Nsenga Salvatora

Pierre Nkulu

Edouard Meerts

Wa Ilunga

Mylor Ngoy Ilunga

David Shutcha

Bauman

Salvatora Nsenga Nkulu
email: messinkulu@gmail.com

Pierre Meerts

Mylor Edouard Ilunga Wa Ilunga1

Ngoy Shutcha

David Bauman

Salvatora Nsenga

Nicolas Barbier

Medicinal Vitex species (Lamiaceae) occupy different niches in Haut-Katanga tropical dry woodlands

Keywords: aluminium, conservation, indicator species, medicinal, miombo, niche, soil, termite mound, tropical dry woodlands, woody species

come

INTRODUCTION

The provision of medicinal plants is one of the important ecosystem services or nature contribution to people supplied by tropical forests and woodlands. Indeed, 70% of the population in sub-Saharan Africa uses plant medicinal resources as the access to the 'modern' healthcare system is very limited due to the lack of suitable infrastructure and the low incomes [START_REF] Foulon | Quel système de financement de l'accès aux soins des populations dans les PED ? Le cas des districts de santé au Burkina Faso[END_REF]OMS 2011). In addition, medicinal plants are also known to develop value chains and contribute to the income of some households, especially those in rural areas (OMS 2011). Unfortunately, the African and global IPBES assessments have shown a decline in the supply of medicinal resources due to direct and indirect factors [START_REF] Archer | Summary for policymakers of the regional assessment report on biodiversity and ecosystem services for Africa of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services[END_REF].

The same situation occurs in the Haut-Katanga province (D.R. Congo) where medicinal woody plants, widely used by the urban and rural dwellers, are declining rapidly because of the reduction of woodland or their intensive exploitation [START_REF] Cabala | Dynamique des écosystèmes forestiers de l' Arc Cuprifère Katangais en République Démocratique du Congo. Causes, transformations spatiales et ampleur[END_REF]. This is the case for Vitex fischeri Gürke var. fischeri, Vitex mombassae Vatke, and Vitex madiensis Oliv. subsp. milanjiensis (Britten) F.White (Lamiaceae) around Lubumbashi (the main city in Haut-Katanga province). These three medicinal species occur in dry woodlands and their leaves are used by the local population to treat certain diseases [START_REF] Amuri | Hypoglycemic and antihyperglycemic activities of nine medicinal herbs used as antidiabetic in the region of Lubumbashi (DR Congo)[END_REF]. Pharmacological and phytochemical studies using modern scientific approaches have demonstrated that these congeneric species possess anticancer, antibacterial, antifungal, and antidiabetic properties [START_REF] Meena | Pharmacological and phytochemical evidences for the extracts from plants of the genus Vitex -A review[END_REF][START_REF] Amuri | Hypoglycemic and antihyperglycemic activities of nine medicinal herbs used as antidiabetic in the region of Lubumbashi (DR Congo)[END_REF]). Nevertheless, recent surveys showed that the traditional practitioners are facing increased scarcity of Vitex materials around Lubumbashi [START_REF] Amuri | Hypoglycemic and antihyperglycemic activities of nine medicinal herbs used as antidiabetic in the region of Lubumbashi (DR Congo)[END_REF]. The cause of the increased scarcity is due to woodland destruction and degradation caused by intense wood harvest for charcoal, mining activities, anarchical urbanisation, and shifting cultivation [START_REF] Cabala | Dynamique des écosystèmes forestiers de l' Arc Cuprifère Katangais en République Démocratique du Congo. Causes, transformations spatiales et ampleur[END_REF][START_REF] Sikuzani | Vingtcinq ans de monitoring de la dynamique spatiale des espaces vert en réponse à l'urbanisation dans les communes de la ville de Lubumbashi[END_REF][START_REF] Nge | Diagnostic socioéconomique et environnemental de la chaîne de valeur « charbon de bois » à Lubumbashi (Haut-Katanga, RDC) : perspectives pour une gestion durable des ressources ligneuses de Miombo[END_REF].

The development of a conservation program of the three Vitex species has become a priority to ensure a sustainable supply of these vital ecosystem services and to support the healthcare system in this region. Resource management of these three sympatric medicinal species under high anthropogenic impact is therefore crucial and requires a good understanding of the species' autecology and synecology. Indeed, the development of conservation programs for Vitex is crucial, in a context of steadily decreasing woodland area. Thus, the implementation of an adequate conservation strategy can only be achieved through a thorough understanding of the deterministic processes responsible for the variability of plant communities associated with Vitex species.

Previous studies in the Katangan miombo woodland have highlighted the importance of habitat heterogeneity at multiple spatial scales in tree community assemblages and species coexistence [START_REF] Duvigneaud | La végétation du Katanga et de ses sols métallifères[END_REF][START_REF] Roberts | Labdsv: Ordination and Multivariate Analysis for Ecology. R Package Version 1.3[END_REF][START_REF] Harms | Habitat associations of trees and shrubs in a 50-ha neotropical forest plot: habitat associations of trees and shrubs[END_REF][START_REF] Muledi | Fine-scale habitats influence tree species assemblage in a miombo forest[END_REF][START_REF] Muledi | Tree growth, recruitment, and survival in a tropical dry woodland: the importance of soil and functional identity of the neighbourhood[END_REF][START_REF] Zellweger | Forest microclimate dynamics drive plant responses to warming[END_REF]. [START_REF] Duvigneaud | La végétation du Katanga et de ses sols métallifères[END_REF] highlighted the influence of topography and geological substrate, on the floristic composition of miombo woodlands in Katanga. The phytosociological study of [START_REF] Roberts | Labdsv: Ordination and Multivariate Analysis for Ecology. R Package Version 1.3[END_REF] based on the Zurich-Montpellier approach recognised the existence of distinct species assemblages in relation to edaphic factors. Recently, based on more robust statistical methods, [START_REF] Muledi | Fine-scale habitats influence tree species assemblage in a miombo forest[END_REF][START_REF] Muledi | Tree growth, recruitment, and survival in a tropical dry woodland: the importance of soil and functional identity of the neighbourhood[END_REF] revealed the importance of soil heterogeneity in predicting the distinct assemblages and performances of tree communities within the miombo at a fine spatial scale, with distinct edaphic habitats associated to specific communities and indicator species at scales of ca 100 m.

Despite their key role in traditional tropical medicine, the ecology of Vitex species, their assemblage with other species into communities, and their dependence on specific abiotic conditions (soil, topography) remain unstudied and therefore poorly understood. It is in this context that we try to better understand the ecological mechanisms explaining the distribution of three medicinal Vitex species (V. fischeri, V. madiensis, and V. mombassae) that co-occur in the dry woodlands of Katanga (SE D.R. Congo).

The genus Vitex (Lamiaceae, formerly Verbenaceae) comprises about 250 species found in tropical and subtropical regions [START_REF] Verdcourt | Verbenaceae[END_REF][START_REF] Mabberley | Mabberley's Plant-book[END_REF]. A total of 21 species of Vitex occur in D.R. Congo (Meerts 2018). Three species (V. fischeri var. fischeri, V. mombassae, and V. madiensis subsp. milanjiensis) are widespread in the Haut-Katanga province [START_REF] Meerts | Arbres et arbustes du Haut-Katanga. Jardin botanique de Meise, Meise. Meerts P ([END_REF]. Vitex mombassae is widely distributed in eastern and southern tropical Africa; V. fischeri var. fischeri occurs mostly in eastern tropical Africa, while V. madiensis subsp. milanjiensis has a Zambezian distribution. All three species occur in dry tropical woodlands and secondary shrub savanna, especially in Brachystegia-Julbernardia miombo woodlands (Meerts 2018;[START_REF] Paton | Labiatae. Introduction. Sous familles I. Viticoideae et II. Premnoideae. In: Flore d' Afrique centrale, Nouvelle Série[END_REF].

Here, we address the following specific questions: (a) Do the three Vitex species occur in distinct plant communities and occupy different ecological niches? (b) What are the ecological factors discriminating the different communities?

MATERIAL AND METHODS

Study area

The study was conducted on the plain of Lubumbashi (Haut-Katanga province in the D.R. Congo), covering an area of 2500 km 2 (11°25'57.23"S, 27°16'3.11"E; 11°53'18.98"S, 27°15'56.31"E; 11°53'21.63"S, 27°44'25. 86"E; 11°25'37.70"S, 27°44'29.59"E), with the post office downtown used as the centroid (Supplementary file 1). The climate is CW6 [START_REF] Peel | Updated world map of the Köppen-Geiger climate classification[END_REF]) with an average annual temperature of 20.3°C and a 1200 mm average annual rainfall between November and April.

Considered as the centre of endemism in the northern part of the Zambezian region, the plain of Lubumbashi is dominated by the humid miombo woodland [START_REF] Kuper | Africa's hotspots of biodiversity redefined[END_REF][START_REF] Cabala | Dynamique des écosystèmes forestiers de l' Arc Cuprifère Katangais en République Démocratique du Congo. Causes, transformations spatiales et ampleur[END_REF], i.e. a particular type of dry tropical woodlands characterised by trees of 15 to 20 m tall, mainly from the Fabaceae family, dominated by the ectomycorrhizal genera Brachystegia, Julbernardia, and Isoberlinia [START_REF] Duvigneaud | La végétation du Katanga et de ses sols métallifères[END_REF][START_REF] Roberts | Labdsv: Ordination and Multivariate Analysis for Ecology. R Package Version 1.3[END_REF][START_REF] Malaisse | .) Tropical forest ecosystems, a state-of-knowledge report prepared by[END_REF][START_REF] Werger | The Sudano-Zambezian region[END_REF][START_REF] Malaisse | Se nourrir en forêt claire africaine. Approche écologique et nutritionnelle[END_REF]. Miombo comprises semi-deciduous woodlands with a continuous herbaceous layer mostly composed of seasonal C4 grasses and sedges, and is subjected to periodic bushfire [START_REF] Frost | Responses of savannas to stress and disturbance[END_REF][START_REF] Malaisse | Se nourrir en forêt claire africaine. Approche écologique et nutritionnelle[END_REF]. Miombo woodlands do not occur in permanently or seasonally waterlogged soil, where they are replaced by riparian forest and dambo savannahs, respectively [START_REF] Roberts | Labdsv: Ordination and Multivariate Analysis for Ecology. R Package Version 1.3[END_REF][START_REF] Meerts | Arbres et arbustes du Haut-Katanga. Jardin botanique de Meise, Meise. Meerts P ([END_REF].

The geological substrate of the region is dominated by dolomitic shale and siltstone of the neoproterozoic Nguba and Roan groups [START_REF] Batumike | Petrology and geochemistry of the Neoproterozoic Nguba and Kundelungu Groups, Katangan Supergroup, southeast Congo: implications for provenance, paleoweathering and geotectonic setting[END_REF], and soils are ferralitic, deep, with poorly differentiated horizons and diffuse or gradual transitions. Red and yellow ferralitic soils are the most common, characterised by an acidic pH and low nutrient content [START_REF] Ngongo | Evaluation du milieu physique de la cuvette Zaïroise pour l'agriculture[END_REF][START_REF] Ngongo | Guide des sols en R.D. Congo[END_REF].

Vegetation inventory strategy

A total of 200 1-ha plots were defined along four main roads (Kasenga, Kafubu, Kipushi, and Likasi), covering the different degraded states of the landscape (miombo woodland, degraded miombo, shrub savannah, postcultivation wasteland, and agricultural land) as described by [START_REF] André | Quantification of anthropogenic effects in the landscape of Lubumbashi[END_REF] based on vegetation crown cover and height. On each road axis, 50 one-hectare quadrats were systematically delineated (at an equidistance of 1 km) along the five secondary roads. The sampling strategy structured by the road network (Supplementary file 1) is adapted from [START_REF] Chidumayo | Distribution and abundance of a keystone tree, Schinziophyton rautanenii, and factors affecting its structure in Zambia, southern Africa[END_REF] and is partly due to logistical constraints. All plots were carefully scanned for the presence of Vitex individuals. In each 1-ha plot where at least one species of Vitex was present (i.e. 114 1-ha plots), a 10 m radius circular plot was installed, with its centre corresponding to the highest density of Vitex in the quadrat. In each of these circular plots, all trees and shrubs with a diameter at breast height (DBH) ≥ 10 cm were inventoried and identified to species level. The identification of woody species associated with Vitex was carried out using standard floras (Flora Zambeziaca and Flore d' Afrique centrale), the determination keys of [START_REF] Meerts | Arbres et arbustes du Haut-Katanga. Jardin botanique de Meise, Meise. Meerts P ([END_REF] and the Upper Katanga tree checklist [START_REF] Meerts | An annotated checklist to the trees and shrubs of the Upper Katanga (D.R. Congo)[END_REF]. The three Vitex species were identified with Meerts (2018) and [START_REF] Paton | Labiatae. Introduction. Sous familles I. Viticoideae et II. Premnoideae. In: Flore d' Afrique centrale, Nouvelle Série[END_REF]. The nomenclature of the families was adapted from APG IV (2016). The coverages of the shrub and tree layers (Cs and Ct, respectively) were estimated using the transect intercept method [START_REF] Fiala | Comparison of five canopy cover estimation techniques in the western Oregon Cascades[END_REF]. All further analyses are based on the species composition of the 114 circular plots with at least one Vitex individual.

Soil and topographic variables

Soil samples were systematically collected from each 10 m radius plot (0-20 cm soil layer). The soil samples were analysed according to the standard protocol of [START_REF] Pansu | Handbook of soil analysismineralogical, organic and inorganic methods[END_REF]. The bioavailable mineral elements (Al, Ca, K, P, Fe, Mg, Mn, Cu, and Zn) were extracted with 1 M ammonium acetate-EDTA at pH 4.65. Elemental concentrations were determined by ICP-OES with CCD detector (Varian, Vista MPX). Soil organic matter was measured by the loss on ignition method at 550°C. Soil pH was determined with an electrode pH meter in a soilwater solution (5 g soil to in 25 ml water).

Plot elevation was obtained in the field with a GPS (Edge 830), and height above the nearest drainage (HAND) was obtained for all circular plots at a one arcsecond (ca 30 m) resolution (100 river head threshold cells) from the digital elevation model SRTM [START_REF] Donchyts | Global 30m Height Above the Nearest Drainage (HAND)[END_REF], using Google Earth Engine [START_REF] Gorelick | Google Earth Engine: planetaryscale geospatial analysis for everyone[END_REF].

Data analysis

Absolute abundance (number of individuals of a species in a habitat) and relative abundance (ratio of the number of individuals of a species in a habitat to the total number of individuals of the species across all habitats) were calculated per habitat following [START_REF] Cottam | The use of distance measures in phytosociological Sampling[END_REF].

We used multivariate statistical methods focused on both prediction and explanation to model the relationships between species and ecological variables and define species assemblages and specialist and indicator species of particular habitats. First, we used Multivariate Regression Trees (MRT; [START_REF] De | Multivariate regression trees: a new technique for modeling species-environment relationships[END_REF] to define abiotic habitats that best predicted discontinuities in species assemblages and tested for the specialist and indicator nature of all species within the resulting habitats using the IndVal index [START_REF] Dufrêne | Species assemblages and indicator species: the need for a flexible asymmetrical approach[END_REF]. We also applied a constrained ordination approach (redundancy analysis, RDA) to test the abiotic variables which we hypothesised could explain the observed variation in species composition among the studied plots. The candidate explanatory variables to be included in both the MRT and RDA were chemical soil variables, plot elevation (E), height above the nearest drainage (HAND), average tree height (H), and the tree and shrub ground cover (Ct and Cs, respectively), used to describe vegetation structure. Elevation was used as a proxy for geological variations in the region (see [START_REF] Batumike | Petrology and geochemistry of the Neoproterozoic Nguba and Kundelungu Groups, Katangan Supergroup, southeast Congo: implications for provenance, paleoweathering and geotectonic setting[END_REF], whereas HAND was used as a proxy for local topography-related water constraints on the woody community [START_REF] Nobre | Height above the nearest drainage -a hydrologically relevant new terrain model[END_REF], as miombo woodland composition is known to vary with topography, due to its effect on soil depth and drainage [START_REF] Duvigneaud | La végétation du Katanga et de ses sols métallifères[END_REF]). However, miombo woodlands do not occur in waterlogged soils where they are replaced by other vegetation types (e.g. riparian forest and dambo savannahs). Within miombo woodlands, an important source of variation in soil water availability is termite mound activity (i.e. higher available water reserves in mounds), a factor included in our design, since we sampled vegetation both on and off termite mounds. Tree height and ground cover of tree layer were used as proxies of recent anthropogenic disturbance (logging, charcoal, agriculture), because recently disturbed plots have sparser and lower woody cover [START_REF] Sikuzani | Amplification of anthropogenic pressure heavily hampers natural ecosystems regeneration within the savanization halo around Lubumbashi City (Democratic Republic of Congo)[END_REF]. See Supplementary file 2 for variation range of all ecological variables.

To avoid unstable models due to collinearity issues, we calculated pairwise Pearson correlations across all explanatory variables and, for the variable pairs with r > +0.7 and r < -0.7, one of the two variables was removed from the model [START_REF] Dormann | Collinearity: a review of methods to deal with it and a simulation study evaluating their performance[END_REF]) (see Supplementary file 3). The resulting set of variables used in the MRT and RDA were: Ct, Cs, HAND, E, Al, K, P, Fe, Mg, Mn, Co, and Cu. Variables removed from correlated pairs were reconsidered in the result discussion section.

Testing for distinct habitats and indicator species

Multivariate Regression Trees (MRT) is a form of constrained clustering method [START_REF] De | Multivariate regression trees: a new technique for modeling species-environment relationships[END_REF] that we used to optimise the detection of distinctive habitats and associated woody plant communities related to Vitex species. MRT sequentially split community data on the basis of a threshold value for one of the environmental variables, to be optimised, and minimises within-cluster dissimilarity [START_REF] De | Multivariate regression trees: a new technique for modeling species-environment relationships[END_REF]. MRT optimises predictive power using cross-validation, unlike constrained ordinations such as redundancy analyses that emphasise explanatory power, hence ensuring a reduced set of environmental variables best predicting species assemblages into a number of distinct habitats. The final tree partition was therefore selected by minimising the relative error [START_REF] Borcard | Numerical Ecology With R. Second Edition[END_REF].

The indicator species associated with each habitat were defined using the IndVal index. This index is calculated by considering both the fidelity and specificity of the species [START_REF] Dufrêne | Species assemblages and indicator species: the need for a flexible asymmetrical approach[END_REF]. Specialist species were defined as those whose relative abundance in a given habitat was greater than 70% (criterion adapted from [START_REF] Harms | Habitat associations of trees and shrubs in a 50-ha neotropical forest plot: habitat associations of trees and shrubs[END_REF]. MRT and IndVal index calculations were performed using the packages mvpart [START_REF] De | mvpart: Multivariate Partitioning[END_REF] and labdsv [START_REF] Roberts | Labdsv: Ordination and Multivariate Analysis for Ecology. R Package Version 1.3[END_REF].

Explaining community composition variability

Redundancy analysis (RDA) was used as a complementary approach to the combination of MRT and IndVal indices, to explain the variability of plant community composition from the heterogeneity of ecological factors. Explanatory variables were standardised to mean zero and unit standard deviation prior to the analysis. The statistical significance of the overall association between the community matrix and the environmental variable matrix, as well as each of the constrained RDA axis, were tested using 999 permutations of the model residuals [START_REF] Anderson | An empirical comparison of permutation methods for tests of partial regression coefficients in a linear model[END_REF][START_REF] Borcard | Numerical Ecology With R. Second Edition[END_REF]). The adjusted R 2 was used to measure the unbiased amount of variation of the community matrix explained by the explanatory matrix [START_REF] Legendre | Numerical Ecology. Third Edition[END_REF].

A Hellinger transformation was performed on the matrix of tree species abundance per plot using the R package vegan [START_REF] Oksanen | l'argumentation en faveur du renforcement des systèmes pour des meilleurs résultats sanitaires[END_REF] values and the weights given to rare species [START_REF] Legendre | Ecologically meaningful transformations for ordination of species data[END_REF][START_REF] Borcard | Numerical Ecology With R. Second Edition[END_REF]. The RDA was performed using the rda function from the R package vegan [START_REF] Oksanen | l'argumentation en faveur du renforcement des systèmes pour des meilleurs résultats sanitaires[END_REF].

Comparison of habitats' abiotic features

We used permutation ANOVAs to test for differences in each environmental variable among the habitats defined by the MRT approach in order to better characterize them. To do so, the PERMANOVAs consisted of one environmental variable (e.g. Ca content) being modelled as a function of the factor 'habitat' (four levels, one per habitat; see Results), where each observation consisted of the corresponding variable value in one plot. If the ANOVA for a given environmental variable indicated that the variable differed significantly among habitats (based on 999 permutations), permutation t-tests were used to compare each pair of habitats and define the pairs that differed significantly. Permutation ANOVAs and t-tests were performed using the R functions anova.1way (Legendre 2007) and t.perm (Legendre 2005). These permutation tests were chosen to accommodate the unbalanced nature of the sampling design (i.e. varying number of plots per habitat; see details in Supplementary file 4) as well as any deviations from the assumptions of parametric tests in the model residuals. We used the false discovery rate correction to adjust p values and control for the inflated type I error rate that typically arises from multiple tests [START_REF] Benjamini | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF]. A level of significance of 0.05 (prior to correction) was used throughout the study. All analyses were performed in the statistical environment R v.4.0 (R Core Team 2021) (see Supplementary file 4 for the R code).

RESULTS

Habitats and indicator species

A total of 82 species were inventoried, belonging to 32 families and 62 genera (Supplementary file 5). We inventoried 286 individuals of Vitex across all plots. Cluster partitioning of the MRT analyses in relation to environmental variables identified four habitats with an explanatory power of 11% (Fig. 1). Habitats were defined by three environmental variables (Mg and Al soil contents, and elevation). The discrimination of H1 (15 plots) and H4 (23 plots) was carried out on the basis of the Mg concentration at a threshold of 140 µg.g -1 . Above this Mg concentration, H1 and H2 (6 plots) were separated based on soil Al concentration at a threshold value of 220 µg.g -1 . The elevation at a threshold of 1230 m separated H3 (69 plots) H4 (Figs 1,2). Significant variation among the habitats was found for the following variables: tree cover, elevation, Al, Fe, K, Mg, Mn, pH, and P (Supplementary file 6).

H1 was characterised by a very distinct floristic composition (Supplementary file 5), with 29 species only occurring in this habitat. Fabaceae were rare (9%) in this habitat. The species were grouped into two categories according to leaf phenology: deciduous (Cassia abbreviata, Lannea discolor, and Zanthoxylum chalybeum) and evergreen (Allophylus africanus, Boscia mossambicensis, Markhamia obtusifolia, and V. fischeri). Markhamia obtusifolia and Combretum molle were the most abundant species.

In the other three habitats, the Fabaceae was the most frequent family (48%). H2 had a low number of species (30 species) with an average tree height of 8.5 m. The average cover of the tree and shrub layer was 53% and were the most abundant species in H2. However, H3 and H4 were characterised by a dense shrub layer (52%) and a sparse tree layer (28%) with 66 and 54 species, respectively. The trees observed in H3 and H4 had an average height of 6.5 m. Brachystegia boehmii and Albizia adianthifolia were the abundant species in H3 and B. spiciformis and Ochna schweinfurthiana in H4.

Significant indicator species associated with each habitat were identified by permutations of the IndVal index in the habitat where the species was more abundant (Supplementary file 5). A total of 15 indicator species were detected in habitat H1, H2, and H4 (Table 1). No indicator species was found for H3. Two species of Vitex were indicative of distinct habitats. Vitex fischeri was indicative of H1 and V. mombassae of H4. In addition, V. fischeri had high absolute abundance in H1 and V. mombassae in Both species had low abundance values in H2 and H3. In contrast, V. madiensis was equally frequent in H2, H3 and H4, and absent in H1 (Table 2).

A total of 45 specialist species were detected in all habitats. Vitex fischeri was a specialist in H1 and V. mombassae in H4. In contrast, B. boehmii, B. spiciformis, Julbernardia paniculata, and V. madiensis were generalists of H2, H3, and H4 (Supplementary file 5).

Relationship between species and environmental variables

A constrained ordination (RDA) was used to further describe and quantify the relation between community composition and environmental heterogeneity. The RDA between environmental variables and species abundances indicated a significant explanation of species composition variability by the environment (p < 0.001), although the overall explanatory power was relatively limited (adjusted R 2 = 0.111). The first two constrained axes explained 10.6% of the total variability. Axis 1 expressed on the constrained coordinated (7.6% of the variance) had major contributions from Mg, pH, K, Mn, and organic matter content (negative correlations), and Al and E (positive correlations) (Fig. 3A). Axis 2 (3% of variance) was mostly correlated with E (positively) and P (negatively), separating plots of lower elevation and higher P soil concentrations from higher elevations plots of lower P elevations (Fig. 3A).

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The projection of the 4 MRT habitats in the ordination plane showed that habitats H1 and H2 were clearly different from the other two (H3 and H4). H1 was characterised by soil with high Mg concentration on axis 1, and H2 was located on fertile soils with high P concentration on axis 2. However, H3 and H4 formed a continuum located in intermediate altitude areas on poor soils with high Al concentrations (Fig. 3A). Vitex fischeri and V. mombassae were indicators of specific habitats (H1 and H4, respectively), while V. madiensis had a wide ecological range, occurring in H2, H3, and H4 (Fig. 3B).

DISCUSSION

Plant community of the three Vitex

The development of a conservation strategy requires a good knowledge of the species autecology. In this paper, we characterised the autecology of three congeneric, sympatric species of the genus Vitex that co-occur in the dry woodlands of southern D.R. Congo. The three species are important conservation targets due to their medicinal properties. In this paper, we assessed if they are associated to distinct plant communities and if their distribution can be accounted for by variation in environmental factors. The three species were found to occur in four habitats. We first examine if these habitats correspond to previously described vegetation units.

Habitat 1 corresponds to termite mound vegetation. The Katangan dry tropical woodlands are characterised by the presence of Macrotermes mounds, which harbour a highly distinctive vegetation [START_REF] Malaisse | .) Tropical forest ecosystems, a state-of-knowledge report prepared by[END_REF][START_REF] Mushagalusa | Edaphic specialization in relation to termite mounds in Katanga (D.R. Congo): a reciprocal transplant experiment with congeneric tree species[END_REF]. We found Vitex fischeri to be a specialist of such vegetation, while the other two species almost never occur in it. Noticeably, the ectomycorrhizal tree genera characteristic of miombo woodlands (Brachystegia, Isoberlinia, Julbernardia) are lacking altogether in the termite mound community.

The other two species occur in the woodlands surrounding the termite mounds. Such woodlands are characterised by a high frequency of Fabaceae (Julbernardia paniculata, Albizia adianthifolia) and, in particular, the abundance of several species of Brachystegia. Based on the phytosociological classification of Schmitz (1971), the three communities comprising V. madiensis and V. mombassae belong to the Xerobrachystegion alliance. Vitex communities occur on soils that are neither extremely dry as suggested by the absence of species considered by [START_REF] Duvigneaud | La végétation du Katanga et de ses sols métallifères[END_REF] as indicative of shallow soils on rock outcrops (such as Brachystegia microphylla) nor with drainage impeded by lateritic crust as indicated by the lack of B. utilis and B. stipulata.

Modelling species-habitat associations

Our work further supports previous results pointing to the importance of topography and soil heterogeneity for the assemblage and dynamics of tree communities in miombo woodlands [START_REF] Chidumayo | Species structure in Zambian miombo woodland[END_REF][START_REF] Kanschik | Dry miombo -ecology of its major plant species and their potential use as bio-indicators[END_REF][START_REF] Mapaure | Small-scale variations in species composition of miombo woodland in Sengwa, Zimbabwe: the influence of edaphic factors, fire and elephant herbivory[END_REF][START_REF] Backéus | Tree communities and structural dynamics in miombo (Brachystegia-Julbernardia) woodland, Tanzania[END_REF][START_REF] Munishi | Plant communities and tree species associations in a miombo ecosystem in the Lake Rukwa basin, Southern Tanzania: implications for conservation[END_REF][START_REF] Mwakalukwa | Floristic composition, structure, and species associations of dry miombo woodland in Tanzania[END_REF][START_REF] Muledi | Fine-scale habitats influence tree species assemblage in a miombo forest[END_REF][START_REF] Muledi | Tree growth, recruitment, and survival in a tropical dry woodland: the importance of soil and functional identity of the neighbourhood[END_REF].

Here, we used complementary multivariate constrained analyses (MRT and RDA) to model soil-plant relationships associated with the three sympatric congeneric Vitex species at a regional scale. We highlighted four distinct habitats as well as associated specialist and indicator species and provided a detailed characterisation of the physico-chemical features of these habitats.

In this study, the four habitats presented statistically different values of nine environmental variables (see Supplementary file 6) and the discriminatory environmental variables of the habitats associated with the Vitex species were Mg, Al, and elevation. It is evident that even at fine spatial scales, heterogeneity in the edaphic and topographic environment facilitates the specialisation of species to contrasting habitats [START_REF] Harms | Habitat associations of trees and shrubs in a 50-ha neotropical forest plot: habitat associations of trees and shrubs[END_REF][START_REF] Muledi | Fine-scale habitats influence tree species assemblage in a miombo forest[END_REF]. Thus, V. fischeri was a specialist and indicator in H1 characterised by high Mg and low exchangeable Al content (Supplementary file 6). The strong affinity of this species to high termite mounds was demonstrated by [START_REF] Mushagalusa | Edaphic specialization in relation to termite mounds in Katanga (D.R. Congo): a reciprocal transplant experiment with congeneric tree species[END_REF]. Compared to the surrounding matrix soils, termite mounds contain higher levels of clay-exchangeable base cations and the core of the mound constitutes a reservoir of available water [START_REF] Turner | Termites, water and soils. Agricola[END_REF][START_REF] Mujinya | Clay composition and properties in termite mounds of the Lubumbashi area, D.R. Congo[END_REF]. In a reciprocal transplant experiment, [START_REF] Mushagalusa | Edaphic specialization in relation to termite mounds in Katanga (D.R. Congo): a reciprocal transplant experiment with congeneric tree species[END_REF] showed that termite mounds specialists are drought-sensitive species that do not resist the dry season when growing off termite mounds.

The other two Vitex species (V. madiensis and V. mombassae) were associated to base cation-poor soils with high aluminium concentration. Vitex madiensis was mainly a generalist of H2, H3, and H4. These results corroborate the observations of [START_REF] Chidumayo | Responses of Miombo to Harvesting: Ecology and Management[END_REF] who reported the presence of V. madiensis in several different habitats in the Zambezian region. In contrast, V. mombassae was a significant indicator and specialist of H4 located at lower elevations compared to H1, H2, and H3. The influence of elevation is surprising considering the narrow variation range in the study region (1178-1377 m) and could be mediated by geomorphological context. [START_REF] Batumike | Petrology and geochemistry of the Neoproterozoic Nguba and Kundelungu Groups, Katangan Supergroup, southeast Congo: implications for provenance, paleoweathering and geotectonic setting[END_REF] suggested that geological variability on a regional scale may impact the pedology of the Lubumbashi plain, but also its topography. [START_REF] Duvigneaud | La végétation du Katanga et de ses sols métallifères[END_REF] pointed to topographical patterns in the floristic composition of Katangan dry woodlands, possibly due to variation in drainage. However, our results do not show a significant contribution of drainage on the structuring of the four habitats (Supplementary file 6). The significant effect of elevation could be mediated by geological background, because the different bedrocks tend to occupy slightly different elevation ranges (Supplementary files 7 and 8). Therefore, soil factors not analysed in this study and related to lithology, such as soil granulometry, could play in role in defining the ecological niche of V. mombassae.

The model generated from the MRT based solely on variability in soil chemistry, elevation, HAND, and structural variables predicted 10% and explained 11% of the spatial distribution of tree communities, while the RDA explained 9.9% using the same environmental variables. The explanatory and predictive powers observed in our study are lower than the range of variation (19% to 49%) of community-habitat associations in tropical forests (e.g. [START_REF] Mapaure | Small-scale variations in species composition of miombo woodland in Sengwa, Zimbabwe: the influence of edaphic factors, fire and elephant herbivory[END_REF][START_REF] Jones | Explaining variation in tropical plant community composition: influence of environmental and spatial data quality[END_REF][START_REF] Legendre | Partitioning beta diversity in a subtropical broadleaved forest of China[END_REF][START_REF] Chang | Better environmental data may reverse conclusions about nicheand dispersal-based processes in community assembly[END_REF][START_REF] Punchi-Manage | Effects of topography on structuring local species assemblages in a Sri Lankan mixed dipterocarp forest[END_REF][START_REF] Vleminckx | Impact of fine-scale edaphic heterogeneity on tree species assembly in a central African rainforest[END_REF][START_REF] Muledi | Fine-scale habitats influence tree species assemblage in a miombo forest[END_REF]. The proportion unexplained by our variables could be attributed to stochastic processes [START_REF] Legendre | Numerical Ecology. Third Edition[END_REF], or unmeasured environmental variables [START_REF] Diniz-Filho | Spatial autocorrelation analysis allows disentangling the balance between neutral and niche processes in metacommunities[END_REF][START_REF] Baldeck | Soil resources and topography shape local tree community structure in tropical forests[END_REF]). In addition, the woodland landscape of the Lubumbashi plain is subject to periodic anthropogenic activities that cause new filters responsible for species dispersal limitation [START_REF] Cabala | Dynamique des écosystèmes forestiers de l' Arc Cuprifère Katangais en République Démocratique du Congo. Causes, transformations spatiales et ampleur[END_REF]. These results suggest that the age of the forest land use succession plays an additive role in the community assemblages of these three Vitex species. In this paper, anthropogenic disturbance was indirectly assessed through the height and total cover of the woody layer. These variables do not appear to be key drivers of the discrimination of the four habitats, and all three Vitex species occur across a broad range of landscape degradation. A future paper will specifically examine the response of Vitex populations to anthropogenic disturbance.

These results emphasise the importance of a detailed knowledge of species ecology to design species-specific conservation strategies, for congeneric species occurring sympatrically in the same landscape. The designation of protected areas should include the different habitats needed to conserve the three species. In particular, V. fischeri is associated to termite mounds, a habitat that is threatened by brick manufacturing. Vitex mombassae occurs mostly at lower elevations which are under pressure by shifting agricultural practices, while V. madiensis occupies a broader range of habitats, except termite mounds.

Future work on these Vitex species will be necessary to further understand their ecology as well as how it translates into potential differences in terms of secondary compounds of medical interest. Examples of future questions are whether the broad-niched V. madiensis is phenotypically more variable than the other two species, and whether populations occurring in different habitats show a different compositions of pharmacologically relevant compounds.

Figure 1 .

 1 Figure 1.Habitats and corresponding community assemblages as defined by multivariate regression trees. The dominant species (highest absolute abundance) in each habitat are ranked in decreasing order of abundance and the indicator species are in bold with an asterisk indicating a significant p value (p < 0.05; 4999 permutations). Optimised habitat discrimination was performed using the following environmental variables: elevation (E), aluminium concentration (Al), and magnesium (Mg). Statistical values for the MRT analysis included: relative error = 0.891, cross-validation error = 1.03, standard error = 0.0249. n represents the number of plots within each habitat.

Figure 2 .

 2 Figure 2. Spatial distribution of sampling points. The 4 MRT habitats are represented by different symbols. Topographic relief highlighted by different colours. The centre of the city of Lubumbashi is indicated by a grey disc.

Figure 3 .

 3 Figure 3. Ordination diagram showing the result of the redundancy analysis (RDA) in the constrained space. A. Projection of environmental variables and plots. Only significant variables (p < 0.05) are displayed; adjusted R² value = 0.098. The 4 habitats of the MRT are represented by different colours (H1: blue, H2: yellow, H3: grey, and H4: orange). B. Projection of species with goodness of fit ≥ 0.1 in the ordination plane formed by axes 1 and 2. Indicator species are coloured in the same way as their respective habitat and species in black have a goodness of fit = 0.1. The indicator species of H2 are not shown because they have goodness of fit values below 0.1. Elevation (E), shrub cover (Cs), tree cover (Ct), organic matter (OM). See Supplementary file 5 for species acronyms.

  before performing the multivariate analyses, to standardise the data by reducing the relative importance of the highest abundance

		Mg > 140 µg/g	Mg < 140 µg/g
	Al < 220 µg/g	Al > 220 µg/g	E > 1230 m	E < 1230 m
	H1	H2	H3	H4
	n = 15	n = 6	n = 69	n = 23
	Markhamia obtusifolia*	Brachystegia boehmii	Brachystegia boehmii	Brachystegia spiciformis
	Combretum molle*	Brachystegia spiciformis	Albizia adianthifolia	Ochna schweinfurthiana
	Phyllocosmus lemaireanus	Albizia adianthifolia	Julbernardia paniculata	Diplorhynchus condylocarpon
	Vitex fischeri*	Vitex madiensis	Ochna schweinfurthiana	Syzygium guineense
	Haplocoelum foliolosum*	Pericopsis angolensis*		Vitex mombassae*
	Ziziphus mucronata*	Uapaca pilosa*		Pterocarpus angolensis*
	Allophylus africanus*	Piliostigma thonningii*
	Ficus thonningii*
	Cassia abbreviata*
	Zanthoxylum chalybeum*
	Bosnia angustifolia*

Table 1 .

 1 IndVal values of indicator species for habitats associated with Vitex. * p < 0.05; ** p < 0.01; *** p < 0.001.

	Species	MRT-Habitats	IndVal
	Vitex fischeri	1	0.70***
	Haplocoelum foliolosum	1	0.58***
	Allophylus africanus	1	0.50***
	Ziziphus mucronata	1	0.46***
	Combretum molle	1	0.43***
	Markhamia obtusifolia	1	0.40**
	Cassia abbreviata	1	0.27**
	Ficus thonningii	1	0.22*
	Zanthoxylum chalybeum	1	0.20**
	Boscia angustifolia	1	0.18*
	Pericopsis angolensis	2	0.31*
	Uapaca pilosa	2	0.24**
	Piliostigma thonningii	2	0.15*
	Vitex mombassae	4	0.32**
	Pterocarpus angolensis	4	0.26*

43%, respectively. Brachystegia boehmii and B. spiciformis

Table 2 .

 2 Absolute abundance (ABA) and relative abundance (REB) of the three species of Vitex in the four habitats.

			ABA				REB (%)
	Species	H1	H2	H3	H4	H1	H2	H3	H4
	Vitex fischeri	39	3	3	0	88	6	6	0
	Vitex madiensis	0	58	59	51	0	35	35	30
	Vitex mombassae	0	7	13	56	0	9	17	74

ACKNOWLEDGEMENTS

We thank Jonathan Muledi (UNILU) for his help in identifying species in the field, as well as Thomas Drouet (ULB), Nausicaa Noret (ULB), and Alexandre Van Baekel (ULB) for their invaluable help with the soil analyses. We are grateful to Pierre Ploton for his help with Google Earth Engine. This research was funded by the Académie de Recherche et d'Enseignement Supérieur (ARES, Fédération Wallonie-Bruxelles, Belgium; project PHYTOKAT; project leaders Pierre Duez (UMons) and Salvius Bakari Amuri (UNILU)). DB was supported by the Wiener-Anspach Foundation, the Belgian American Educational Foundation, and the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 895799.

SUPPLEMENTARY FILES

Supplementary file 1

Geographic map showing the spatial distribution of plots in the study area. Link: https://doi.org/10.5091/plecevo.89394.suppl1

Supplementary file 2

Variation of environmental variables in the whole dataset. Link: https://doi.org/10.5091/plecevo.89394.suppl2

Supplementary file 3

Pearson correlation coefficients between environmental variables. Link: https://doi.org/10.5091/plecevo.89394.suppl3 Supplementary file 4 R code. Link: https://doi.org/10.5091/plecevo.89394.suppl4

Supplementary file 5

Abundance of woody species associated with the three Vitex species in the four habitats. Link: https://doi.org/10.5091/plecevo.89394.suppl5

Supplementary file 6

Comparison of environmental variables between the four habitats. Link: https://doi.org/10.5091/plecevo.89394.suppl6

Supplementary file 7

Distribution of the four habitats in relation to geological substrate. Link: https://doi.org/10.5091/plecevo.89394.suppl7

Supplementary file 8

Box plots of elevation as a function of geological substrate. Link: https://doi.org/10.5091/plecevo.89394.suppl8