Leucheria cantillanensis (Nassauvieae, Asteraceae), a new species endemic to Central Chile

Abstract A new species, Leucheria cantillanensissp. nov., endemic to the coastal mountain range of Central Chile, is described. By using both nDNA and cpDNA, phylogenetic relationships of the new species were investigated. This new species belongs to the acaulescent/subacaulescent clade of Leucheria, which is congruent with the morphology of the species. A detailed description, distribution map, insights about its habitat, conservation status, and illustrations are provided. An updated key for acaulescent/subacaulescent species of Leucheria from Central Chile is also given.


Introduction
The ecosystems found in central and southern Chile are one of 35 world biodiversity hotspots, owing to their combination of great diversity and high levels of endemism, and a past and ongoing loss of habitat and biodiversity (Myers et al. 2000;Mittermeier et al. 2005). Central Chile features a Mediterranean-type climate, although important climatic heterogeneity can be found due to latitudinal and altitudinal gradients (Armesto et al. 2007). This heterogeneity, accompanied by the climatic history throughout the Quaternary, must have contributed to the increased species richness and endemism in the area (Arroyo et al. 1995;Villagrán 1995). Many genera show a high number of species within this area, such as Adesmia DC., Chaetanthera Ruiz & Pav., Mutisia L.f., Oriastrum Poepp., Senecio L., and Leucheria Lag., occurring in all available environments, from the coastal dunes, sclerophyllous forests and matorral to Andean vegetation near 4000 m above sea level (a.s.l.).
The genus Leucheria comprises 49 species (Crisci 1976;Katinas et al. 2008;Katinas et al. 2018;Jara-Arancio et al. 2019) distributed in Peru, Bolivia, Chile, and Argentina on the continent, plus the Falkland Islands. Most species are concentrated within the Patagonian-Andean and the Subantarctic phytogeographic domains (Cabrera and Willink 1973). The region of Chile with the greatest diversity of Leucheria overlaps with the Central and southern Chile biodiversity hotspot, with most species richness and endemism occurring within this area (Moreira-Muñoz et al. 2012). The available specimen coverage of Leucheria in Chile is reasonably good, with more than 1200 collected specimens distributed between the most important herbaria in the country, SGO and CONC. However, their collection localities are clustered mainly on accessible regions and along main highways, such as border crossings between Chile and Argentina and Bolivia, ski centres, or the Pan American Route 5 in the North of Chile. Furthermore, many species are poorly collected (e.g. Leucheria apiifolia Phil., Leucheria glabriuscula Reiche, Leucheria graui Katinas, M.C. Tellería & Crisci), and many other species have important geographic gaps among their collections (e.g. Leucheria achilleifolia Hook. & Arn., Leucheria polyclados (J. Remy) Reiche). Since the most comprehensive revision of the genus (Crisci, 1976), at least two new species (Katinas et al. 2008;Katinas et al. 2018) and a new variety (Ratto et al. 2014), which was later elevated to species level (Jara-Arancio et al. 2019), have been described.
In the context of the ongoing taxonomic revision of the genus, unusual specimens of Leucheria were collected at the Reserva Natural Altos de Cantillana (MMA 2018). The locality is known as one of the 72 priority sites for the conservation of biodiversity in Chile and the top in priority within the Metropolitan Region (CONAMA 2004). Altos de Cantillana is located in the coastal mountain range of the Metropolitan Region of Chile, a paradoxically species-rich area with high levels of endemism, but poorly collected and with only a few updated floristic catalogues (Romero and Teillier 2009;García 2010;Flores-Toro and Amigo 2013;Romero-Gárate and Teillier 2014). This work aims to describe a new species of Leucheria and investigate its phylogenetic affinities based on molecular data. We also provide a distribution map as well as information on its habitat and phenology, and a provisional assessment of its conservation status.

Herbarium and fieldwork
During the austral summer of 2019, a botanical exploration was made to the coastal mountain range of the Metropolitan Region of Chile, specifically to the Reserva Natural Altos de Cantillana (Fig. 1). Specimens of Leucheria that could not be assigned to any of the accepted species for the genus were found. Herbarium specimens were collected, together with leaf material preserved in silica gel, as well as material preserved in alcohol 70%. Herbarium specimens were deposited to CONC and SGO herbaria. A systematic examination of herbarium specimens of Leucheria found at CONC and SGO, as well as online digital images of specimens available on E, K, and P, was carried out. The descriptions and keys were prepared after examining all available specimens. Terminology for describing floral parts follows Simpson (2010) and Beentje (2016).

Conservation status
The assessment of the conservation status of the species was made using the International Union for Conservation of Nature (IUCN 2017) criteria. The extent of occurrence (EOO) and area of occupancy (AOO) were calculated using GeoCat (Bachman et al. 2011).
Taxon sampling for phylogenetic analysis DNA sequences for nDNA (ITS), as well as cpDNA intergenic spacers (rpl32-trnL and trnF-trnL) were obtained from GenBank (www.ncbi.nlm.nih.gov/Genbank) for all species of Leucheria recognized in the phylogenetic reconstruction of the genus  DNA extraction, amplification, sequencing, and phylogenetic analyses Total genomic DNA was extracted from silica-dried material collected in the field from the type specimen using the Qiagen DNeasy Plant Mini Kit (QIAGEN, Santiago, Chile) following the manufacturer's instructions. Genomic DNA was used to amplify by PCR the internal transcribed spacer region (ITS) and the chloroplast trnL-trnF (Taberlet et al. 1991) and rpl32-trnL (Shaw et al. 2007) intergenic spacers. We amplified all regions in 25 μl PCR reactions following thermocycling procedures used in Jara- Arancio et al. (2017). Sanger sequencing was performed in the Plataforma de Secuenciación y Tecnologías Ómicas, Pontificia Universidad Católica de Chile, using the ABI PRISM 3500 xl Genetic Analyzer (Applied Biosystems). GenBank accession numbers for all DNA sequences are given in Suppl. material 1.
The assembled sequences were aligned using the ClustalW algorithm in Geneious Prime 2019.1.1 (https://www.geneious.com). Phylogenetic analyses were carried for both Maximum-likelihood (ML, Felsenstein 1981), using RAxML-AVX3 version (Stamatakis 2014) included in RAxMLGUI v.2.0 beta (Silvestro andMichalak 2012, Edler et al. 2019) and Bayesian inference (BI) using MrBayes x64 v3.2.7 (Ronquist et al. 2012) respectively. The best-supported model of nucleotide sequence evolution for each partition was determined based on the Akaike Information Criterion (AIC) using MrModeltest v2 (Nylander 2004). For the combined analysis with BI, three partitions were used corresponding for each region, in which evolutionary models for each one were: SYM+I+G in ITS; GTR+G in rpl32-trnL and GTR+I+G in trnL-trnF. Maximum likelihood analyses were run using the GTRGAMMA approximation, which approximates to a GTR model. Substitutions parameters were estimated independently for the nuclear and plastid partitions. The analysis included 1000 ML slow bootstrap replicates with 100 runs. Bayesian analyses were conducted under the respective best fit models for each partition, with two independent runs for 15 million generations, sampling every 10000 generations. Time series plots and effective sample size (ESS) were analyzed using TRACER v.1.7 (Rambaut et al. 2018) in order to check convergence for each run. The first 3 million generations were discarded as burn-in.

Leucheria cantillanensis
Distribution and habitat. Leucheria cantillanensis seems to be endemic to the Cantillana Mountain Range, which is part of the coastal mountain range of central Chile. It grows in shaded crevices of rocky outcrops near 2000 m a.s.l. with SW orientation (Fig. 7). It is known thus far only from the type locality (Fig. 1). L. cantillanensis occurs associated with other rupicolous taxa such as Calceolaria andina Benth.
Phenology. Collected flowering and fruiting in December. Etymology. The specific epithet refers to the coastal mountain range where the species was found, Altos de Cantillana.
Conservation status. Leucheria cantillanensis is assessed here as Critically Endangered (CR) under the IUCN categories and criteria B2ab(i,ii,iii). Criterion B2 was selected because its Area of Occupancy is <10 km 2 (4 km 2 ). Criterion "a" was selected because it is known to exist at only a single location, with only one subpopulation. Criterion b(i,ii,iii) was selected because we expect a continuing decline of suitable area for the species to exist in since it is only found at the highest elevations within the mountain coastal range with very specific soil types and exposition. The quality of its habitat has also been deteriorating over time. The overall precipitation and snow cover in the Cantillana plateau has decreased dramatically over the past 20 years, affecting not only the Andean relict flora but all the vegetation in the area. Fog events which compensate for the drought over the summer, are not as common as before. Leucheria cantillanensis is present in the private reserve "Reserva Natural Altos de Cantillana".
Key to acaulescent/subacaulescent species of Leucheria of Central Chile (31°-34°S) The caulescent herbs with lignified taproots and annual species are excluded. It is important to observe the belowground structures in fresh and dry specimens in order to correctly assign the species to this group. For the habit, please refer to the insets in Figure 5.

Discussion
The ability to establish infrageneric relationships using classic chloroplast markers and ITS within Leucheria is demonstrated in the present study. We were able to corroborate our initial conjectures about the phylogenetic position of Leucheria cantillanensis within the genus. Based on belowground structures (rhizome and roots), we assumed this species belonged to the acaulescent/subacaulescent group found by Jara- Arancio et al. (2017). This group is characterized by plants with a basal and compact rosette, with one or few monocephalic (sometimes with more capitula) scapes. In the first phylogenetic study with molecular data, Jara-Arancio et al. (2017) found that the independent acaulescent evolutionary lines (L. candidissima and L. salina lines) proposed by Crisci (1976) were paraphyletic, as they should include the species L. achillaeifolia and L. nutans in order to be monophyletic. We believe that this finding is highly supported by morphologic characters, as all the species within this clade have the same belowground structures, such as horizontal rhizomes covered by old leaves, and rounded in crosssection, long, dark roots. These characters are distinctly different from the other clades found by Jara-Arancio et al. (2017), which can be either annual or perennial plants with a lignified taproot. Leucheria cantillanensis evidently belongs to the acaulescent/ subacaulescent group, as it has a creeping rhizome that grows between rock crevices and long dark roots (Figs 4,6). Among the genus Leucheria, L. cantillanensis can be easily distinguished by its unique combination of vegetative (Figs 5, 6) and sexual characters (Figs 3, 4, 6), as described in detail in the diagnosis. From the biogeographic point of view, it is interesting to note that the closest species of L. cantillanensis are mainly found at high elevations in the Andes range (except for L. suaveolens, which is endemic to the Falkland Islands). Although we do not have a time-calibrated phylogeny for Leucheria, it is possible to hypothesize, based on Villagrán (2001) and Villagrán and Armesto (2005), that the species diverged from this mainly Andean clade during the repeated glacial/interglacial cycles during the Quaternary. Cold-adapted species occupied lower elevation during glacial periods, and a subsequent shift upwards towards high elevations in both Andean and coastal mountain ranges during warmer periods likely provided the opportunity for allopatric speciation.
Leucheria cantillanensis is an exclusively rupicolous species. Plant communities on rocky places are characterized by having high levels of endemism caused by the high specialization that plants require to thrive in these specific habitats (Porembski et al. 1994;Larson et al. 2005). Currently, only one population of L. cantillanensis is known. The area of Cantillana mountain range with elevations >2000 m comprises an area of near 15 km 2 (EULA-Chile 2004) but the specific rocky outcrops and exposition where the species is found, dramatically reduces the suitable habitat for the species. What is more, during the last decade, Central Chile has experienced a severe deficit in precipitation, the so-called Mega Drought (Garreaud et al. 2020), which has caused major damage and changes to the vegetation in the area (Miranda et al. 2020). Observations by the Park rangers over the past years reveal a decrease in snow cover in the mountain plateau and a reduction of fog and cloud events, an important source of water for rupicolous taxa. In this scenario, it is possible to infer a continuous decline in the quality of its habitat and a projected decline of suitable habitat and population size in the near future. Ex situ conservation measures could be a cost-efficient method to preserve the species, but more information about its distribution, ecology and population size is needed.
The discovery of this new species, which is restricted to mountain tops in the Cantillana Mountain Range in Central Chile, highlights the importance of this site in terms of its unique biodiversity. Cantillana harbours several taxa endemic to the Mediterranean region of Chile Teiller 2009, Romero-Gárate andTeillier 2014) and several new species of insects, lizards and amphibians have been described over the recent years (Vaz-de-Mello and Halffter 2006;Núñez 2007;Zúñiga-Reinoso and Cid-Arcos 2013;Charrier et al. 2015) in the area. Rock communities have been rarely studied in Chile (García 2010). Further botanical surveys in rocky outcrops may reveal a hidden diversity of plants in this biodiversity hotspot and will provide important information needed for the management and conservation of L. cantillanensis.