Calceolariaflavida (Calceolariaceae) a new endemic species to central Chile

Abstract A new species of Calceolariasect.Cheiloncos endemic to central Chile is described. A comparison with the morphologically similar species Calceolariaasperula and Calceolariapetioalaris is made, and a key as well as detailed images to differentiate them is provided. The species is only known from the Natural Sanctuary Cerro El Roble, which is part of the coastal mountain range of central Chile and can be considered as Critically Endangered (CR) under the IUCN categories and criteria.


Introduction
Calceolaria Linnaeus is the largest genus within Calceolariaceae with approx. 250 species distributed from Mexico to Southern Chile and Argentina (Molau 1988;Cosacov et al. 2009). The centre of diversity of the genus is found in Peru (Molau 1988). The genus includes herbs and shrubs characterised by opposite leaves and bilabiate corollas with a saccate lower lip with an infolded lobe normally bearing the elaiophore, a highly specialised, oil-producing structure involved in pollination (Vogel 1974).
The latest and most comprehensive revision of Calceolaria for Chile since Reiche (1911), published by Ehrhart (2000), was followed by the publication of new species segregated from the C. integrifolia complex (Ehrhart 2005), the revision of Calceolaria section Calceolaria (Puppo and Novoa 2012) and by Calceolaria philippii Eyzaguirre (2014). In Chile, there are 61 currently recognized species of Calceolaria, ten of which are further separated in a total of 30 subspecies (Rodriguez et al. 2018). Out of a total of 81 taxa, 60 (74%) are endemic to Chile. Calceolaria in Chile presents a wide distribution, from the latitudes of Arica y Parinacota region (18°35'S) to Magallanes region (54°50'S), and from the coast to the high elevations of the Andes (0-4300 m). Unsurprisingly, the area of most diversity is central Chile, from the Coquimbo region to the Araucania region (Ehrhart 2000). High levels of endemism in plants are common in the biogeographic area of central Chile which is recognized as a biodiversity hotspot (Myers et al. 2000;Arroyo et al. 2004).
Infrageneric classification within Calceolaria has been a subject of several works (Bentham 1846;Wettstein 1891;Kränzlin 1907;Pennell 1951). Molau (1988), working on the monograph of the tropical species of Calceolaria, restructured previous classifications and divided Calceolaria into three subgenera. Calceolaria subgenus Calceolaria comprises mainly species found in tropical regions, while Calceolaria subgenus Cheiloncos (Wettstein) Pennell and Calceolaria subgenus Rosula (Descole & Borsini) Molau comprise mostly temperate species. Subsequently, Ehrhart (2000), organized the species native to Chile in four sections: Calceolaria sect. Calceolaria (one species, Calceolaria pinnata L. subsp. pinnata), Calceolaria sect. Kremastocheilos Witasek (one species, Calceolaria uniflora Lam.), Calceolaria sect. Tenella C. Ehrhart (one species, Calceolaria tenella Poepp. & Endl.), and Calceolaria sect. Cheiloncos Wettstein (47 species). The latter section, comprising most of the species present in Chile, was further divided into 14 informal Greges, based on vegetative and reproductive characters. More recently, molecular (Andersson 2006) and combined molecular and morphological studies (Cosacov et al. 2009), confirmed the subgeneric classification and some of the sections proposed by Molau (1988) as monophyletic, while most of the sections were found to be polyphyletic, and sections Tenella and Kremastocheilos sensu Ehrhart (2000) had little support. Due to the lack of resolution and the low sampling of Chilean species in Andersson (2006) and Cosacov et al. (2009), the classification proposed by Ehrhart (2000), particularly for Calceolaria sect. Cheiloncos, is yet to be supported by studies with more extensive sampling.
The aim of this work is to describe a new species of Calceolaria, endemic to central Chile, assess its conservation status and provide a key for correct identification.

Methods
Between the austral Spring of 2018-2020, several botanical explorations were carried out in the coastal mountain range of central Chile, between the limits of Valparaiso region and the Metropolitan region, in the Natural Sanctuary "Cerro El Roble", 75 km northeast of Santiago's urban area (Fig. 1). Specimens of Calceolaria that could not be assigned to any of the described species of the genus were found flowering in two sites close to the summit (1722-1729 m and 1766 m). The climate of the study site is classified as Mediterranean type with a rainfall regime characterized by an annual mean precipitation of 656 mm, a water deficit of 897 mm, and a 7-month dry season (Donoso et al. 2010). The soil is mainly composed of weathered granitic rocks (Brüggen 1950). The vegetation of this area is characterized by a relict deciduous forest dominated by Nothofagus macrocarpa (Nothofagaceae), and surrounded by sclerophyllous forest and scrub of Quillaja saponaria (Quillajaceae) and Lithraea caustica (Anacardiaceae) (Latorre-Beltrán 2012). At lower elevations, on the bottom of creeks with permanent flooding by groundwater, dense swamp forests of Drimys winteri (Winteraceae) and Luma chequen (Myrtaceae) can be found. At the summit, a relict andean scrub dominated by Chuquiraga oppositifolia (Asteraceae) and Azorella prolifera (Apiaceae) is found (Ministerio del Medio Ambiente 2018).
Specialised literature on systematics and taxonomy of Calceolaria was consulted (Witasek 1905;Valenzuela 1969;Ehrhart 2000;Ehrhart 2005). Herbarium specimens were collected and deposited at SGO (Lavandero 372; Lavandero & Santilli 201027). A systematic examination of selected specimens of Calceolaria found at SGO, EIF, CONC, as well as online digital images of specimens available on E, PH and US (acronyms following Thiers 2021) was carried out to search for more collections that could be morphologically coincident with the species. Herbarium specimens with similar morphology were found at SGO identified as Calceolaria asperula and Calceolaria aff. asperula. A thorough examination and dissection of the type specimen of Calceolaria asperula Phil. (SGO 055831) was performed, due to discrepancies with the schematic representation of the flower and the description of the species by Ehrhart (2000) and to confirm the identity of the new species.
The description and key were prepared after examining all available specimens. Description was made based on terminology following Ehrhart (2000) and Ehrhart (2005).
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).

Results
Following the morphological comparison of the plant collected with the specimens found in the consulted herbaria, we reached the conclusion that the individuals found in Cerro El Roble represent a new species. The new species is vegetatively similar to Calceolaria asperula Philippi and to Calceolaria petioalaris Cavanilles, both species endemic to Central Chile, belonging to Calceolaria sect. Cheiloncos, group B, Grex X (C. dentatae) and Grex XI (C. petioalaris) sensu Ehrhart (2000), respectively. The three species have in common the growth form and other vegetative characters. They are perennial herbs with a lignified base, without any woody shoots aboveground that last from one growing season to the next one, with non-branching shoots aboveground, new shoots early in the season with very short internodes, giving them the appearance of a rosette-like structure (these internodes elongate later in the season and the rosette disappears), and ovate leaves and serrate margins, covered in glandular hairs.
Nevertheless, both leaf texture and indumentum and flower morphology differ considerably among the three species (Figs 2-4). The secondary and tertiary venation of the new species is visibly impressed on the adaxial side and prominent on the abaxial side of the lamina (Fig. 2C-D). The leaf indumentum is formed by long and densely arranged glandular and eglandular trichomes, which gives a glutinous and sticky tex-ture. Freshly collected material can hardly be separated from the paper in which it is dried. The leaf texture and indumentum is similar to Calceolaria asperula ( Fig. 2A-B), but the latter has a deeply impressed venation on the upper surface, forming deep cavities, giving the most rugose aspect of the three species. Calceolaria petioalaris, has a venation slightly impressed on the adaxial side and slightly prominent on the abaxial side, with leaf indumentum composed of short glandular and eglandular trichomes, which give a less glutinous and sticky texture; freshly collected material can easily be separated from the paper in which it is dried ( Fig. 2E-F). The flower lips of the new species are rounded in shape, saccate, and the upper lip is narrower and longer than half the length of the lower lip ( Fig. 3C-D), while the flower lips of C. petioalaris are squared, flat and almost equal in width, and the upper lip is shorter than the lower lip ( Fig. 3E-F). The length of the stamens of the new plant and C. petioalaris is similar, while C. asperula presents much shorter filaments (Fig. 4A, C, E). The new species shows an elaiophore similar to C. petioalaris and different from the one of C. asperula which has an elaiophore made of dispersed oil producing trichomes (Fig. 4B, D, F), a character that is unique among Calceolaria found in Chile (Ehrhart 2000).
The dissection of the type specimen of Calceolaria asperula (SGO 055831) showed that the lips differ in size, being the upper lip less than half the size of the lower lip (Suppl. material 1). This contrasts with the schematic representation of the flower of Calceolaria asperula found in Ehrhart (2000). The dissection also confirms that the elaiophore of Calceolaria asperula, is formed by dispersed oil producing trichomes (Suppl. material 1: Fig. S1D-E). Diagnosis. C. flavida is most similar to C. asperula and C. petioalaris in growth habit and in having leaves of similar shape covered in glandular hairs. C. flavida can easily be distinguished from C. asperula in having pale yellow corolla (vs. bright yellow), the upper lip longer than half the length of the lower lip (vs. upper lip shorter than half the length of the lower lip), anthers much shorter than filaments and opening towards the distal part of the upper lip (vs. anthers as long as filaments and opening toward the style) and an elaiophore with densely arranged oil-producing trichomes (vs. dispersed oil producing trichomes). It can be distinguished from C. petioalaris by its reddish stems (vs. green), secondary and tertiary veins of the adaxial side of leaf lamina visibly impressed (vs. secondary and tertiary veins of the adaxial side of leaf lamina slightly impressed), pale yellow corolla (vs. bright yellow), upper lip narrower than lower lip seen from above (vs. upper lip as wide as lower lip), lips rounded in shape (vs. squared), saccate upper lip (vs. flat), and style inserted in corolla (vs. exserted).
Habitat and distribution. C. flavida seems to be endemic to the Natural Sanctuary Cerro El Roble (33°00'S 71°01' W), which is part of the coastal mountain range of central Chile (Fig. 1) (Fig. 6A).
Phenology. The species was found flowering between October and January. Etymology. TThe specific epithet flavida is a singular, feminine, nominative Latin adjective alluding to pale yellow colour of corolla.
Conservation status. C. flavida can be considered as Critically Endangered (CR) under the IUCN categories and criteria B1ab(iii). The criterion B1 was selected because its extent of occurrence is < 100 km 2 (0.995 km 2 ). The criterion "a" was selected because it is known to exist at only one location (=1). The criterion "b(iii)" was selected because there is a projected decline in the area, extent and quality of habitat. Climate change and the persistent drought that has been affecting Central Chile represent a threat to plants that grow in the region. Starting in 2010, the Chilean territory between the Coquimbo and Araucanía Regions has experienced a rise in temperature and a precipitation deficit of approximately 30% causing visible deterioration of nonirrigated vegetation as well as increasing the likeability of forest fires (Garreaud, 2015). Stems green; leaf margin dentate with sharp teeth, indumentum of short glandular hairs; leaves with secondary and tertiary veins slightly impressed on the adaxial side; corolla bright yellow, upper lip as wide as lower lip seen from above, lips squared in shape, bright yellow, flat; style exserted from corolla..... C. petioalaris -Stems reddish; leaf margin dentate, with smooth teeth, indumentum of long glandular hairs; leaves with secondary and tertiary veins visibly impressed on the adaxial side; corolla pale yellow, upper lip narrower than lower lip seen from above, lips rounded in shape, pale yellow, saccate; style inserted in corolla.......C. flavida

Discussion
Initial confusion existed regarding the identity of Calceolaria asperula. In the protologue of C. asperula, Philippi (1895) only gives the diameter of the inferior lip in a short description without mentioning the upper lip. Ehrhart (2000) only illustrates the taxon with a schematic representation of the flower, showing lips of almost equal size, being the upper lip slightly smaller than the lower, and only gives the size of the upper lip seen from above, being 5.5 mm in diameter approximately. Ehrhart (2000) describes it as a species with a unique combination of characters such as the anthers opening towards the style and an elaiophore made of dispersed oil-producing hairs. The dissection of a flower from the type material of C. asperula (SGO055831) (Suppl. material 1) shows Ehrhart's (2000) description to be mostly accurate regarding vegetative morphology and elaiophore structure, but the upper and lower lip description is incomplete and imprecise, making the schematic representation of the flower doubtful. This imprecision in Ehrhart's schematic representation of the flower might explain why specimens of Calceolaria flavida found in SGO were formerly identified as C. asperula or, in some cases, as Calceolaria aff. asperula.
The classification proposed by Ehrhart (2000) for the species of Calceolaria present in Chile, although not yet confirmed by molecular evidence, is however very useful for grouping species based on both vegetative and reproductive characters, the latter having higher weight for the classification at lower levels. Within Calceolaria sect. Cheiloncos, Calceolaria flavida falls into group B, by having an upper lip at least 1/3 as long as the lower lip and anthers shorter than the filaments. At the Grex level, it could be classified within two Greges, Grex X (comprising C. densifolia Phil., C. dentata Ruiz & Pav., C. flavovirens C. Ehrhart, C. lepida Phil., C. morisii Walp., C. nitida Colla, C. polifolia Hook, C. asperula Phil. and C. purpurea Graham) and Grex XI (comprising C. petioalaris Cav. and C. latifolia Benth.). Among these species, Calceolaria flavida has clear affinities with two species, C. asperula and C. petioalaris, based on leaf shape and growth habit, leaving out all the other species within these two Greges presenting a shrubby habit. The most useful characters to differentiate the new species from the morphologically most similar species C. asperula and C. petioalaris, proved to be flower related, showing the importance of these stable characters for the taxonomy of Calceolaria. From the ecological and geographical perspective, these three species can be clearly distinguished. Calceolaria petioalaris is the only one among the three species that associates with meso-hydrophytic conditions, growing most of the time near water courses such as small streams or ravines from the coast up to mid-elevations of the Andean cordillera (50-1800 m), between the Coquimbo and Maule Regions (Ehrhart 2000). García, a rupicolous taxon found only on rocky outcrops of the coastal Cordillera between 32°42'S -33°12'S at elevations of 1600-2100 m (García 2010). La Campana National Park, adjacent to Natural Sanctuary Cerro El Roble, is also home to the narrow endemic Calceolaria campanae Phillipi, which grows between the rock crevices near the summit of Cerro La Campana. This pattern of diversity found in Calceolaria is not unusual for the Chilean flora. Several genera share the same pattern of high diversity and endemism in Central Chile, such as Senecio L., Chaetanthera Ruiz & Pav., Haplopappus Cassini, Leucheria Lagascae, Oxalis L. and Adesmia D.C. Fuentes et al. 1995). A combination of high climatic heterogeneity due to latitudinal and altitudinal gradients (Armesto et al. 2007), plus the climatic history of the Quaternary, particularly glaciations and the presence of coastal refugia, are the probable drivers for the higher diversity and endemism in this region Villagrán 1995;Hinojosa and Villagrán 1997;Villagrán and Hinojosa 1997).
The origin and present distribution of C. flavida could be related to the series of expansions/contractions and isolation of the vegetation belts in the Coastal Cordillera due to the glacial/interglacial cycles. Since there is no updated phylogeny of Calceolaria, no relationships could be inferred for C. flavida. Based on its morphology and following the preliminary phylogenetic studies (Cosacov et al. 2009), it could be hypothesized that it belongs to the subgenus Cheiloncos sect. Rugosae along with the most morphologically similar species Calceolaria asperula and Calceolaria petioalaris, all endemic to central Chile. A well-resolved phylogeny of the genus could help clarify the relationships among these species and establish a better understanding of the complex evolutionary history of Calceolaria in central Chile.
Since Calceolaria flavida appears to be a narrow-endemic and our preliminary assessment classifies it as Critically Endangered (CR), further surveys in the Coastal Cordillera of central Chile are needed in order to fully understand its distribution and population size.