spasm requiring tracheostomy after hydroxyapatite injection laryngoplasty in a patient with spasmodic dysphonia and presbyphonia.

Case report: An 83-year-old man had been treated for Ad-SD by botulinum toxin injections every six months for 14 years. Due to severely disabling glottic leak, autologous fat injection laryngoplasty was then successfully performed. Six months later, following recurrence of severe hypophonia, hydroxyapatite injection was performed, subsequently complicated by immediate acute respiratory distress secondary to adductor laryngeal spasm requiring reintubation and tracheostomy. The postoperative course was marked by gradual recovery of vocal fold mobility with decannulation on Day 12. Discussion: This case allows a discussion of the possible pathophysiological mechanisms responsible for adductor laryngeal spasm. Vocal fold medialization procedures should be performed with caution in patients with Ad-SD.

INTRODUCTION

Adductor spasmodic dysphonia (Ad-SD) is a rare condition, defined as focal laryngeal dystonia inducing irregular and unpredictable vocal fold spasms resulting in a shaky voice.

The standard treatment for Ad-SD, for more than 30 years, consists of botulinum toxin injection into the thyroarytenoid muscles [START_REF] Rumbach | Outcome Measurement in the Treatment of Spasmodic Dysphonia: A Systematic Review of the Literature[END_REF]. Thyroarytenoid muscle atrophy, a common cause of dysphonia in older people, is often treated by vocal fold medialization using autologous fat or hydroxyapatite (HA) [START_REF] Bradley | What is the optimal treatment for presbyphonia?: Optimal Presbyphonia Treatment[END_REF]. We report the first case of severe laryngeal spasm requiring tracheostomy after HA injection for severe glottic leak in a patient with Ad-SD for more than 20 years.

CASE REPORT

An 83-year-old man had been treated for 14 years with botulinum toxin (an average of one injection every 6 months) for acoustically characteristic adductor spasmodic dysphonia confirmed by nasal laryngoscopy. This treatment was effective, allowing temporary improvement of voice quality. With age, this patient had also developed thyroarytenoid muscle atrophy responsible for a whispering voice (Fig. 1a.), justifying, three months after the last botulinum toxin injection, an injection of autologous fat (2.5 mL on each side). This procedure was performed under general anaesthesia in December 2017 by an experienced senior surgeon, allowing marked functional improvement with no effect on Ad-SD (Fig. 1b) for several months.

As a result of partial resorption of the fat and recurrence of presbyphonia in March 2018 (Fig. 2a.), hydroxyapatite (1.5 mL on each side) was injected six months after the last botulinum toxin injection. This procedure was performed under general anaesthesia and under good conditions (Fig. 2b. and Fig. 2c.). After extubation, the patient presented acute respiratory distress in the recovery room with severe inspiratory dyspnoea, laboured breathing, hypercapnia and desaturation, requiring emergency re-intubation and tracheostomy. Direct laryngoscopy performed under general anaesthesia triggered severe laryngeal spasm with tight constriction of the ventricular folds, masking the glottis. (Fig. 3). Laryngeal spasm was relieved by deep general anaesthesia allowing easy intubation before tracheostomy. Nasal laryngoscopy on postoperative days 1 and 3 demonstrated complete laryngeal spasm with hyperadduction of the vocal folds, and absence of a visible airway. Small abduction movements were observed on day 5; subnormal vocal fold mobility was observed on day 7, followed by complete vocal fold mobility on day 12, allowing decannulation. A short course of intravenous methylprednisolone 120 mg was administered on days 2 and 3 and then stopped due to its obvious lack of efficacy.

DISCUSSION

This case report describes the first case of severe laryngeal spasm after hydroxyapatite injection requiring tracheostomy, which should encourage otorhinolaryngologists to exercise caution when using synthetic materials for injection laryngoplasty in patients with Ad-SD.

Transient side effects, such as a whispering voice, aspiration or even dyspnoea, can occur after botulinum toxin injection for the treatment of Ad-SD [START_REF] Novakovic | Botulinum toxin treatment of adductor spasmodic dysphonia: Longitudinal functional outcomes[END_REF]. These side effects usually resolve spontaneously over several days, allowing a completely satisfactory functional result, justifying botulinum toxin injection as the treatment of choice for Ad-SD for more than 30 years.

Several studies have also shown that treatment of presbyphonia by HA injection is an effective option, allowing long-term improvement of voice quality [START_REF] Kwon | Calcium hydroxylapatite injection laryngoplasty for the treatment of presbylaryngis: Long-term results[END_REF]. DeFratta et al. [START_REF] Defatta | Complications of Injection Laryngoplasty Using Calcium Hydroxylapatite[END_REF] described the main complications after HA injection that can occur if the substance is not injected strictly into the thyroarytenoid muscles. These authors described the absence of or decreased mucosal vibration and the presence of granulomas on the free edge as major complications, and oedema, erythema and ecchymosis of the vocal folds as minor complications, but no cases of laryngeal spasm requiring tracheostomy have been previously reported in the literature. This case, presenting a combination of Ad-SD and hydroxyapatite injection, raises a number of questions: i) why was no laryngeal spasm observed after autologous fat injection, despite the injection of a much larger volume than that of hydroxyapatite? (ii) Is this phenomenon similar to paradoxical vocal fold adduction syndrome? (iii) Although Ad-SD is considered to be due to a central mechanism, can it also be related to peripheral disorders mediated by laryngeal efferents, as suggested by recent studies [START_REF] Murry | Spasmodic Dysphonia: Let's Look at That Again[END_REF][7]?

The pathophysiological mechanisms responsible for spasmodic dysphonia have been regularly revised over the last 50 years [START_REF] Murry | Spasmodic Dysphonia: Let's Look at That Again[END_REF]. Moore [START_REF] Moore | Organic Voice Disorders[END_REF], as early as 1971, noted that spasmodic dysphonia was a neurological disorder and not a psychological disorder, as had long been believed. Since the work by Robe [START_REF] Robe | A study of spastic dysphonia. Neurologic and electroencephalographic abnormalities[END_REF], it is generally accepted that spasmodic dysphonia is the result of neuronal dysfunction. Berardelli [START_REF] Berardelli | The pathophysiology of primary dystonia[END_REF] also showed that focal dystonia involves basal ganglia dysfunction as well as sensory disorders.

Ad-SD is currently defined as a multifactorial condition combining, among other things [START_REF] Ludlow | Research priorities in spasmodic dysphonia[END_REF]: a genetic predisposition and environmental factors, dopaminergic dysfunction and dystonic movement disorders, and decreased inhibition of laryngeal adductor muscles to superior laryngeal nerve stimulation. According to Ludlow [START_REF] Ludlow | Research priorities in spasmodic dysphonia[END_REF], the superior laryngeal nerve is stimulated via multiple sensory receptors in the larynx, in which the role played by the various neurotransmitters that modulate the motor response has yet to be determined. Superior laryngeal nerve section in patients with Ad-SD appears to induce significant improvement of symptoms, clearly demonstrating the presence of a peripheral vocal fold proprioception defect.

The present case report also suggests a peripheral mechanism: injection of a substance (hydroxyapatite) known to induce major mucosal immobility reactions in a patient whose laryngeal sensory receptors transmit aberrant information (hyperstimulation, lack of inhibition, ...) [START_REF] Ludlow | Research priorities in spasmodic dysphonia[END_REF][START_REF] Berardelli | The pathophysiology of primary dystonia[END_REF], resulted in severe, non-inflammatory laryngeal spasm. This hypothesis is supported by the inefficacy of corticosteroid therapy.

We do not believe that the patient's disorders were due to excessive vocal fold medialization, as nasal laryngoscopy on postoperative days 1 and 3 clearly demonstrated the laryngeal spasm (invisible glottis), while abduction movements were observed on day 5, revealing the glottis, and finally decannulation was able to be performed on day 7. It would have been impossible for HA to have been resorbed in only 7 days. Laryngeal spasm was therefore responsible for the patient's dyspnoea.

CONCLUSION

Hydroxyapatite injection laryngoplasty in a patient with spasmodic dysphonia must be performed very cautiously with close monitoring, as it can cause severe laryngeal spasm. This case is consistent with data from the literature, which tend to indicate that Ad-SD is a condition combining a central dopaminergic disorder with alteration of peripheral laryngeal receptors. Further studies are needed to confirm these hypotheses.

Conflicts of interest

Fig. 1a .

 1a Fig. 1a. Thyroarytenoid muscle atrophy before autologous fat injection.

Fig. 1b .

 1b Fig. 1b. Laryngeal closure immediately after autologous fat injection.

Fig. 2a .

 2a Fig. 2a. Subtotal fat resorption (6 months later).

Fig. 2b .

 2b Fig. 2b. Intraoperative appearance after hydroxyapatite injection (in adduction).

Fig. 2c .

 2c Fig. 2c. Intraoperative appearance after hydroxyapatite injection (in abduction).

Fig. 3 .

 3 Fig. 3. Laryngeal spasm on recovery after hydroxyapatite injection.

The authors have no conflict of interest to declare.