Through extensive and fine-grained field work we explored the links between functional traits related to population health status and vegetation and soil parameters. We found that large (> 20 individuals) populations of C. calceolus display a specific assemblage of measurable characteristics that discriminate them from small (< 10 individuals) populations, indicating that it is possible to assess the health of a population of a rare plant species by measuring a specific set of functional traits. While we could not highlight a direct link between vegetation alliances and soil types and population health status, we show that the unique combination of companion plants, and several edaphic parameters, such as SOM, CaCO3, pH, and P, could be used to potentially assess the optimal sites to implement (re)-introduction actions for this emblematic and patrimonial orchid species.
Functional trait variation across small and large populations – We found that, based on our classification, small and large populations display different functional signatures, in which, large populations of C. calceolus displayed significantly higher values for most measured plant traits, compared to small populations. This indicates that classic plant functional traits (Díaz et al. 2016; Wright et al. 2004) could be used to characterize the quality of C. calceolus populations (Adler et al. 2014). In plant evolutionary ecology and conservation, the predicted relationship between plant population size and fitness has been amply observed (Reed 2005). For instance, Leimu et al. (2006) performed a meta-analysis on 105 studies focusing on correlations between plant population size, fitness and genetic variation. They highlighted that all these correlations were significantly positive. Moreover, rather interestingly for our study, the same research also showed that these positive relationships were more pronounced for rare than for common species, an effect thus likely heightened when large population size differences exist in nature. Relationships between populations size and fitness may happen for two reasons: (i) an extinction vortex causing a decrease in genetic variation leading to inbreeding depression (Ruegg and Turbek 2022), a reduced mate availability or random genetic drift that consequently reduce populations fitness; or (ii) a difference in habitat quality (Ellstrand and Elam 1993; Fischer and Matthies 1998; Leimu et al. 2006). From a conservation point of view, these hypotheses are even more central in the case of an endangered species such as C. Calceolus. Indeed, small populations, which can be crucial for a species survival, are more vulnerable to stochastic events and fluctuations (Honnay and Jacquemyn 2007; Reed 2005). The recovery time after a perturbation is lengthened by a reduced fitness and will make the population more prone to extinction when supplementary perturbations happen (Reed 2005). In fact, smaller populations appear to be less able to adapt to new environmental changes because of the loss of adaptative genetic variation through genetic drift (Reed et al. 2003; Willi et al. 2007). In addition, reduced pollinator activity in small populations of rare species generally contributes to reduced plant fitness (Leimu et al. 2006). For the C. calceolus populations studied here, further investigations should be made to understand whether the relationship between population size and fitness is due to inbreeding depression or to habitat quality. Genetic studies performed in Europe on C. calceolus populations showed that this species has a relatively high genetic diversity compared with rare taxa and taxa with the same life history (Brzosko 2002; Brzosko et al. 2002). On the other hand, signatures of a bottleneck effect and recent founder events were identified in Estonia, and genetic and genotypic diversity variables were significantly correlated with population size in Poland (Brzosko et al. 2011; Gargiulo et al. 2018). Finally, in addition to genetic studies and for conservation purposes, it would be necessary to understand the minimum size of C. calceolus populations so as not to be threatened by the extinction vortex and then ensure that populations stay under this threshold.
Vegetation communities associated with C. calceolus – Across our sampling, we found that C. calceolus grows on 11 vegetation alliances, with those where C. calceolus mostly occurred being xerothermophilic beech forests (Cephalanthero-Fagenion) and the low elevation mesophyll beech forest (Galio-Fagenion) – as observed by Käsermann and Moser (1999), but we couldn’t find a recurrent pattern between vegetation composition and C. calceolus population size. It is essential to highlight that vegetation alliances were difficult to assess because C. calceolus often grows in somewhat hybrid environments (i.e., transition zones) that do not always fit traditional classification methods (Delarze et al. 2015). Consequently, we were only able to associate our vegetation inventories with the closest vegetation alliances found in the literature (see Table 1). All identified vegetation alliances were forests, except for the pre-forest shrub stage (Sambuco-Salicion). Together, these findings indicate that C. calceolus prefer to grow at the forest edges, or in the ecotones, of mixed-stands forest type, therefore preferring intermediate levels of direct solar radiations (i.e., not in full light, but neither in full understory shading) (Rusconi et al. 2022). Along these lines, Hurskainen et al. (2017) highlighted that removing trees to create forest gaps favoured C. calceolus populations significantly. These observations point to the very complex ecology of this orchid and the delicate balance of light parameters it requires to grow (Kirillova and Kirillov 2019). Accordingly, because open forests are disappearing in Switzerland, recovery of C. calceolus populations might be impacted. In this regard, specific forest management plans should be implemented to favour this species optimal light requirements (Bornand et al. 2016). However, based on our findings, vegetation composition per se is not a sufficiently strong marker for the identification of suitable (re)introduction sites, and other parameters should be accounted for.
The use of indicator species for finding suitable habitats – Through species indicator analyses, we found that large and small C. calceolus populations were best discriminated by four species, positively by Ajuga reptans, Juniperus communis, Equisetum telmateia, and Gymnadenia conopsea, while negatively by Sesleria caerulea and Carex sempervirens. Interestingly, the ecological characteristics of A. reptans (the most discriminant species for large populations of C. calceolus) reflect the ecological needs of C. calceolus: clear forests with average humidity and average soil nutrients (Landolt et al. 2010). However, A. reptans would not be a good indicator species for finding C. calceolus suitable habitats, as it is even more broadly distributed than C. calceolus in Switzerland, growing in the sub-alpine regions (Lauber et al. 2018). The three other species positively discriminating large populations also shared similar or close ecological requirements to C. calceolus and the same elevation optimum (Landolt et al. 2010). The two species discriminating small populations had the same soil nutrient requirements than C. calceolus, but their ecological optima are at higher elevations (Landolt et al. 2010). Therefore, a combination of some of the observed positively and negatively discriminating species could be used to find suitable habitats for C. calceolus. Such an approach could be confirmed using a combination of fieldwork for assessing population fitness (as was done here) and species distribution modelling (Guisan et al. 2006).
Soil characteristics associated with C. calceolus population health status – Based on the soil horizon profile analysis, we observed that C. calceolus grows on three, or two depending on the soil nomenclature, soil types, which corresponds to the observations made by Käsermann and Moser (1999). In opposition with the results of the vegetation types, and based on the total number of existing soil references (110 in Baize (2009) and 32 soil groups in IUSS (IUSS Working Group 2015)), we thus found that C. calceolus grows on a very limited range of soil types, attesting that C. calceolus preferentially grows on calcareous or dolomitic substrate. Moreover, the physicochemical characteristics results corroborate what is generally found in the literature: C. calceolus grows in soil with neutral to alkaline pH (Rusconi et al. 2022) with the presence of calcium carbonates (in the form of CaCO3 or CaMg(CO3)2) (Käsermann and Moser 1999) and with on average a high concentration of soil organic matter (Kļaviņa and Osvalde 2017). The observations regarding the humus forms, Mull-to-Moder, also support the notion that the rooting of this plant is where biological activity is relatively intense and where the organic matter is well and rapidly integrated into the soil matrix (Zanella et al. 2018). Moreover, through multivariate comparative analysis, we found that soil parameters that most strongly influenced C. calceolus functional traits were pH, HR, P, Corg and SOM. In this regard, our results contradict those found by Kļaviņa and Osvalde (2017), in which pH did not affect population vitality. A precise characterization of the edaphic niche of endangered species (as we did in this study) is crucial for implementing conservation plans and identifying suitable (re)introduction sites. Specifically for C. calceolus, we encourage to perform soil physicochemical analysis to verify that the preselected zone has the following edaphic properties: presence of CaCO3 or CaMg(CO3)2, neutral to alkaline pH, about 15% of organic matter and a CEC of about 40 cmol/kg.
In conclusion, with this work, we provide an additional step toward a better understanding how the selection of (re)introduction sites for the conservation of endangered plant species can be resolved. Specifically, we argue that beyond the use of classic approaches for site selection, such as building species distribution models (Guisan et al. 2013; Pecchi et al. 2019; Prasad et al. 2016), or, on the opposite, using only practitioner-informed current or past occurrence knowledge (Rusconi et al. 2022), might not provide the most accurate predictions for finding optimal sites to enhance or protect the target species. Instead, a fine-grained analysis of multiple targeted ecosystem variables is generally needed (Prasad et al. 2016; Richardson et al. 2009). Moreover, the analyses derived from our fieldwork also highlighted a direct link between plant and population life history traits and in situ measurable functional traits. In return, we then highlighted that narrow ranges of edaphic factors best correlated with unique sets of the measured plant traits, ultimately corroborating the importance of above-belowground links in plant ecology (Wardle 2002), and conservation biology.