1 INTRODUCTION
Alpine environments, dominated by perennial herbs, face severe impacts from global climate change (Seddon et al. , 2016). Global warming has led to significant changes, such as the encroachment of woody subalpine plants, narrowing of alpine ecosystems (Capers & Stone, 2011) or the increase of diversity in European summits (Steinbauer et al. , 2018). While the loss of alpine habitat could be compensated by glacier retreatment (Whittaker, 1993; Losapio et al. , 2021), the snowline has been wholly lost in the lower and southern mountain regions where alpine plants are currently refuged in their ridges and peaks facing potential local extinction (Rumpf et al. , 2022). Global warming also affects alpine plant reproduction, including plant-pollinator interactions (Inouye, 2020) and seed germination (Mondoni et al. , 2012). Moreover, human disturbance in mountainous areas, such as civil infrastructures or recreation resources, leads to alpine habitat fragmentation and quality decline (Winkler, 2020; Chardon et al. , 2023). Therefore, alpine ecosystems and their species are considered vulnerable to environmental changes (Schwager & Berg, 2019).
By 2100, 36-55% of the alpine species in European mountains are predicted to lose more than 80% of their habitats (Inouye, 2020). However, limited information is available regarding the current conservation status of many European alpine species, such as those included in the Androsace L. section Aretia (L.). SectionAretia includes narrow endemics with low dispersal ability (Anderberg & Kelso, 1996), with 34 recognized species (Boucher et al. , 2021) mainly distributed in the ”European Alpine System” (Ozenda, 1995). Only a handful of Aretia species have undergone threat assessments (Fasciani & Pace, 2015; Eustacchio et al. , 2023). In Spain, Androsace cantabrica (Losa & P. Monts.) Kress has been included in the list of priority species for conservation (Moreno Saizet al. , 2008).
Androsace cantabrica is an endemic species to the central region of the North Iberian Cordillera Cantábrica (Fig. 1A; Kress, 1997). It is a perennial, monoecious, and allogamous plant with small, densely clustered rosettes. The stem is usually less than 5cm long, and the flower corolla is deep pink (Figs. 1B and C; Kress, 1997).Androsace cantabrica is known to occur in seven localities; however, population size estimates are only known in four of them (Fig. 1A, in red), with less than 6,000 individuals estimated across 20 1x1km UTM quadrats (Baudet et al. (2004). This species is found on siliceous or acidic substrates in mountainous areas above 2000m, typically in ridges, and often associated with low shrubs or pastures (Tejero et al. , 2022). The central distribution core is centred around the Tres Mares area, partially overlapping with the Alto Campoo ski resort. All population sites have traditionally been subjected to controlled burning to promote pasture development. Global warming will likely affect its reproductive output, like Tejero et al. (2022) observed lower germination rates in experiments with warmer temperatures. Baudet et al. (2004) proposed to categoriseA. cantabrica as ”Endangered” in the Spanish Red List, later confirmed by Moreno Saiz et al. (2008). However, A. cantabrica is not an accepted species name (https://powo.science.kew.org/, accessed 14th July 2024); instead, it is treated as a synonym of A. adfinis subsp. adfinis Biroli. This taxonomic uncertainty has conservation implications because the European conservation framework does not consider it a species, thus not categorised as threatened. Scientific evidence is urgently needed to resolve this taxonomic conflict as a first step to provide effective conservation (Godfray et al. , 2004).
Molecular phylogenetics has emerged as a crucial tool for addressing taxonomic challenges (de Queiroz & Gauthier, 1992), and its resolution has greatly enhanced with the advent of high-throughput sequencing methodologies (Campos et al. , 2023). However, attempts to clarify species boundaries in this complex of Androsace species were mostly based on traditional molecular approaches. Previous morphological studies suggested that A. cantabrica was closely related toA. laggeri A.Huet (Kress, 1997). Genetic data using different molecular markers, such as the plastid trn L-F region and the internal transcribed spacer (ITS, Schneeweiss et al. , 2004), amplified fragment length polymorphism (AFLPs, Dixon et al. , 2008), and double digest restriction-site associated DNA (ddRAD-seq, Boucher et al. , 2021), proposed that A. cantabrica andA. adfinis are sister taxa, forming a cantabrica-adfinis clade sister to a clade formed by A. halleri L. and A. laggeri (/halleri clade hereafter), but with low bootstrap support. However, a more recent phylogenetic tree reconstructed with full plastome sequences suggests that the clade formed by A. cantabrica and A. adfinis is not sister to the /halleri clade (Smyčka et al., 2022). Therefore, the evolutionary relationships among the Iberian Androsace section Aretia taxa and A. adfinis remain uncertain and still need to be resolved.
Targeted sequencing using the universal Angiosperms353 probe set can generate hundreds of homologous low-copy nuclear loci sequences, establishing it as a powerful tool in plant evolutionary studies (Johnson et al. , 2019). This approach is cost-effective and allows the use of herbarium materials in phylogenomic analysis (Breweret al. , 2019). Nuclear genes can yield a distinct phylogenetic topology compared to plastid genes (Stubbs et al. , 2023). By combining these two genomic sources, researchers can explore reticulate evolution and potential hybrid origins more effectively (Vriesendorp & Bakker, 2005). However, most previous studies using Angiosperms353 data have primarily focused on clade boundaries at the genus, family, and order levels (e.g., Nepenthes (Nepenthaceae), Murphy et al. , 2020; Gentianales, Antonelli et al. , 2021; Primulaceae, Larson et al. , 2023), with few addressing species-level taxonomic conflicts (e.g., Campos et al. , 2023). In addition to its application in phylogenomics, Angiosperms353 data can be utilised in population genetic studies (Slimp et al. , 2021), which is invaluable for designing effective conservation plans for threatened species (Liu & Zhao, 1999; Xiong et al. , 2024). Compared to RAD-Seq (Davey & Blaxter, 2010), Angiosperms353 offers a more cost-effective alternative with reduced missing data, and it can be used in plants with different genome sizes (Slimp et al. , 2021). However, to our knowledge, the application of Angiosperms353 in practical conservation genetics has yet to be reported.
Our research objectives are threefold: to clarify the taxonomic status of A. cantabrica using nuclear Angiosperms353 loci and plastid data; to evaluate its threatened status and IUCN category; and to provide conservation recommendations for A. cantabrica based on population genetics analysis. This approach will also enable us to evaluate the effectiveness of Angiosperms353 in conservation genetics research.