﻿Nicotianarupicola sp. nov. and Nicotianaknightiana (sect.Paniculatae, Solanaceae), a new endemic and a new record for the flora of Chile

﻿Abstract Nicotianaknightiana is recorded for the first time for the flora of Chile. A new species of Nicotiana, endemic to the coast of the Coquimbo region is described and illustrated. Molecular analysis placed the new species within the N.sect.Paniculatae, as sister to N.cordifolia, an endemic to Juan Fernandez islands. The new species can be considered critically endangered (CR) according to the IUCN categories due to its restricted and fragmented distribution, small population number, and the threat that urbanization and mining activities represent for the conservation of the biodiversity of the area.


Introduction
Nicotiana L. is one of the largest genera in the Solanaceae with 75 recognised species (Clarkson et al. 2017) including the important crop plant Nicotiana tabacum L. Nicotiana is naturally distributed in America and Australia, with one species from southwest Africa (Hunziker 2001). Evidence of homoploidy and polyploidy showed that hybridization has been a major driver of speciation in the genus (Clarkson et al. 2010).
Since Reiche's (1903) treatment, no modern revision of Nicotiana in Chile has been published. According to the last catalogue of the flora of Chile (Rodriguez et al. 2018), the country is home to 12 native accepted species of Nicotiana distributed from Arica y Parinacota region to Magallanes region and Juan Fernandez Archipelago, and two introduced species, Nicotiana tabacum, in Juan Fernandez and Easter Island, and Nicotiana glauca Graham in North and Central Chile. In his monograph of the genus, Goodspeed (1954) provided a taxonomic classification in which he recognised three subgenera further divided into 14 sections. Most of his sections have been confirmed as monophyletic by molecular analyses (Aoki and Ito 2000;Chase et al. 2003;Clarkson et al. 2004;Mehmood et al. 2020). Two Chilean species are included in Nicotiana sect. Paniculatae Goodsp.: the endemics Nicotiana cordifolia Phil. found in Juan Fernandez Archipelago (Philippi 1856) and Nicotiana solanifolia Walp., that grows between Tarapacá and Coquimbo regions (Walpers 1844) and has been traditionally used in the economic and cultural life of the Atacama coastal communities (Ballester et al. 2016). According to Goodspeed (1954), N. sect. Paniculatae share cordate-ovate leaves, evenly spaced along the stem, a long and narrow cylindrical thyrse with thickened central axis, a calyx with regular, broadly triangular teeth, a short and distinct tube proper with a several times longer throat, a greenish to yellowish corolla, stamens of almost equal length, filaments curving immediately above their insertion (except N. cordifolia and N. raimondii J. F. Macbr.), and anthers bending towards the stigma.
The aim of the present work is to record Nicotiana knightiana Goodsp. for the first time for the flora of Chile and describe a new species of Nicotiana endemic to Chile, determining its phylogenetic position and conservation status.

Herbarium and fieldwork
Fieldwork was carried out during November 2020 in Fuerte Lambert and during March 2020 in the proximity of the rivermouth of Elqui river, within the city of La Serena, Coquimbo region (Fig. 1). Specimens were collected and deposited in SGO herbarium. Physical and digital specimens of Nicotiana, including types, were revised at SGO, EIF, CORD, L, E, SI, F, NYBG, UC and US to reach a confident identification and check for possible previous collections of the species found in the field (acronyms according to Index herbariorum; http://sweetgum.nybg.org/science/ih/). Additionally, the citizen science platform iNaturalist (www.inaturalist.org) was consulted to search for possible observations of any of the two species. Terminology of the descriptions followed Goodspeed (1954). Accordingly, the corolla will be differentiated in three parts: the narrowest basal part of the corolla tube (the tube proper), the part of the corolla between the portion where the corolla tube broadens and up to the limb (throat), and the limb. The corolla tube is defined as the tubular portion of the corolla, from the receptacle up to the limb. Taxon sampling for phylogenetic analysis DNA sequences for cpDNA intergenic spacers trnF-trnL, trnS-trnG, and genes ndhF and matK were obtained from GenBank (www.ncbi.nlm.nih.gov/Genbank) for all species of Nicotiana used in Clarkson et al. (2004). Sequences for the new species were generated in the present study. As outgroups we used Symonanthus aromaticus (C.A.Gardner) Haegi, and Mandragora officinarum L.
DNA extraction, amplification, sequencing, and phylogenetic analyses Total genomic DNA was extracted from silica-dried material collected in the field using the Qiagen DNeasy Plant Mini Kit (QIAGEN, Santiago, Chile), following the manufacturer's instructions. Genomic DNA was used to amplify by PCR the following chloroplast regions: trnL-trnF using the primers c and f (Taberlet et al. 1991), trnS-trnG using the primers trnS(GCU) and 3'trnG (UUC) (Hamilton 1999;Shaw et al. 2005), ndhF using the primers 972F and 2110R (Olmstead and Sweere 1994), matK using the primers pairs matK-TR and matK_4F, trnK-710F and matK-1848R, and matK_1F and matK_4R (Johnson and Soltis 1995;Aoki and Ito 2000;Bremer et al. 2002). We amplified all regions in 25 μl PCR reactions using the following thermocycling conditions: initial denaturation of 95 °C for 5 min; 35 cycles at 95 °C for 1 min, a specific annealing temperature for 1 min at 50 °C (60 °C for trnS-trnG), 72 °C for 1 min; and a final elongation period of 72 °C for 15 min. Sanger sequencing was performed using the same primers used during amplification in the case of regions matK and trnL-trnF, the same primers used during amplification plus the primers ndhF_1318 and ndhF_1603R (Olmstead and Sweere 1994) in the case of region ndhF, and the primers trnS(GCU) and trnG(UUC) (Hamilton 1999) for the trnS-trnG region. 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 MAFFT v7.450 algorithm (Katoh et al. 2002;Katoh and Standley 2013) (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 BI analysis, four partitions were used corresponding to each region, in which evolutionary models for each one were: GTR+G for ndhF, trnL-trnF and trnS-trnG, and GTR+I+G for matK. Maximum likelihood analyses were run using the GTRGAMMA approximation. The analysis included 1000 ML slow bootstrap replicates with 500 runs. Bayesian analyses were conducted under the respective best fit models for each partition, with two independent runs for 4 million generations, sampling every 1000 generations. Time series plots and effective sample size (ESS) were analysed using TRACER v.1.7 (Rambaut et al. 2018) in order to check convergence for each run. The first 1 million generations were discarded as burn-in.

Conservation assessment
The assessment of the conservation status of the new species was made using the International Union for Conservation of Nature criteria (IUCN 2017). The extent of occurrence (EOO) and area of occupancy (AOO) were calculated using GeoCat (Bachman et al. 2011).

Results
We could not find any described species of Nicotiana for Chile that matched the morphology of the plants from Elqui river and Fuerte Lambert. A specimen matching the morphology of the species from Elqui river was found at SGO, collected in 2021 in the city of Huasco Bajo, Atacama, approx. 160 km north of La Serena. Plants from Elqui river are 2 m-long, somewhat ineffectively rooted, with branching short perennial shrubs, a pale yellow-green corolla, a dark green limb and eglandular indumentum (Fig. 2, Fig. 3C-D). This species was identified as N. knightiana, a member of Nicotiana sect. Paniculatae known from coastal southern Peru. Two geo-referenced and misidentified observations of N. knightiana in Chile are available on iNaturalist, one from November 2018 in the proximity of our collection site (https://www.inaturalist. org/observations/19777218) and one from May 2021 at approx. 6 km southwards (https://www.inaturalist.org/observations/80390445) ( Fig. 1). A specimen matching the morphology of the species from Fuerte Lambert was found at EIF, collected in 2006 in the whereabouts of Chungungo, Coquimbo, aprox 50 km north of Fuerte Lambert. Plants from Fuerte Lambert are perennial, rupicolous shrubs, with glandular indumentum, a characteristic congested inflorescence, and small, yellow flowers with an almost glabrous corolla ( Fig. 3A-B, Fig. 4). Both species have capsules that produce a large number of seeds but the ones of N. knightiana are more rounded in shape compared to those of the new species (Fig. 5).

Molecular phylogenetic analyses
The concatenated DNA matrix contained 4427 nucleotide characters (1554 matK, 1074 ndhF, 932 trnL-trnF and 867 trnS-trnG), representing 60 ingroup and 2 outgroup accessions. Both BI and ML analyses yielded congruent topologies. The topology of the phylogenetic tree constructed in this study is congruent with the clades found by Clarkson et al. (2004) (Fig.6). Overall, the support given by Bayesian posterior probabilities are higher than bootstrap values given by ML analyses. Nicotiana sections Tomentosae   Description. Robust annual or short-lived shrub up to 3 m with many new stems at different stages of development arising from a lignified horizontal stem poorly anchored to the soil. Stems herbaceous, green, tomentose. Leaves ovate, undulate, base rounded to subcordate, apex obtuse to acute; bigger leaves 13 × 10 cm, indumentum similar to the stem but much denser on the abaxial side which confers a whitish colour, hairs simple, eglandular, pluricellular, brochidodromous venation, petioles a third or half as long as the leaves. Inflorescence a broad thyrse or lax panicle, 40 cm. Pedicels 0.5-1 cm in mature fruits, covered in glandular hairs. Calyx up to 7 mm, cylindric, tomentose, teethup to 2 mm, triangular. Corolla 20-23 mm excluding the limb (tubular part), tube proper 4 mm, throat 16 mm, pale yellow-green, covered in short, eglandular, hairs, limb bottom 3 mm wide, dark green, same indumentum as tube proper, notched into 5 lobes shorter than 1 mm. Stamens extending below the limb, 19 mm except one slightly shorter, filaments adnate to the tube proper, then free, pubescent in the proximal 6 mm, then glabrous and slightly curved, with stamens bending toward the stigma. Capsule 6-8 mm, ovoid. Seeds mainly subrotund, 0.5-0.7 mm, brown, surface reticulate. Embryo straight. Distribution and habitat. Nicotiana knightiana grows naturally in the coast of southern Perú in roadsides, pastures and rocky ravines bottoms. It was found in Chile, within the city of La Serena, Coquimbo region, in the proximity of the rivermouth of Rio Elqui, and in the proximity of Avenida Los Pescadores. It was also found growing in the city of Huasco Bajo, Atacama region (Fig. 1). It grows in a dense Tessaria absinthioides (Hook. & Arn.) DC. scrub, associated with Myoporum laetum G. Forst., Figure 6. Phylogeny of Nicotiana resulting from Maximum Likelihood analysis of the plastid regions matK, rps16, trnS-trnG and trnL-trnF. Numbers above and below the branches represent the Posterior probabilities from the BI analysis and bootstrap values from the ML analysis, respectively. The species whose sequences were obtained in the present study are highlighted in bold, while section Paniculatae including N. rustica is highlighted in grey. Diagnosis. Nicotiana rupicola is most similar to N. solanifolia, from which it differs by its congested panicle (vs. lax panicle), its short and glabrous corolla up to 18 mm (vs. corolla of 35-50 mm, pubescent), non-retroflexed corolla limb (vs. retroflexed corolla limb), mature capsule included or slightly exserted from calyx, 6-10 mm (vs. more than half the length excluded from calyx at maturity, 12-18 mm).
Description. Perennial shrub up to 2 m with many stems arising from a lignified horizontal stem. Stems lignified, light brown, glabrous. Leaves orbicular to ovate, flat to slightly ondulate, margins slightly revolute, base rounded to cordate, apex retuse to obtuse; bigger leaves 10 × 8.5 cm, reducing their size towards the apex, densely covered in two types of hairs in both sides: simple, straight, pluricellular, up to 1 mm long, and glandular (capitate), straight, pluricellular, 50-600 μm long, brochidodromous venation, petioles a quarter to half as long as the leaves, 0.5-2.5(-5.0) cm long. Inflorescence a compact panicle, up to 35 cm; pedicels up to 0.5 cm, same indumentum as the leaves. Calyx up to 10 mm, cylindric, same indumentum as the leaves, teeth up to 3.5 mm, triangular. Corolla 17-18 mm excluding the limb (tubular part), tube proper up 5.5-6 mm, throat up to 12 mm, yellow, glabrous, limb 4 mm wide, yellow, glabrous or with scattered hairs, notched into 5 lobes. Stamens extending below the limb, similar length; filaments adnate for the first 5 mm to the tube proper, then free, pubescent at the base, then glabrous and slightly curved, with the distal portion bending toward the stigma. Capsule 6-10 mm long, ovoid.
Seeds mainly angular, laterally compressed, 0.4-0.6 mm long, dark brown, surface reticulate. Embryo unknown. Chromosome number unknown. (Fig. 3; Fig. 4B-D; Fig. 8) Distribution and habitat. Nicotiana rupicola is endemic to Chile where it is currently known from two locations, Fuerte Lambert and Chungungo, both in the region of Coquimbo (Fig. 1). In Fuerte Lambert it grows among the rocks in a cliff near the ocean together with Alstroemeria magnifica Herb., Cistanthe grandiflora (Lindl. Conservation status. Nicotiana rupicola can be considered as Critically Endangered (CR) under the IUCN categories and criteria B1ab(iii); D. The criterion B1 was selected because its extent of occurrence is < 100 km 2 (8 km 2 ). The criterion "a" was selected because the distribution is highly fragmented. The criterion "b(iii)" was selected because there is a projected decline in the area, extent and quality of habitat. This area is constantly threatened by the expansion of urbanization that is affecting central-north coastal Chile. One of the locations is currently found at less than 300 m from the residential area of Coquimbo and the habitat is being altered by numerous and increasing amounts of formal and informal paths and human activity (camping, garbage, etc.). Moreover, mining activities within the extent of occurrence, especially Minera Dominga, which pretends to settle between the two known localities, will more likely affect possible unknown populations and the quality of the habitat. The criterion D was selected because we observed less than 50 individuals around the two known localities.
Key to the species of Nicotiana sect. Paniculatae found in Chile  (Fig. 4).
It is worth mentioning that N. solanifolia has been erroneously reported for the Coquimbo region based on a collection held at K (Cuming 860), which is the type of Nicotiana breviloba Jeffr., considered by Goodspeed (1954) as a synonym of N. solanifolia. Johnston (1936) considered that Cuming's collections with collection number between 853 and 911 were obtained north of Huasco, Atacama region. This is consistent with the collection localities of the specimens of N. solanifolia revised in the present work which show that the distribution of N. solanifolia and of N. rupicola do not overlap. The southernmost collection of N. solanifolia is found at approx. 100 km north of the northernmost collection locality of N. rupicola.
Molecular analyses showed that plants from Elqui river are likely correctly identified as N. knightiana and that our initial conjectures about the phylogenetic position of N. rupicola as part of the sect. Paniculatae were confirmed (Fig. 6). Our topology retrieves Clarkson's et al. (2004) results of two separate clades within the sect. Paniculatae that reflect geography, one including species from Peru and one including the two endemics N. solanifolia from the North of Chile and N. cordifolia from Juan Fernández Archipelago (Fig. 6). Species of Chilean sect. Paniculatae share long tubular flowers and long-petiolate leaves. Surprisingly, N. rupicola results in being more closely related to the Juan Fernández species N. cordifolia than to the continental species Nicotiana solanifolia, despite their morphological affinity.
An important question regards whether N. knightiana has to be considered native or introduced to Chile. Either the species was never noticed or collected during the last two centuries of botanical expeditions, and presents naturally disjunct populations, being almost 1500 km apart from the closest population found in Peru, or it was recently introduced in Chile by anthropogenic means. The earliest evidence of its presence dates back to 2018 (iNaturalist) and it seems to have been established and possibly expanded to the surrounding area in sites with similar ecological conditions to the river mouth of Elqui river, such as Huasco Bajo. The production of abundant and small seeds, together with the ability of some species to grow in a broad range of open and disturbed habitats, is considered as a common adaptation that ensures high probability of dispersal and establishment. Such is the case for Nicotiana paniculata, N. glauca, and N. plumbaginifolia Viv. that are considered invasive species (Gallo et al. 2008;Gairola et al. 2016;Rodríguez-Caballero et al. 2020;Alharthi et al. 2021) Nicotiana rupicola presents a restricted distribution limited to a small portion of the coastal area of northern Chile, where it grows on two locations on easily accessible coastal rocky cliffs at less than 300 m from urbanization. The population from Fuerte Lambert is situated in an area where urban expansion has caused major damage to the vegetation. The coastal area between Tongoy and Coquimbo is catalogued as a site of interest for the conservation of woody and succulent species due to its high diversity and rates of endemism (Squeo 2000). The area is home to various threatened species such as: Carica chilensis (Planch. ex A. DC.) Solms, Myrcianthes coquimbensis (Barnéoud) Landrum & Grifo, Porlieria chilensis I.M. Johnst. Controversially, it is an area particularly affected by intense expanding urbanization that seriously threatens the conservation of the local biodiversity. Additionally, a new mining facility and a discharge port (Minera Dominga) will be constructed within its extent of occurrence, close to its northernmost known locality. It is likely that several more individuals of N. rupicola are present in the area, since the abiotic and biotic conditions are similar to the currently known localities. None of these individuals were considered during the environmental impact assessment of the project. It is of great importance for the conservation of the species to search for more individuals and localities, and to raise the attention to much needed conservation measures for the species and the unique ecosystem where it is found.