Molecular phylogenetics and morphology reveal the Plettkea lineage including several members of Arenaria and Pycnophyllopsis to be a clade of 21 South American species nested within Stellaria (Caryophyllaceae, Alsineae)

Abstract: Caryophyllaceae with a cushion-like life form occur with a large number of species at the higher altitudes of the Andes (3500–5000 m) and have evolved convergently in several different lineages. Based on molecular phylogenetic analysis it is shown that members of the former genera Plettkea and Pycnophyllopsis, but also certain species previously classified as Arenaria constitute a subclade nested within the monophyletic genus Stellaria. Both plastid (trnK-matK-psbA + trnL-F) and nuclear (nrITS) trees converged on such a highly supported ‘Plettkea’ clade. Morphologically, the members of the ‘Plettkea’ subclade of Stellaria are further characterized by reduced to completely absent petals and seeds with a more or less conspicuous tuberculate testa. This clade is described as S. sect. Plettkea (Mattf.) Montesinos & Borsch. Species-level relationships within S. sect. Plettkea are also congruently inferred by plastid and nuclear genomic compartments, with three further sublineages recognized: Altogether, our detailed taxonomic revision showed that the ‘Plettkea’ clade in fact constitutes an Andean radiation of 21 species within Stellaria, four of which are described as new to science. Earlier treatments indicated just a few species with a putative placement. The results of this investigation underscore the importance of fieldwork and integrated molecular-morphological approaches to assess the species diversity in Andean plant groups. In addition to the phylogenetic analysis, we provide a taxonomic backbone including all names and types, descriptions and information on distribution and ecology and a key for identification. Regarding the next relatives of the S. sect. Plettkea clade, our plastid trees depict the ‘Nitentes’ clade of Stellaria as sister, whereas nrITS instead suggests a sister group relationship of the ‘Nitentes’ with the speciose ‘Larbreae’ clade. Our inferred relationships of major clades further deviate from published molecular trees by indicating an early branching position of the ‘Petiolares’ clade. Citation: Montesinos-Tubée D. B. & Borsch T. 2023: Molecular phylogenetics and morphology reveal the Plettkea lineage including several members of Arenaria and Pycnophyllopsis to be a clade of 21 South American species nested within Stellaria (Caryophyllaceae, Alsineae) Version of record first published online on 21 December 2023 ahead of inclusion in December 2023 issue. Resumen: Las Caryophyllaceae, con forma de vida en matas o almohadillas, se encuentran en las mayores altitudes de los Andes (3500–5000 m) con un gran número de especies y han tenido una evolución convergente en diversos linajes. Análisis filogenéticos moleculares, recuperan un subclado anidado dentro del género monofilético Stellaria, constituido por los miembros previamente clasificados en Plettkea y Pycnophyllopsis y especies clasificadas hasta ahora en Arenaria. Tanto los árboles de plástidos (trnK-matK-psbA + trnL-F) como los nucleares (nrITS) convergen en un clado de ‘Plettkea’ altamente apoyado estadísticamente. Morfológicamente, los miembros del subclado ‘Plettkea’ de Stellaria se caracterizan, además, por compartir pétalos reducidos o completamente ausentes y semillas con un tegumento tuberculado más o menos conspicuo. El subclado se valida como S. sect. Plettkea (Mattf.) Montesinos & Borsch. Las relaciones a nivel de especie dentro de esta sección también se infieren congruentemente por compartimentos genómicos plastidiales y nucleares, con tres sublinajes. En conjunto, nuestra detallada revisión taxonómica mostró que el clado Plettkea constituye una radiación andina de 21 especies dentro de Stellaria, cuatro de las cuales se describen como nuevas para la ciencia. Tratamientos anteriores indicaban sólo unas pocas especies con una ubicación putativa. Los resultados de esta investigación destacan la importancia del trabajo de campo y la integración de métodos moleculares y morfológicos para evaluar la diversidad de especies en los grupos de plantas andinas. Además del análisis filogenético, proporcionamos una columna vertebral taxonómica que incluye todos los nombres y datos de los tipos nomenclaturales. Se proporcionan descripciones e información sobre distribución y ecología así como una clave para identificarlas. Con respecto a los parientes próximos del clado de S. sect. Plettkea, nuestros árboles de plástidos muestran al clado ‘Nitentes’ de Stellaria como hermano, mientras que nrITS sugiere más bien una relación de grupo hermano de los ‘Nitentes’ con el clado ‘Larbreae’. Las relaciones inferidas de los clados principales se desvían aún más de los árboles moleculares publicados previamente, al indicar una posición de ramificación temprana del clado ‘Petiolares’.


Introduction
Members of the family Caryophyllaceae constitute important elements of the flora in the Andes, mostly at higher altitudes.All together the diversity of Caryophyllaceae in the interandean dry valleys, the altiplano, Patagonian plateaus, the paramos, and high mountain peaks may be estimated with c. 280 species and new species continue to be described (e.g.Timaná 2017;Montesinos-Tubée & al. 2018, 2020).Species of different genera show adaptations as cushion plants such as Arenaria L., Pycnophyllopsis Skottsb.or Pycnophyllum J. Rémy, some of which are well known for their large cushions like Pycnophyllum molle J. Remy.These cushion plants, especially the exclusively Andean Pycnophyllum spp.and Pycnophyllopsis spp.grow at altitudes of 4000-5500 m (Catorci & al. 2014;Montesinos-Tubée 2015).They are not easily distinguishable in the field and also comprehensive phylogenetic analyses were so far lacking, limiting insights on the naturalness of these genera.Apart from details in the cushion growth form, the species of Pycnophyllum are dioecious, whereas this feature is less consistent in Pycnophyllopsis in which most species are dioecious with pistillate and staminate individuals but not all (P.smithii Timaná and P. weberbaueri Muschler are hermaphroditic; Timaná 2017; Sharples & Tripp 2019).In Pycnophyllopsis the leaves bear scarious margins and possess a sparse indumentum, both of which are absent in Pycnophyllum which tends to have glabrous leaves.In the case of Pycnophyllum the species included (P.bryoides (Phil.)Rohrb., P. molle J. Remy, P. spathulathum Mattf.) in the overall molecular tree of the Caryophyllaceae by Greenberg & Donoghue (2011) depicted this genus in the monophyletic tribe Polycarpeae, and thus in a major lineage of the family distant from both the Alsineae and Arenarieae clades.
Even though there are treatments by J. L. Zarucchi and R. E. Gereau in the Catalogue of the flowering plants and gymnosperms of Peru (Brako & Zarucchi 1993), and F. O. Zuloaga & C. A. Zanotti in the Catálogo de las plantas vasculares del Cono Sur (Zuloaga & al. 2008), no comprehensive taxonomic revisions or monographs have been elaborated for most of the larger genera of the Caryophyllaceae in South America like Arenaria, Cerastium L., Drymaria Willd.ex Schult.or Stellaria L. since Williams (1898) and Macbride (1937).To some extent Pycnophyllum is an exception as it was the focus of more recent studies (Timaná 2005(Timaná , 2017)), although only including a few new ITS sequences analysed with parsimony in a broad context of Caryophyllaceae with sequences available at the time (Timaná 2005).The representation of South American Caryophyllaceae in molecular phylogenetic analyses is so far rather scarce.
Based on a few species, Greenberg & Donoghue (2011) showed that Pycnophyllum and Drymaria belong to the 'Polycarpeae' subclade of the broadly paraphyletic subfamily Paronychioideae.To the contrary, most other genera comprising Andean species are part of the Alsinoideae.The circumscription of the subfamily Alsinoideae and of its tribes has varied over time, and the first more comprehensive molecular phylogenetic analysis by Harbaugh & al. (2010) revealed that not only the subfamily but also most of the tribes were para-or polyphyletic.This extends to many other genera as evident through the broader taxon sampling in Greenberg & Donoghue (2011).The pre-phylogenetic classification by Bittrich (1993) included 23 genera with an estimated 700 species in the tribe Alsineae.The re-definition of the tribe by Harbaugh & al. (2010) to make it monophyletic just contained the genera Cerastium L., Holosteum L., Lepyrodiclis Fenzl, Moenchia Ehrh., Myosoton Moench, Odontostemma Benth.ex G. Don, Plettkea Mattf., Pseudostellaria Pax, and Stellaria but not Arenaria and relatives.The latter were classified in the tribe Arenarieae along with the genera Brachystemma D. Don and Moehringia L. Arabi & al. (2022) presented phylogenetic trees inferred from nrITS and from combined nrITS + chloroplast rps16 sequence data.The authors found two subclades of the monophyletic Alsineae, one including the genera Mesostemma Vved., Shivparvatia Pulsalkar & D. K. Singh, Odontostemma, Pseudostellaria, Stellaria ("Alsineae A"), and the other comprising Nubelaria M. T. Sharples & E. A. Tripp, Hartmaniella M. L. Zhang & Rabeler, Rabelera M. T. Sharples & E. A. Tripp, Dichodon (Bartl. ex Rchb.)Rchb., Holosteum, Moenchia, and Cerastium ("Alsineae B").The monotypic genus Pseudocerastium C. Y. Wu & al., an endemic of the Anhui and Hubei provinces in China, was found to be nested within Cerastium (Yao & al. 2021) and consequently merged with that genus.Recently, Xue & al. (2023) described the three further monotypic genera Hesperostellaria Gang Yao & al., Reniostellaria Gang Yao & al. and Torreyostellaria Gang Yao & al. of the Alsineae.The genus Plettkea, which was not sampled by Arabi & al. (2022), is a further Andean taxon of the Alsineae that forms small cushions.However, Sharples & Tripp (2019) had already included one of its members, Plettkea tetrasticha Mattf., in their RAD analysis of the genus Stellaria along with nine South American species of Stellaria (from the 'Petiolares' clade), and depicted it nested within Stellaria.Mattfeld (1934) described Plettkea as different from Pycnophyllopsis using the single-seeded closed fruit (capsule) and bipartite petals in combination with strongly perigynous flowers as diagnostic characters and, in contrast, considered Pycnophyllopsis to differ by having only slightly perigynous flowers with a broadened disk.Although Mattfeld (1934) noted that the 1-seeded capsule would be a difference of both Pycnophyllopsis and Plettkea from Stellaria, he believed that this feature could have originated multiple times in the Caryophyllaceae (e.g. in Pycnophyllum).In his view, the 1-seeded capsule would not be a "very strong" character to serve as indicator for the overall affinities of the two genera (Matt feld 1934).Mattfeld considered the two completely al.2021).Recently, Wei & Ronse De Craene (2019) investigated the development of petals in Caryophyllaceae.Although their publication did not specifically present results on Stellaria, the most likely explanation seems a reduction and loss of petals is linked with an acceleration of stamen initiation, thus leading to reduced space for the development of petals (Louis Ronse De Craene, pers. comm.).We would therefore assume gradual petal reductions in our study group.The finding of numerous evolutionary transitions between petaly and apetaly, both within and across multiple genera (Sharples & al. 2021), adds to the convergent evolution of life forms in different unrelated lineages of the Caryophyllaceae.Interestingly, the presence or absence of petals or their reduction was not used in pre-phylogenetic definitions of genus concepts, while both Arenaria and Stellaria were said to contain species groups lacking petals (e.g. in Pax & Hoffmann 1934).
Apart from Stellaria, several species that show highly reduced petals (sometimes simplified to being apetalous) were previously included within the genus Arenaria, such as A. alpamarcae A. Gray, A. andina Rohrb., A. aphanantha Wedd., A. crassipes Baehni & J. F. Macbr., A. engleriana Muschl., A. nitida (Bartl.)Rohrb.and A. pedunculosa Wedd..These South American members of Arenaria were placed variously in A. subg.Dicranilla (Fenzl) Fenzl, A. subg.Leiosperma (F.N. Williams) McNeill and A. subg.Eremogoneastrum F. N. Williams.And even more interestingly, several of them such as A. alpamarcae were reported to form small cushions and grow at high altitudes in the central Andes.However, no comprehensive phylogeny of Arenaria was available.Considering the high level of homoplasy in petal evolution, we could not take the presence or absence of petals as a solid indicator for assigning a species to any of the genera Arenaria in tribe Arenarieae or Pycnophyllopsis and other genera of the tribe Alsineae.
In the context of ongoing work aiming at a new species-level taxonomic backbone for the family Caryophyllaceae as part of the Caryophyllales Taxonomic Expert Network (see Borsch & al. 2015;Arias & al. 2018) sequence data are being generated for speciose genera that lack comprehensive recent revisions such as Arenaria.As a first step, phylogenetic work has been directed toward including as many species as possible across Alsineae, Arenarieae and other tribes, while keeping the sequencing effort per sample at a minimum (Mansion & al. 2012), which then allowed us to formulate specific questions on the hypothesized clades.More specifically, fieldwork was initiated in 2015 with the intention of gathering a significant number of the species of the Caryophyllaceae from the central Andes to be included in this phylogenetic approach, considering the high and still insufficiently known species diversity in the area with significant levels of convergent evolution of life forms adapted to high mountain habitats.Based on this first set of trnL-F and ITS sequence data, all members of the genera Plettkea free styles as diagnostic for the Alsineae whereas Pycnophyllum with the stigmas fused for most of their length was believed to be only distantly related (tribe Pycnophylleae).Consequently, Mattfeld (1934) transferred Pycnophyllum macrophyllum Muschler and P. weberbaueri to Plettkea, thus assuming the convergence of both the cushion plant life form and of the 1-seeded capsule fruit.Bittrich (1993) accepted Plettkea as different from Pycnophyllopsis, considering the flowers being strongly perigynous, pentamerous, or tetramerous, and with episepalous stamens.In contrast to this, Timaná (2017) merged Plettkea with Pycnophyllopsis to then consist of eight species, one of which (Pycnophyllopsis smithii) was newly described based on a specimen from Huarochirí (Peru, Dpto.Lima).Timaná (2017) did not analyse the phylogenetic relationships of these two genera.
The genus Stellaria consists of herbs, commonly known as chickweeds or starworts that occur in a wide range of ecosystems, from low to high altitudes on nearly all continents (Sharples & Tripp 2019).Pax & Hoffmann (1934) attempted the last worldwide species-level classification and estimated the genus to contain over 100 species.Sharples (2019) and Sharples & Tripp (2019) re-circumscribed Stellaria to be monophyletic based on their RAD trees that yielded much better resolution than the tree of Greenberg & Donoghue (2011) for Stellaria and relatives.Sharples & Tripp (2019) segregated S. holostea L. into the new genus Rabelera, morphologically differing from Stellaria by the square and ciliate stems, elongated sessile and lanceolate leaves, leafy bracts and by the petals that are only bilobed for one half of their length.Another distant lineage, S. arisanensis (Hayata) Hayata and allies was described as the new genus Nubelaria.For the monophyletic core of Stellaria, Sharples & Tripp (2019) found five major clades that they named informally as 'Insignes' (species from North America and Asia), 'Larbreae' (being the most diverse clade with species in temperate Asia, Europe and North America), 'Nitentes' (three species from Central and North America), 'Plettkeae' represented by one species from the central Andes and 'Petiolares' (South America, represented by nine species).Although three of the names ('Insignes', 'Larbreae', 'Petiolares') were among the unranked divisions that first appeared in Fenzl's (1840) treatment of Stellaria, Sharples and Tripp (2019) did not equate their clades to a formal infrageneric classification.
Most species of Stellaria bear showy petals but approximately one-fourth of the genus is characterized by petal reductions, ranging from absent to partial or reduced petals as shown by Sharples & al. (2021).According to their reconstruction of character evolution, petal loss or reduction evolved independently in multiple lineages, including the branch to Plettkea where this is an obvious feature, even when looking at the plants in the field.The evolution of apetaly can be linked to abiotic pollination (Culley & al. 2002) or it could be associated with the evolution of autogamy (Pieper & al. 2016;Klepikova & and Pycnophyllopsis along with a number of taxa currently classified as Arenaria and further specimens that could not be identified using the available Floras, were recovered as part of a strongly deviant clade including the members of core Stellaria sensu Sharples & Tripp (2019) and Arabi & al. (2022).
The specific objectives of this investigation were to (1) reconstruct phylogenetic relationships within the clade of the Caryophyllaceae comprising Stellaria, Plettkea, Pycnophyllosis and the entities formerly classified as Arenaria but belonging here (2) evaluate specieslevel relationships in Plettkea and related taxa and (3) to provide an updated species-level treatment of Plettkea and related taxa including morphological descriptions, a consistent classification of all known entities with the correct nomenclature and a first assessment of species distribution using available material.

Sampling strategy and selection of material
Fieldwork was undertaken in the highlands of the central Andes between 2015 and 2022 covering all species of Caryophyllaceae.Herbarium specimens were collected with duplicates and corresponding leaf tissue was dried in silica gel.Collecting focused on eight Andean departments of Peru (c.200 localities), allowing us to cover most of the type localities of previously described species in Arenaria, Pycnophyllopsis and Stellaria as hitherto classified.In addition, herbarium material was evaluated from B and several other herbaria from which material was loaned to B (GOET, L, LPZ, O and PRC) and sampled for molecular analysis in case the respective taxon was not found in the field.The following institutions were visited: CONC, CUZ, F, GOET, HSP, HOXA, HUT, K, LP, LPB, LPZ, MO, MOL, NY, P, PRC, SI, US and USM (herbarium codes according to Thiers continuously updated).
Following a first round of phylogenetic assignment of the material to major clades using short sequences (plastid trnL-F and nrITS) that could easily be generated for many samples following the approach of Mansion & al. (2012), a fraction of these samples including species from all three genera mentioned above turned out to belong to a very distinct subclade of a Stellaria clade.Further research then focused on these samples.To determine the precise position of this subclade, the sampling was extended to represent all major lineages of Stellaria, guided by the trees of Sharples & Tripp (2019).

Compilation of all previously published names / taxa
A list of Arenaria names was received from the WFO Data Centre in February 2018 and imported into the EDIT Platform.This import included names accepted in the WFO backbone and their synonyms therein (WFO 2018).For Plettkea, Pycnophyllopsis and Stellaria, the names were obtained from the World Checklist of Vascular Plants dataset received from the Royal Botanic Gardens, Kew in December 2019 (Kew WCVP 2019).In both cases, the import included accepted names and synonyms and each of these taxonomic states was preliminarily assigned to entities that were considered to be part of the Plettkea lineage based on molecular data.With the objective of ensuring that we gathered all names published in our study group, the relevant treatments of South American Caryophyllaceae were also consulted, especially Williams (1898), Mattfeld (1922), Macbride (1936), Zarucchi & Gereau in Brako & Zarucchi (1993) and Beck & al. (2014) for the central Andes.Furthermore, the Catálogo de las plantas vasculares del Cono Sur was considered (Zuloaga & al. 2008) and the recent review by Montesinos-Tubée & Teillier (2022) for Chile.Further names were entered into the TEN's database using the EDIT Platform for Cybertaxonomy (Berendsohn 2010), if not already present.The EDIT Platform is an open-source software with tools and services covering all parts of the taxonomic workflow.Moreover, we cross-checked this data set with Tropicos. org (2023).We then used JSTOR Global Plants (JSTOR 2023) to search for type material associated with the respective names to verify information in the protologues and discover additional type specimens.

Identification of plant material and elaboration of the taxonomic treatment
In the absence of a modern Flora or identification keys, type and other herbarium specimens cited in the original works were examined for a spectrum of morphological characters including those mentioned in the original descriptions to compare with our recently collected material.This process included further specimens of our Andean study group at the cited herbaria to assess the amount of morphological variation and to group the available specimens into morphologically distinct entities including the type specimens.It must be noted that many of the herbarium specimens had so far remained unidentified to species.In a second step, these entities were compared with the specimens included in lineages recovered by molecular phylogenetic analysis in order to check consistency in the presence of morphological character states.In general, in terms of species delimitation the molecular trees are taken as a hypothesis of what the closest relatives of a species are, and thus the differences in morphology these taxa will be discussed (in the notes of the taxonomic treatment).Also, the geographic location of the type specimens was checked in order to see if these localities were included within a hypothesized range of a respective taxon at species level.Due to the often very small number of specimens available for the respective entities, we did not employ any morphometric approach.

Assessment of morphological characters
Habit characters were mostly examined during fieldwork and recorded for the collected specimens.All other characters were assessed from herbarium specimens with Olympus SZX10 and NSZ-405 1X-4.5Xstereo microscopes.For new to be described species, macro photos were taken in addition to the text description.The colour of the distinct organs was assessed with a colour chart.Seed morphology was evaluated under a Hitachi FE-SEM (Field Emission Scanning Electron Microscope) at BGBM.Furthermore, field photographs of the collected and analysed specimens were taken.In Fig. 1

DNA extraction, PCR amplification and sequencing
Genomic DNA was isolated from silica-gel dried or herbarium materials using the triple extraction Cetyltrimethylammonium bromide (CTAB) procedure by Borsch & al. (2003).
The chloroplast trnL-F region was amplified with universal primers trnTc and trnTf (Taberlet & al. 1991).Reaction conditions entailed 5.0 ul of 10X Taq Reaction Buffer, 3.0 ul of 25 mM MgCl2, 5.0 ul of 5 M betaine, 2.0 ul of 10 pm/ul of the forward and reverse primer, 6.25 ul of dNTP (each 1.25 mM) and 0.25 ul Hot Start Taq polymerase made up to a total of 50 ul with ultrapure water.The following temperature and timing were applied for the PCR amplifications: denaturation at 95 °C for 2 min; 30 cycles consisting of 94 °C for 30 s, 52-54 °C for 30 s, 72 °C for 90 s; and 72 °C for 10 min; 4 °C hold.Due to microsatellites, primers trnL460F (Worberg & al. 2007) and trnTd (Taberlet & al. 1991) were employed as additional sequencing primers.For matK we amplified the whole region spanning the trnK 5′ exon to the psbA gene, thus covering the trnK group II intron, the matK CDS and the trnK-psbA spacer.This region was generally amplified in two halves using primers trnKF + CARYmatK1440R (upstream) and CARmatK480F + psbA5R (Steele and Vilgalys 1994).For degraded DNAs from herbarium samples, the region was amplified in quarters using primer pairs trnKF + CARmatK978R (5′-TTT GGT TAG AAW AAT TAG CCG-3′; designed here), CARmatK745F (5′-CTA TCC ACT TAT CTT TCA GG-3′; designed here) + CARYmatK1440R (Schäferhoff & al. 2009), CARmatK480F (GBOL Project, T. Borsch, pers.comm.)+ CARmatK910R (5′-AAT GAC TGC AAA TCC TTC TGA-3′; designed here), and CAR-matK1040F (5′-AGT CAA ATG TTA GAA AAT GC-3′; designed here) + psbA5R.Due to several mismatches that prevented amplification the new primer CARmatK1033F (5′-GGTACGGAGTCAAATGTTAG-3′) was designed for Stellaria radians to replace CARmatK1040F.Most of these primers were also used for sequencing.CAR-matK537R (5′-TTTAGATTATTCCAATTATG-3′) was designed as an additional specific sequencing primer to fill gaps between long microsatellites in some species.The nrITS region was generally amplified with the universal primers ITS4 and ITS5 (White & al. 1990).Since this led to the amplification of fungal DNA in a number of samples, the Caryophyllaceae-specific primer CAR-ITS5 (5′-AGGATCATTGYCGAAACCTG-3′) was designed to replace ITS5 in these cases.PCR products were then run over a long gel and excised to remove unspecific fragments and purified over a GenepHlow™ Gel/PCR Kit from geneaid (article no.DFH300).Concentrations were measured using the Nanodrop -Spectrophotometer nd-1000-v3.7.Cycle sequencing was then carried out by Macrogen Europe in Amsterdam (the Netherlands) with the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI 3730xl DNA Analyzer (96-capillary sequencer).

Multiple sequence alignment, indel coding and tree inference
Pherograms were assembled with PhyDE 0.9971 (Müller & al. 2010) and errors in base calling, which occurred as a slippage effect in particular after long microsatellites, were corrected manually.Multiple sequence alignment followed a motif approach to recognize microstructural mutational events including one to several nucleotides at once (Kelchner 2000;Morrison 2009) following the alignment rules as laid out in Löhne & Borsch (2005).Simple indel coding (Simmons & Ochoterena 2000) was done through SeqState (Müller 2005).Maximum parsimony was implemented in PAUP* version 4.0a169 (Swofford 2002) using random addition with 1000 replicates, TBR Branch swapping an MULTREES in effect.PAUP* was also used for Jackknifing with 36.788 % of characters deleted in each of 10,000 replicates.The program jModeltest 2.1.10( Darriba & al. 2012) was used to determine the best-fitting evolutionary models using the Akaike Information criterion to select them.For the trnK intron and trnK-psbA spacer as well as the trnL-F region TIM1+G were found, for the matK CDS the TVM+G model and for ITS (SYM+I+G).Bayesian trees were inferred with MrBayes v. 3.2 (Ronquist & al. 2012), implementing four independent runs with four chains each and 5 million MCMC generations.Convergence and effective sample size (ESS) were checked with Tracer v. 1.7 (Rambaut & al. 2018).The burn-in was set at 2500 and every 1000 th generation was sampled to calculate a majority rule consensus tree.The detailed specifications in the MrBayes block can be seen in the matrices provided for the plastid and ITS partitions (see results).Maximum Likelihood calculations were done with RAxML v.8.2.12 on XSEDE in CIPRES using the GTR model for substitutions and the BIN model for indel characters.Tree-Graph 2.15.0-887 beta (Stöver & Müller 2010) was used to visualize and annotate the trees based on the Bayesian majority rule consensus, and depicting confidence values for nodes from all three tree inference methods.Scleranthus annuus L. was selected to root the trees to a relatively distant taxon outside the Alsineae and Arenarieae tribes as its putative sister (Arabi & al. 2022).This way, the circumscription of Stellaria proposed by Sharples & Tripp (2019) could be further tested with sequence data and an extended taxon sampling including many species hitherto not represented in any phylogenetic analysis.

Sequence data sets
The plastid data set contained 51 samples.The trnL-F and trnK-matK sequences of 49 samples (all 23 from the Plettkea clade) were generated in this investigation and two (DB38206 Rabelera holostea (L.) M. T. Sharples & E. A. Tripp and DB38311 Stellaria cf.alsine Grimm) were taken from the German Barcode of Life (GBOL) project.The multiple sequence alignment of the trnL-F region contained 1345 positions, from which the first 32 and the last 36 were trimmed (Supplementary appendix S1).Seven short mutational hotspots were excluded because of poly-AT microsatellites (poly GC in pos.1256-1267 in Arenaria) and a part of the trnL p8 loop (pos.364-455) constituted by a long polyA/T satellite and AT-rich elements for which no homology could be assessed between the Stellaria clade (including Plettkea) and the other lineages.The trnL-F region contributed 1105 characters to the final matrix of which 420 (34.9 %) were variable and 220 (18.3 %) were informative, plus 99 indel characters of which 37 were informative.The multiple sequence alignment of the trnK-matK partition (Supplementary appendix S1) had a length of 3124 positions, comprising the complete trnK intron with the matK CDS, the trnK 3′ exon, the trnK-psbA intergenic spacer and 40 nucleotides of the psbA gene.Ten short mutational hotspots (mostly microsatellites and regions comprising short AT-rich repetitive elements without clear homology) were excluded from the trnK intron partition.The matK-trnK region contributed 2935 characters, of which 1086 (37 %) were variable and 535 (18.2 %) informative.The indel partition contributed 117 of these characters from which 38 were informative.The complete combined plastid matrix used for analyses is provided in Supplementary appendix S2.
The ITS dataset included 49 sequences, from which 47 were generated in this study and 2 were obtained from the GBOL project to complement the ITS partition for the same samples.The alignment contained 802 nucleotide positions (Supplementary appendix S3), all of which were used in the matrix for tree inference.Simple indel coding resulted in additional 78 binary characters (Supplementary appendix S4).

Trees inferred from the plastid partition
The Bayesian majority rule consensus tree is shown in Fig. 3 and depicts a well resolved and statistically supported backbone of the Alsineae.Three sublineages (A, B, C) are found within a Plettkea clade, although these three and sublineages defined by two other species (Stellaria galianoi Montesinos & Borsch, S. macbridei Montesinos & Borsch) remain in a polytomy.An identical picture is given by the maximum parsimony strict consensus tree summarized from 5780 shortest trees with a score of 2267 (Supplementary appendix S5).The ML tree with the best likelihood score (Supplementary appendix S5) shows these species in inconsistent, unsupported positions as sisters to subclades C and A, respectively.The sample representing the 'Nitentes' clade (DB38311) is found with high support as sister to the Plettkea clade by all tree inference methods (Fig. 3).A further exploratory tree with the sequences available from GenBank of S. cryptantha (Mattf.)M. T. Sharples & E. A. Tripp (trnL-F and partial matK) and several species of the other major Stellaria clades (c.800 nt fragments of matK) can also be found in Supplementary appendix S6 (and matrix in S7).

Trees inferred from nrITS
The ITS Bayesian majority rule tree (Fig. 4) provides slightly more resolution within a well-supported Plettkea clade with sublineage B plus Stellaria utcubambensis Montesinos & Borsch (CAR433) and sublineage C being sisters, although not very well supported.The lineage with the two samples of S. galianoi then appears as sister to this clade albeit with significant support (0.98 PP) for the respective node only by Bayesian inference.The respective positions of the representatives of the 'Larbreae' (S. pungens Brongn., S. ruscifolia D. F. K. Schltdl.) and 'Nitentes' clades sensu Sharples & Tripp (2019) differ inconsistently between the Bayesian and the ML tree (Supplementary appendix S8) while the first branching posi- tion of the 'Petiolares' followed by the 'Insignes' clade is congruently found in both, although not very well supported by Bayesian PP or Likelihood BS.An exploratory analysis with available published ITS sequences with a much better sampling of the 'Larbreae' and 'Nitentes' clades (Supplementary appendix S9 and matrix in S10) shows both in a sister group relationship with high support, contrary to the ITS tree in Fig. 4. Nevertheless, the first branching position of the 'Petiolares' clade in Stellaria remains unaffected by taxon sampling (Fig. 4, Supplementary appendix S9).

Overall relationships in Stellaria and position of the Plettkea clade
In comparison with previous phylogenetic analyses of Plettkea that were only represented by two species, one in the RAD study by Sharples & Tripp (2021), and another in the tree of Caryophyllaceae by Greenberg & Donoghue (2011) we could include 14 of the 20 species of the Plettkea lineage into a phylogenetic analysis of both plastid and nrITS regions (Fig. 3, 4) which resulted in a maximally supported clade throughout all analyses that was recovered as deeply nested within the monophyletic genus Stellaria according to the circumscription of Sharples & Tripp (2019).An additional species of the Plettkea clade, S. laevis (Bartl.)Rohrb.(AC912) was recovered with ITS (Supplementary appendix S9) within the Plettkea clade.The RAD trees of Stellaria presented by Sharples & Tripp (2019), depicted five major clades.The only specimen from the Plettkea clade represented therein is S. tetrasticha (Mattf.)M. T. Sharples & E. A. Tripp, annotated by the authors as "Plettkeae".It appeared as sister to the 'Larbreae' and 'Nitentes' clades, whereas the 'Insignes' clade (represented by S. radians L., S. pubera Michx.and S. sessiliflora Y. Yabe) was found sister to the 'Petiolares' clade.
The relationships of the species-rich 'Larbreae' clade (Sharples & Tripp 2019) to the 'Nitentes' clade and the Plettkea clade remain unclear.Whereas our plastid topology provides a well-supported hypothesis of the 'Nitentes' being sister to the Andean Plettkea clade, ITS seems to favour a sister group relationship of the 'Larbreae' and 'Nitentes', also shown by the best-scoring ML tree of the RAD data in Sharples & Tripp (2019).Future phylogenomic analyses using hybseq data should be employed to test for a possible reticulate event that could have occurred before the divergence of the Plettkea crown group in the Andes.The more broadly sampled ITS tree (Supplementary appendix S9) indicates that Stellaria alsine as currently accepted contains at least two different evolutionary lineages, while further samples with this annotation in GenBank rather seem to be misidentified (in the 'Petiolares' clade).Sharples (2023) proposed in a short paper that appeared during the revision of this manuscript, that the name S. undulata Thunb.(≡ S. alsine var.undulata (Thunb.)Ohwi) would apply to the lineage of specimens resolved in the 'Larbreae' clade, so that the specimens shown in our Fig. 3 and 4 in the 'Nitentes' clade are true S. alsine.Nevertheless, a detailed analysis of species limits and distribution of this entity is still lacking.A further question arises regarding the phylogenetic position of the 'Petiolares' clade which also contains a significant number of South American species of Stellaria (e.g. S. cuspidata Willd.ex D. F. K. Schltdl., S. recurvata Willd.ex D. F. K. Schltdl., S. sp., S. weddellii Pedersen; Fig. 3, 4) and which may constitute another south American radiation within Stellaria.
Whereas ITS data indicate a first branching position of the 'Petiolares' clade, the analyses of RAD data by Sharples & Tripp (2019) yielded different results depending on the selection of loci and the genetic distances covered by the taxon set.Their analysis based on more loci showed a tree incongruent to our ITS phylogenies (Fig. 4, Supplementary appendix S9).Also, our plastid trees are different, providing evidence for a possible paraphyly of the 'Petiolares' clade with respect to S. radians ('Insignes' clade).However, a detailed plastid phylogenomic analysis needs to be undertaken to recover the true history of the plastid genome as the current plastid sequences may not provide a sufficient character sampling of this genomic compartment to properly recover the respective nodes.The sister group relationship of the 'Petiolares' and 'Insignes' lineages in the ITS tree (Fig. 3) is supported only through Bayesian posterior probabilities but not by ML-BS and MP-JK and thus depends on the inference method.

Composition and species-level relationships within the Plettkea clade
The Plettkea clade gains maximum support in all trees (Bayesian Inference, Maximum Parsimony, Maximum Likelihood inferred from plastid and nuclear partitions) and appears as a crown group on a rather long internal branch within Stellaria, suggesting that it forms an Andean radiation within this genus.In our trees the clade contains 13 taxa (represented by 23 samples).Considering that this is the first thorough sampling of this Andean plant group, and that so far, no comprehensive taxonomic revision existed, several entities turned out to represent new species, and changes are required in nomenclature.These are presented in the taxonomic treatment section (below).For simplicity and better readability, we already use the correct names here in the discussion and to annotate species in the phylogenetic trees.The trees inferred from plastid and nrITS sequence data are largely congruent and depict three sublineages (A, B, C) in addition to branches representing three more isolated species (Stellaria galianoi, S. utcubambensis, S. macbridei, respectively).According to Timaná (2017) eight species were classified as Pycnophyllopsis, all of which belong to the Plettkea clade as part of the genus Stellaria (Fig. 3, 4).The first is P. muscosa Skottsb., which is the type species of the genus Pycnophyllopsis and is considered to stand out by a "stellate calyx rather than the typical oblong, nearly cylindrical oblong calyx that characterizes all the other species in this genus [Pycnophyllopsis]" (Timaná 2017).then P. keraiopetala Mattf., P. cryptantha (Mattf.)Molinari, P. laevis (Bartl.)Timaná, P. macrophylla (Muschl.)Molinari, P. smithii Timaná, P. tetrasticha (Mattf.)Timaná and P. weberbaueri (Muschl.)Timaná.According to our molecular trees, the genus Pycnophyllopsis would therefore be polyphyletic within the Plettkea clade, considering that P. keraiopetala and P. weberbaueri belong to sublineage C and MP-JK in the plastid tree and this is congruently found in the ITS tree (86.6 % MP-JK and 59.3 MP-JK, respectively).All four specimens share the glabrous, petiolate leaves and the subglobose calyx, and occur in similar habitats, indicating that the molecular differences show a pattern of phytogeographic differentiation within this species.Although node support is not very high, S. pedunculosa (Wedd.)Montesinos & Borsch is congruently inferred as sister to the S. andina clade with both genomic compartments.Stellaria apurimacensis Montesinos & D. Cornejo constitutes the earliest branch of subclade B, consistently in both the plastid and nuclear trees.The species is repre-true spine-like apical portions rather than the usual compact or sprawling forms found in the sister sublineage B which also occur in S Peru and NW Bolivia.
Sublineage B is found in all trees and comprise Stellaria andina (Rohrb.)Montesinos & Borsch and relatives.This species is best represented in our molecular analysis with three samples from Bolivia (CAR377, B101149337; CAR634, B101149331; CAR638, B101149337) and one sample from Peru (CAR664, B100745260).These form a well-supported clade (1.0 BI-PP and 100 % MP-JK) in which a single sequence from Peru appears as sister to the three sequences from Bolivia (0.82 BI-PP and 51.1 % differs by a reduced stamen number.In terms of habit as seen in the field, both species, however, appear to be very similar and resemble cushion-forming species of Pycnophyllum.In terms of the taxon concepts at species level we agree with Timaná (2017) who accepted Pycnophyllopsis keraiopetala as different from P. weberbaueri.The latter of which is sampled by two individuals (CAR653 and CAR670) that appear as sisters.
The plastid tree recovered three entities in an unresolved position within the Plettkea clade that do not belong to any of the three subclades (A, B, C); this is corroborated by ITS.Stellaria macbridei (a new name for Arenaria crassipes) is the first of these and represented by two sequences in our trees [CAR430 (B100766225) and CAR630 (B100761527)] that form a well-supported clade with 1.00 BI-PP and 98.3 % MP-JK (plastid tree) and 99.9 % MP-JK in the ITS tree.Both sequences were obtained from two isolated populations near the boundary of the Huánuco-Áncash departments, in highland slopes.These plants are characterized by having subcoriaceous or membranous leaves with a hirsute midrib, a character not found in the other species of the clade.The second isolated entity is S. galianoi.The sequences represent the sented here by one specimen (CAR619) that is the type.Whereas our molecular data provide confidence for the position of S. apurimacensis in subclade B (1.0 BI-PP and 99.7 MP-JK in matK-trnK-psbA + trnL-F and 89.2 MP-JK in ITS) the support for the clade comprising S. andina and S. pedunculosa is rather low and appears to be connected to its short internal branch.Nevertheless, considering that these two other species of subclade B appear to be clearly monophyletic, our phylogenetic results underscore the status of S. apurimacensis as a distinct species.
Sublineage C (Fig. 3, 4) is constituted by samples of two species previously classified as Pycnophyllopsis.These are Stellaria keraiopetala (Mattf.)Montesinos & Borsch (one specimen CAR639, B101149336) sister to the lineage of the two sequenced specimens of S. weberbaueri (Muschl.)Montesinos & Borsch (1.0 BI-PP and 99.9 % of MP-JK in the plastid tree and 87.9 in MP-JK with ITS.The ITS tree depicts maximum support for subclade C (100 % MP-JK) whereas the branch leading to the respective node in the plastid tree is rather short (0.54 BI-PP, 52.8 MP-JK).Sublineage C entails two morphologically rather different species.Apart from the deviating dioecious reproductive system in S. weberbaueri (Timaná 2017)   All of these radiations are relatively young, starting 1.5-2 mya in Lupinus (Hughes & Eastwood 2006) or 3.5-4 mya as in Gomphrena (Ortuño Limarino & Borsch 2020).Whereas the Andean clade of Gomphrena has two sublineages, one in central Bolivia and S Peru and the other in central to south Bolivia and northern Argentina, species diversification in the Plettkea clade to some extent seems to reflect the biogeographic subdivision of the Amotape-Huancabamba zone (Weigend 2004) in Peru.Stellaria sect.Plettkea subclade A (Stellaria alpamarcae, S. congesta and relatives) occurs in the northern half of Peru (from the departments of Lima and Junín northward) whereas subclades B and C (Fig. 3, 4) seem to be confined to areas further southeast of the Amotape-Huancabamba zone, reaching into Bolivia.It will be interesting to further test if an early branching position of S. galianoi (from department of Cusco in the southwestern part of the distribution of the Plettkea clade) in subclade B as suggested by the ITS tree (Fig. 4) will be substantiated by adding further molecular characters.The Plettkea clade will therefore be an interesting study group to better understand the biogeographical patterns in the central Andes.It is to be hoped that the key provided in our study will help to discover further specimens of S. sect.Plettkea for an improved mapping of species distributions.Notes -Until now there was no formal infrageneric name for the Plettkea clade.Sharples & Tripp (2019) annotated the lineage of Stellaria tetrasticha, corresponding to the Plettkea clade, in their trees with "Plettkeae" albeit without any reasoning for that name.The first infrageneric classification system by Fenzl (1840) recognized four entities, one of which, "Eustellaria", was further subdivided.However, no specific nomenclatural rank was designated, until Pax & Hoffmann (1934) specified these entities as sections and subsections, respectively.We therefore suggest continuing the use of the sectional rank for the subdivision of Stellaria.No entity corresponding to a section Plettkea was included by Fenzl (1840) or Pax & Hoffmann (1934), so it is described here as a section of Stellaria.
As Sharples (2019) and Sharples & Tripp (2019) already pointed out, more work needs to be done to develop a consistent infrageneric classification for Stellaria, which includes testing if existing sectional/subsectional names are validly published.Considering that S. graminea was designated as the neotype for the monophyletic genus Stellaria (Tikhomirov 2016), and that the name "Eustellaria" is not permitted under Art.21.  A. pycnophylla Rohrb.However, the authors overlooked that these species do not match the original diagnosis by Fenzl (1840), who stated that the species of Dicranilla (Fenzl) Rchb.possess smooth, shiny, black seeds.On the contrary, members of the Andean Plettkea clade have tuberculate, brown seeds.Molecular phylogenetic data (T.Borsch, unpublished) further show that A. dicranoides belongs to the core Arenaria clade, underscoring that Pax & Hoffmann (1934) created an extended, morphologically heterogeneous and polyphyletic section.McNeill (1962) typified the genus name Dicranilla based on the section with Arenaria dicranoides, and commented on the misinterpretation of the seed characters by Pax & Hoffmann (1934).In summary, there was no name available for the 'Plettkea' clade at the level of section.

Key to the species of
Distribution -The species is endemic to Peru (Cano & Sánchez 2006) and is known to occur in Áncash, Huánuco, La Libertad and Junín departments according to the material revised and at altitudes of 3900-4500 m.
Etymology -The epithet congesta refers to the uniform and compact growth of the species.
Notes -The specimen at GOET has the annotation "rel.Haenk.II, 12 (cum descriptione)", which indicates that the specimen may have been used to make a description.Morphological description -See Timaná (2005).

Stellaria cryptantha (
Distribution -The species is found in highland passes in Andean mountainous ranges between the departments of Puno and Lima in Peru at altitudes of 4800-5200 m. Notes -According to Timaná (2017), this distinctive species, has tetramerous flowers and 2 free styles.The species, originally described by Mattfeld (1934) as Plettkea cryptantha, is characterized by decussate sepals with the inner pair shorter and non-ciliate as opposed to the outer pair.Molinari-Novoa ( 2016) described the new combination Pycnophyllopsis cryptantha (Mattf.)Molinari, without studying any type specimen; the holotype was destroyed and no neotype had yet been selected.Timaná (2017)   ter to CAR431 (Supplementary appendix S6) that is the representative of S. cryptantha in our analysis.Distribution and ecology -Peru, Cusco, Urubamba.The species inhabits continuously moist environments dominated by tussock grasslands and bryophyte communities located on high passes that divide the inter Andean valleys, between the highland tropical forests and the puna scrublands in the upper basins near the Urubamba River lower slopes.The altitudinal range is 4230-4400 m and is restricted to this region.The exhaustive field collection of Caryophyllaceae in most parts of Peru for the past seven years and examination of nearly all available herbarium material did not reveal additional areas where the plant occurs.Nevertheless, further fieldwork in Urubamba is needed better understand the frequency and population structure of the species.Flowering specimens have been collected from March to May; fruiting specimens have been observed between April and June (pers.obs.).Associated taxa are: Gentianella sp.(Gentianaceae), Plantago rigida Kunth (Plantaginaceae), Senecio rhizomatus Rusby (Asteraceae) and Stellaria weddellii Pedersen (Caryophyllaceae), among others.

Stellaria engleriana
Etymology -The specific epithet refers to Prof. Washington Galiano (1950-), for his devoted career on studying the floristic diversity of the Cusco department in S Peru.
Notes -Stellaria galianoi is most likely monophyletic and is phylogenetically rather isolated among other mem-bers of the Plettkea clade (Fig. 3, 4).This species has the longest internodes of vegetative stems across the Plettkea clade, eventually, in the uppermost the internode is long, sometimes as reaching 1.5 cm.This character makes it easy to recognize among the apetalous taxa of Andean Stellaria.The new species differs from the known South American species of Arenaria by its leaf form and by the densely tuberculate seeds.Morphological description -See Timaná (2005).
Distribution -The species is endemic to Bolivia and based on two collections and according to Timaná (2017), it is known to occur at altitudes of 4600-5100 m.
Notes -The neotype located at the Herbario Nacional de Bolivia, Universidad Mayor de San Andrés (LPB) was designated by Timaná since the original material collected by K. Pflanz 223 was lost (B †).According to Timaná (2017), Pycnophyllopsis keraiopetala (now Stellaria keraiopetala) is the only species with trimerous petals and stamens, although tetramerous and pentamerous forms are also found (sometimes all three forms in the same plant).Stellaria keraiopetala is the sister to S. weberbaueri, and both share the habit of dense small cushions with stems covered by overlapping leaves.Stellaria keraiopetala and S. weberbaueri appear to be geographically vicariant species due to its distribution in the highest parts of the Andes in Bolivia and Peru, respectively.Morphological description -See Timaná (2005).

Stellaria laevis
Distribution -The species inhabits highland plateaus of the central Andes in Peru, at altitudes of 4660-4800 m, in the Lima and Junín departments.The environments where the species grows are highland plateau Andean grasslands with rocks, with template and harsh wind conditions.
Notes - Mattfeld (1934) tentatively included the species described by Bartling (1831) under the genus Cherleria into Plettkea.Mattfeld considered the characters mentioned in the original description as he had no access to specimens.Rohrbach (1872) transferred the species to Stellaria without further reference.Timaná (2017) encountered the Haenke specimen at PR and respective duplicates at HAL and GOET and argued that the species belongs to Pycnophyllopsis.The examination of the type specimen at GOET showed the presence of hirsute leaf margins, apetalous flowers supporting the position in the Plettkea clade.In the ITS tree specimen CAR912 of S. laevis is resolved as sister to S. cryptantha, albeit with low support (it was not possible to amplify plastid regions in this specimen).The examination of the type specimen further revealed the similarity with Arenaria bisulca (Bartl.)Fenzl & Rohrb.A species also described by Bartling (1831) under Cherleria, which is therefore put into synonymy in this study.Also, Zanotti & al. (2022) discussed if S. laevis could be closely related to Arenaria bisulca, an opinion to which we agree with due to the exact match of morphological characters.Moreover, S. laevis appears to be morphologically similar to S. andina having several differences such as the habit and growth form (loose mat-forming herb vs. weak herbs S. andina), leaf form and stiffness (rigid in S. laevis vs. weak in S. andina) and minor flower characteristics.Sequence data of ITS resolve S. laevis as part of subclade A within the Plettkea clade of Stellaria (Supplementary appendix S9). ).-Fig.1D.
Distribution and ecology -Stellaria macbridei is endemic to central Peru (Cano & Sánchez Vega 2006) occurring in puna grasslands ecosystems that receive continuous rainfall throughout the year, it is distributed in Áncash, Huánuco, Junín and Lima departments at altitudes of 4200-4800 m.
Etymology -The specific epithet refers to J. F. Macbride (1892Macbride ( -1976)), American botanist who provided the last comprehensive revision for Stellaria and Arenaria from Peru and also was one of the authors who originally described this species.
Notes - Macbride (1936) mentioned that Stellaria macbridei has similarities with S. congesta but differs from that species by the tuber-like root and the small erect habit.Other differences include suboblong leaf form (ovate in S. congesta) and the narrowly lanceolate sepals (lanceolate in S. congesta).The two sequenced specimens are sisters and form a distinct lineage within the Plettkea clade in both the plastid and nuclear trees (Fig. 3, 4) underscoring the identity of S. macbridei as a distinct and eventually monophyletic species.Morphological description -See Timaná (2005).
Distribution -Huascarán National Park in Áncash department, central Peru, at altitudes of 4500-4800 m (Timaná 2017).-Muschler (1911: 458) described Pycnophyllum macrophyllum based on the leaf shape and few relevant flowers characters.Molinari-Novoa (2016) simply transferred the species to Pycnophyllopsis referring to Timaná (2005) who recognized Pycnophyllopsis s.l.(including Plettkea) as distinct from Pycnophyllum without any own analysis of plant material.According to Timaná (2017), Stellaria macrophylla differs from S. cryptantha in the number of floral parts: S. macrophylla is pentamerous with three free styles while S. cryptantha a tetramerous species with two free styles.In addition, the leaves of S. macrophylla are slightly narrower toward the apex, where in S. cryptantha these are more triangular.The only collection known is the type specimen at MOL.This specimen has no seeds which was also confirmed by physical examination (G. Tello, Lima, pers. comm.).Therefore, it is unclear from where Muschler took the information on seeds, which does not even correspond to specimens of the Plettkea clade and therefore could be wrong.Distribution -Based on two collections (DBMT 5395b, 4565), Arenaria pedunculosa is located at the lower slopes SE of Ticsani volcano (Moquegua department), on volcanic pumice soils at altitudes of 4500-4820 m.Flowers and fruits were observed between February and March.In Bolivia the species is known only from the type collection, and a search of material at LPB in 2019 provided no evidence that the species has been collected again.
Notes -Even though the intense survey done at the herbaria mentioned in the methods, no specimens have been found as additional collections for Bolivia, but two specimens have been found from the Moquegua department in Peru.Morphological description -See Timaná (2005).
Distribution -According to Timaná (2017), the species should be distributed in the boundary between the Peruvian departments of Lima and Junín, at altitudes of 4300-4900 m, but should also be present in the Áncash department (Huascaran National Park) at an altitude of c. 4800 m.However, no further specimens in addition to the type appear to be known.

Stellaria spinulosa
Distribution -Stellaria spinulosa occurs in a variety of highland ecosystems across Peru and is considered as the most collected species of the clade (as observed in the material from the different herbaria visited) and extends to Ecuador.Jørgensen & León-Yánez (1999) mentioned the occurrence of the species in Ecuador, which can be confirmed based on an analysis of material stored at MO according to Tropicos.org (2023) and as evaluated by the first author during two visits to MO in 2013 and 2016 and more recently, at PRC, four collections were identified to be S. spinulosa located mostly in central Ecuador, between Chimborazo and Cuicocha.The altitudinal range is from 2800-5000 m according to the specimens evaluated.
Etymology -The epithet refers to the spine-like leaves of the plant, which can be felt as tingling by simple touch; spinulosa is derived from the Latin spinula (small spine) in relation to the thorny and pungent characters of the leaves.The former epithet aphanantha is derived from the Greek aphanes (invisible obscure) and anthos (flower), probably referring to the resemblance of the flower sepals to the leaves.
Notes -The specimen C. Gay 1818 (P00335797) is selected as the lectotype for being most representative with a complete set of morphological characters.As evidenced by this specimen, the species is easily distinguishable from the other members of the clade by the intense yellowish colour of the stems and leaves, the stiff ovate leaf lamina and the nearly sessile flowers with long calyx, the presence of staminoid petals reduced to filaments and the tubercle seeds, which is characteristic for this kind of habit.Morphological description -See Timaná (2005).
Distribution -Central Peru, Áncash department inside the Huascarán National Park on highland peaks at altitudes of 4500-4900 m, where considered a narrow endemism.
Notes -Stellaria tetrasticha was originally described as a member of the genus Plettkea by Mattfeld (1934) and later accepted by Bittrich (1993).The species has a mat form with spreading stems, ovate leaves c. 2 mm long bearing carinate acute apex and sepals with ovateoblong form.Later, Timaná (2017) named Pycnophyllopsis tetrasticha (Mattf.)Timaná based on the morphological affinities found within flower measurements in the neotype he selected.Sharples & Tripp (2019) included a sample of the species in their phylogenetic analysis showing it to be strongly supported as a member of the Plettkea clade.Distribution and ecology -The species inhabits the mountain summits of the tributaries leading to the Utcubamba river in the Amazonas department in N Peru, with an expected growth at altitudes of 3600-3900 m.The species was found in areas where slope burning is an unfortunate practice applied by the local people; such events and climate change could lead to the gradual disappearance of this species.Flowering has been observed during the months of September and November.

Stellaria utcubambensis
Etymology -The specific epithet refers to the Utcubamba river in N Peru that divides Amazonas department from south to north.The new species was found in its uppermost tributaries, and it is very likely that there are further populations on the mountains draining into this river.
Notes -This species is known so far from only one collection, but further populations might be encountered after searching in the field.S. apurimacensis) and the calyx is cylindrical (vs.campanulate in S. apurimacensis).Notes -The species conspicuously deviates from the other members of the Plettkea clade by its dense lanuginose indumentum and by its shrubby habit.Timaná (2005) proposed to classify this species in its own new subgenus, Pycnophyllopsis subg."Coquimbo", but this name, because it appeared in a thesis, was not effectively (and therefore not validly) published according to Art. 30.9 of the Code (Turland & al. 2018).The same applies to the species combination under Pycnophyllopsis and the lectotypification, all of which were not included in Timaná's (2017) paper on the genus.Flower and seed morphology provide evidence for a position in the Plettkea clade.In his thesis, Timaná (2005) depicted a representative of this species in an ITS tree in a lineage with Pycnophyllopsis cryptantha and P. weberbaueri, thus supporting this position.However, these phylogenetic results cannot be reproduced because the sequences are not available and the tree was never properly published.
Distribution -The species inhabits highland plateaus, also known as subnival puna, at altitudes of 4660-5000 m.It is known to occur in the Arequipa and Moquegua departments of Peru, while its occurrence in the Bolivian Andes is not confirmed.
Notes -The species was first described as Pycnophyllum weberbaueri Muschl.by Muschler (1911), presumably based on the cushion growth form, plicate leaves and apical flowers.Later, the species was transferred to the genus Pycnophyllopsis by Timaná (2017), as Pycnophyllopsis weberbaueri (Muschl.)Timaná, based on the consistently pentamerous flowers with three free styles.Muschler (1911) apparently described characters for Stellaria weberbaueri such as true petals, which are actually not present in the specimens examined.Moreover, S. weberbaueri has leaves that do not overlap to be completely imbricate, giving the general impression of a loose cushion with longer internode length.Sharples & al. (2021) reported "Stellaria weberbaueri (Muschl.)M. T. Sharples & E. A. Tripp" in their Appendix 2, but this name has not been validly published anywhere.Diagnosis -This species is similar to Stellaria laevis, from which it can easily be distinguished by its leaf margin, which is ciliate in the lower-middle part of the leaf, whereas in S. laevis the margin is completely covered by thin trichomes.
Distribution and ecology -The species is recorded in northern and central Peru, in the mountains adjacent to the Alto Marañón river in Huari province, Áncash department, and in the Utcubamba high mountains river basins in southern Amazonas department, near the boundary with La Libertad and San Martín departments.An additional specimen to the one sequenced comes from the department of Áncash in Peru: Huari, San Pedro de Chaná 4410 m, 5 May 2018, D. Montesinos & G. San-cho 6147 (B-101249409!, HCSM!, HSP-12974!, HUT-62033!, USM!).The habitat varies in vegetation cover and floristic composition but not in precipitation, as these environments tend to receive over 800 mm/year.The typical ecosystem is humid puna grasslands, with rocky outcrops and highland subhumid forest patches with species of Hesperomeles Lindl.(Rosaceae) and Verbesina L. (Asteraceae).
Etymology -The specific epithet derives from the Greek words xanthos, yellow, and phylla, leaves, referring to the persistent, yellow leaves along the stems of the plant.
Notes -Stellaria xanthophylla shows morphological similarities with S. engleriana but can be differentiated by the following characters: bearing thin trichomes at the base of the lamina (vs.dense trichomes at the base in S. engleriana); sepal form, size and texture (lanceolate to narrowly ovate-lanceolate, 5-7 mm long and 1.5-2 mm wide, surface and margins glabrous or nearly so in S xanthophylla vs. ovate-oblong, 3-4 mm long and 1-1.5 mm wide, with a ciliate lamina surface and margin in S. engleriana).
the cushion type habits of species belonging to the Plettkea clade is shown.Fig. 2 depicts living specimens of species of the 'Petiolares' clade of Stellaria from Peru.
Fig. 3 and 4 include colour bars for species previously recognized under Arenaria and under Pycnophyllopsis (sensu Timaná 2017).This includes the two former species of Pycnophyllopsis that Sharples & Tripp (2019) already merged with Stellaria (S. cryptantha (Mattf.)M. T. Sharples & P. cryptantha and S. laevis to sublineage A (Fig. 3, 4 and Supplementary appendices S6 and S9).Sublineage A in the plastid tree comprises the two individuals of Stellaria alpamarcae (CAR439 and CAR440) in a tritomy together with the only sample of S. engleriana (Muschl.)Montesinos & Borsch (CAR632).The plastid sequence of S. xanthophylla Montesinos & Borsch represented by the single specimen is depicted as sister with 1.0 BI-PP and 83.3 % MP-JK whereas ITS does not resolve relationships among these individuals [CAR429 = S. spinulosa Montesinos & Borsch is inconsistently depicted as part of a polytomy with a general score of 64.3 % to which CAR439 does not belong].The plastid data set further resolves the two specimens of S. congesta Montesinos & Borsch as sisters in the plastid tree that are also just depicted within the broad polytomy of the core of sublineage A with ITS.Stellaria cryptantha (CAR431) is congruently resolved as sister to the other members of the sublineage by the plastid and nuclear genomic partition, and with high support (Fig. 3, 4).This sublineage A is characterized for its occurrence in central Peru, the plants form mats, leaves and sepals with E. A. Tripp and S. tetrasticha (Mattf.)M. T. Sharples & E. A. Tripp) based on RAD data of S. tetrasticha.
, S. keraiopetala PycnophyllopsisFig.4.Bayesian majority rule consensus tree inferred from nuclear ribosomal (ITS) sequence matrix including indels.DNA posterior probabilities are above branches, parsimony jackknife values are below branches left and in italics, and maximum likelihood bootstrap percentages right and in bold.The monophyletic Stellaria sect.Plettkea is deeply nested within the genus Stellaria.Species previously classified within Arenaria and Pycnophyllopsis are annotated with a red or green bar, respectively.
species based on two collections, one of which is the type of the species (CAR438), in a lineage supported by 1.0 BI-PP and 99.5 % MP-JK.Whereas the plastid tree does not allow any conclusions on its relationships with the Plettkea clade, the ITS tree suggests it is sister to subclades B plus C. Stellaria galianoi is characterized by its flexible stems and leaves, long internodes and shady habit, and considered as a unique taxon in the clade for having longer internodes than all other species of the Plettkea clade.The third of these more isolated entities is S. utcubambensis, newly discovered here and is based on a single specimen from the Amazonas department in N Peru [CAR433 (B100766222)].The ITS tree indicates a sister-group relationship of S. utcubambensis to subclade B. This species is unique in the clade since it is the easternmost distributed in the Andes in an isolated canyon that is formed by the Utcubamba river, which leads to the Amazonas in N Peru.
Biogeography of thePlettkea clade Stellaria sect.Plettkea (Mattf.)Montesinos & Borsch represents another radiation of angiosperms at mostly high altitudes in the central Andes.This investigation revealed the existence of a clade of some twenty species that are distributed in the region.Similar examples of central Andean radiations can be found in other genera such as Lupinus (Fabaceae; Hughes & Eastwood 2006), which more recently were shown to consist of geographically structured sub-radiations along the Andes (Contreras-Ortiz & al. 2018) or Gomphrena (Amaranthaceae; Ortuño Limarino & Borsch 2020).