TAXON 2019 Sharples and Tripp • RADseq rejects disjunction in

SYSTEMATICS AND PHYLOGENY

RAD sequencing rejects a long-distance disjunction in Stellaria () and yields support for a new southern Rocky Mountains endemic Mathew T. Sharples1 & Erin A. Tripp1,2 1 Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado 80309, U.S.A. 2 Museum of Natural History, COLO , University of Colorado, Boulder, Colorado 80309, U.S.A. Address for correspondence: Mathew T. Sharples, [email protected] DOI https://doi.org/10.1002/tax.12059

Abstract Whereas the eastern North American–eastern Asian floristic connection represents one of the most widely studied biogeo- graphical relationships in flowering evolution, connections between western North America and Asia have been comparatively rarely investigated, especially through genetic approaches. Stellaria irrigua is one of several that has been treated as an excep- tionally dramatic example of a disjunction between floristically similar, high alpine biotas of the southern Rocky Mountains and south-central Siberia. We here employ numerous new field collections and ddRADseq data to test the hypothesis that S. irrigua—a spe- cies that has been known for over 180 years—represents a long-distance disjunction between the southern Rocky Mountains and central Asia. Extensive fieldwork, review and perusal of herbarium materials, and phylogenomic analyses indicate that S. irrigua is broadly distributed across an amphi-Beringian arc extending from southern and central Asia, east through Beringia, and south throughout moun- tainous regions of western North America. Sampled Asian populations formed two clades, and North American individuals all formed a clade embedded within this broader Asian lineage. Stellaria irrigua is, however, rendered non-monophyletic by a lineage that is embed- ded within the North American populations and is ecologically and morphologically distinctive from S. irrigua. The identity of this newly recognized lineage, which was in prior works attributed to S. irrigua, has been confused since plants of the former were first col- lected in the San Juan Mountains of Colorado in the late 1800s under Arenaria and Alsine. We provide a new name for this taxon, Stellaria sanjuanensis, a charismatic starwort of dry alpine scree slopes of the southern Rocky Mountains. Additionally, two lectotypes are designated, one holotype and one isotype are identified, and two new synonymies are proposed, to help stabilize the and nomenclature of this long-confused species complex. A key to the starworts of the southern Rocky Mountains is also provided, and Stellaria alsine is reported as new to the region. Keywords Altai; biogeography; Caryophyllaceae; disjunction; paraspecies; progenitor-derivative speciation; RADseq; southern Rocky Mountains; Stellaria

Supporting Information may be found online in the Supporting Information section at the end of the article.

■ INTRODUCTION America and South America (Morton, 2005), between Oceania and Patagonia (Webb & al., 1988), as well as between central Stellaria L. (Caryophyllaceae) is a cosmopolitan genus of Asia and the southern Rocky Mountains (Weber, 2003). One up to ~120 species (Morton, 2005). Species in this lineage oc- particularly marked example of long-distance disjunction in cupy a broad range of habitats but are most diverse in upper Stellaria figured starkly in a major biogeographical hypothesis montane environments and are typical elements of arctic-alpine put forward by Weber (2003) to explain floristic connections floras worldwide, especially in western North America and between central Asia and the southern Rocky Mountains, con- eastern Asia, which represent two centers of diversity for the nections first remarked upon by J.D. Hooker (1878). In his hy- genus (Schischkin, 1970; Shilong & Rabeler, 2001; Morton, pothesis, Weber (2003) proposed that this floristic connection 2005; M. Sharples & E. Tripp, in revision). Whereas many comprises multitudes of once more widely ranging species that among the species of Stellaria are restricted to one major phy- subsequently became isolated after the onset of the Pleistocene togeographical area such as the Tibetan Plateau, New Zealand, and consequent contraction of previously continuous northern, or the southern Andes, others have been noted by prior authors Tertiary, boreal habitats. Weber’s hypothesis is compelling but for exhibiting long-distance biogeographical disjunctions. remains little explored by empirical, phylogenetic approaches, These include hypotheses of disjunctions between North especially in comparison to the widely investigated, and much

Article history: Received: 7 Jan 2018 | returned for (first) revision: 19 Apr 2018 | (last) revision received: 10 Feb 2019 | accepted: 21 Feb 2019 Associate Editor: Hans Peter Comes | © 2019 International Association for Plant Taxonomy

1 Sharples and Tripp • RADseq rejects disjunction in Stellaria TAXON 2019

better understood, eastern Asian–eastern North American phylogenomic tool in Caryophyllaceae, and that a revised cir- floristic connection (Li, 1952; Wen & al., 2010; Manos & cumscription of the S. irrigua complex is warranted. Meireles, 2015). Among the most notable of disjunctions between the mountains of central Asia and the southern Rocky Mountains ■ MATERIALS AND METHODS is that of Stellaria irrigua Bunge (Weber, 1961, 2003; Morton, 2005). Stellaria irrigua was described in 1835 from the Altai Field, herbarium, and morphological study. — To Mountains of southern Siberia. All modern floristic treatments facilitate better understanding of Asian and North American of the southern Rocky Mountains have recognized S. irrigua as material attributed to Stellaria irrigua, type material was a species that additionally inhabits alpine scree habitats present studied at the LE Herbarium. Because of the age of the in southern portions of the southern Rocky Mountains (e.g., holotype of S. irrigua (early 19th century) as well as its Douglas, 1992; Weber & Wittmann, 2012; Ackerfield, 2015). depauperate condition, fieldwork to discover additional, Other authors have, however, expressed skepticism as to extant populations of this species in Asia was conducted focus- whether populations in these two geographically disparate ing on the Altai Mountains, particularly in the region where areas are in fact conspecific (Kozhevnikov, 1994; Morton, type material of this species originated (i.e., the mountains 2005), and the Flora of China considers S. irrigua to be Asian around the Chuya River valley). In North America, fieldwork only (Shilong & Rabeler, 2001). in the southern Rocky Mountains was conducted to collect re- Confusion surrounding Stellaria irrigua and its distribu- cent material of this species. On both continents, we attempted tion stems from multiple sources. First, very little material of to study and collect as geographically and morphologically S. irrigua has been collected in Asia since the species was de- broad of a range of representative populations as possible. Be- scribed over 180 years ago; for example, the largest repository sides this species, other Stellaria (and outgroup) samples suit- of temperate Asian material in the world—the able for molecular study were collected in the aforementioned Komarov Herbarium (LE)—contains only a single collection regions and adjacent areas. Newly collected herbarium speci- determined as S. irrigua, the holotype. As such, S. irrigua mens were deposited at the following institutions: COLO, has been considered to be very rare in Asia. Second, the NY, and RM. We supplemented new field collections with species is furthermore considered to be rare in North sampling or study of existing herbarium materials at, or from, America; plants that have been attributed to this name are the following institutions: ALTB, BRIT, CAS, COLO, DAO, restricted to only a few mountain ranges in southern portions K, LE, MO, NDG, NSK, NY, P, RSA, US, and VLA (see Re- of the southern Rocky Mountains, namely the Elk, San Juan, vised Taxonomic Concepts). Herbarium study included exam- and Sangre de Cristo Mountains of Colorado (and far northern ination of type material of all names of species related to New Mexico) where they occur on barren, dry alpine scree S. irrigua, including Alsine polygonoides Greene ex Rydb., slopes of usually volcanic origin. Third, in Bunge’s (1835) Stellaria gonomischa B.Boivin, Stellaria subumbellata Edgew., original description of S. irrigua, he noted that plants occurred S. umbellata, and S. weberi B.Boivin. in “damp and mossy areas” (hence the specific epithet, Because prior research (Mahdavi & al., 2012) along with “irrigated”), in marked contrast to the well-drained scree and our own studies suggested that seed coat morphology may be tuff slopes on which plants occur in the southern Rocky taxonomically informative in Stellaria, we used scanning elec- Mountains. Finally, no molecular data have been generated tron microscopy to document seed morphologies of S. irrigua with which to evaluate this proposed long-distance disjunction. and close relatives from Asia and North America. Seeds were The most comprehensive phylogenetic analysis of removed from mature capsules of fresh specimens and Stellaria to date was that of Greenberg & Donoghue (2011), mounted onto JEOL aluminium SEM stubs, then coated with who sampled ca. 40 species including material of S. irrigua gold using a Cressington 108 Sputter Coater. Seed imaging and its putative closest relative (Stellaria umbellata Turcz.) was conducted using a JEOL JSM-6480 Scanning Electron from Colorado but not from Asia. In the present study, we use Microscope housed in Ramaley Hall at the University of RADseq data combined with new field collections, new field Colorado (Boulder). observations, and new insights from morphology and ecology Molecular analysis. — Double-digest restriction site- to test the hypothesis that S. irrigua is widely disjunct between associated DNA sequencing (ddRADseq) was implemented the southern Rocky Mountains and the Altai Mountains of to reconstruct phylogenetic relationships among 37 samples southern Siberia. Under the phylogenetic expectations of this representing 14 species of Stellaria plus two very closely hypothesis, we would predict to recover the entity known as related outgroups based on Greenberg & Donoghue (2011). Stellaria irrigua in the southern Rocky Mountains to have a sis- These two outgroups—Cerastium beeringianum Cham. & ter taxon relationship to S. irrigua from the Altai Mountains, or Schltdl. and Pseudostellaria jamesiana (Torr.) W.A.Weber & to be embedded within the broader Altai lineage. We addition- R.L.Hartm.—were selected in attempt to minimize locus ally sampled several other species of Stellaria to gauge the effi- dropout due to increased phylogenetic divergence among cacy of using RADseq data to more broadly resolve taxa (Eaton & al., 2017; Tripp & al., 2017), and we rooted phylogenetic relationships within the genus. Our results suggest all trees on the P. jamesiana branch. Our ingroup sampling that RADseq likely represents a powerful and practical of the Stellaria irrigua species complex included: 1 accession

2 TAXON 2019 Sharples and Tripp • RADseq rejects disjunction in Stellaria

of S. irrigua from Asia, 5 accessions of S. irrigua from North this, to explore the effects of missing data on topology and sup- America, and 17 accessions of collections originally attributed port values, we ran additional series of analyses spanning vary- to S. umbellata or S. subumbellata, these from eastern Asia as ing thresholds of missing data. We produced alignments with well as western North America. The remaining 12 accessions varying amounts of missing data by altering the pyRAD pa- represented a diversity of subclades of core Stellaria based rameter that only aligns RAD loci shared by X number of taxa on results of Greenberg & Donoghue (2011) and were in the input data files—the more loci needed to be shared be- included to assess the efficacy of using RAD loci in tween samples to be output, the less resultant missing data reconstructing relationships across the genus as well as to place and the smaller the output alignment. Final parameters differ- results from our investigation of S. irrigua into a broader phy- ing from the default included those pertaining to (1) clustering logenetic context. Voucher information of all samples included within samples (reads clustered at a similarity threshold of in our molecular analyses can be found in Appendix 1. 0.88; minimum coverage of two reads to retain a locus) as well DNA extractions followed the CTAB protocol (Doyle & as (2) clustering between samples (0.88 similarity threshold Doyle, 1987). A double-digest RADseq protocol adapted from and a minimum of four of 37 samples required to share a locus Parchman & al. (2012) and further customized in Tripp & al. for retainment in the final alignment). (2017) was used for library preparations and is briefly described Phylogenetic inference was conducted using RAxML here. Genomic DNA extractions containing an average of v.8.2.9 on the concatenated, final output alignments under a 150 ng/ml of DNA were subjected to double digestion with GTRCAT model of sequence evolution (Stamatakis, 2014). the restriction enzymes EcoRI and MseI. Custom-designed To assess branch support, we implemented rapid bootstrapping barcodes of variable length (7–10 bp) were ligated onto restric- analyses in RAxML with 100 replicates and exported the most tion fragments to facilitate multiplexing of 96 samples in pooled highly supported replicate in each case. All bioinformatic tasks libraries. The barcoded restriction-ligation reaction products from demultiplexing up to phylogenetic inference were con- were stored at 4°C overnight. Products were then PCR ampli- ducted on the JANUS supercomputer (subsequently replaced fied, samples were pooled, and the resultant products were run by SUMMIT) at the University of Colorado (Boulder). Raw on 1% mixed half-and-half multipurpose molecular biology phylogenomic sequence data, in the form of one fastq file for grade agarose/Invitrogen UltraPure 1000 high resolution aga- each individual/tip sampled, are available at https://www. rose gels. DNA smears were size-selected by hand, targeting ncbi.nlm.nih.gov, under SRA Accession #SRP149934. the 200–500 base pair region to improve comparison of homol- ogous fragments across samples. Gel excisions were purified using QIAquick Gel Extraction Kits according to the manufac- ■ RESULTS turer’s protocol. The end-product pooled libraries were submit- ted to the University of Colorado’s BioFrontiers Next- Morphology and ecology. — Bunge’s type specimen of Generation Sequencing Facility (Boulder, Colorado), where Stellaria irrigua from Altai is composed of three juvenile, they underwent a second round of size selection with BluePippin poorly developed individuals plus two disembodied . and were sequenced on an Illumina HiSeq 2000 with a V3 The single mature fruit on this specimen is a capsule 100-Cycle Single Read Sequencing Kit or a NextSeq 500 with with valves that far exceed the (Fig. 1C). Our study of a V2 High-Output 75-Cycle Single Read Sequencing Kit. newly collected field specimens plus existing herbarium Raw reads were first examined using FastQC (Andrews, materials from Asia and North America revealed numerous 2010) to assess data quality and then demultiplexed and fil- collections that share this and other features (see below) and tered using process_radtags, a module within the Stacks pipe- thus can be confidently attributed to the name S. irrigua. These line (Catchen & al., 2013). Flags -c and -q were set as collections indicate that this species is far more common and additional filtering parameters, with an Illumina adapter mis- geographically widespread than previously understood. In con- match of 3 allowed and both cut sites specified. FastQC was trast, material from a previously unrecognized, monophyletic employed a second time to ensure high quality of data post- lineage from the southern Rocky Mountains (see Molecular trimming. Resulting trimmed and demultiplexed reads were Data below) bears capsules with valves that are equal or then used to create a RAD locus alignment for all samples in subequal to the sepals. Numerous additional morphological pyRAD v.3.0.66 via de novo assembly, with any low-quality differences separate these two entities, as shown in Figs. 1–5 bases further excluded from final alignments (Eaton, 2014). and 8 and as described in Table 1. Ecologically, plants of Select samples were separately aligned and visualized against S. irrigua consistently inhabit (during their period of growth) the public Silene latifolia reference genome using SAMtools poorly drained, moist substrata from upper montane mossy to ensure RAD loci were representative of Caryophyllaceae creeksides to damp areas of boreal subalpine forests, to late (Li & al., 2009), but de novo assembly was employed for final summer snow-holding arctic and alpine tundra, both in Asia dataset assembly because it yields datasets with significantly and North America (e.g., Fig. 2B–D). In contrast, plants of less locus dropout (see Tripp & al., 2017 for comparison of the previously unrecognized entity are narrowly restricted de novo to reference-based assembly). Various exploratory pa- and endemic to very dry, well-drained, exposed alpine scree rameters were implemented in pyRAD to yield a set of most slopes of the southern Rocky Mountains (Figs. 2A, 3). Early optimal parameters for downstream analyses. In relation to pressed material of three names relevant to this study—Alsine

3 Sharples and Tripp • RADseq rejects disjunction in Stellaria TAXON 2019

polygonoides, S. irrigua, and S. umbellata—displays some of Sister to the southern Rockies clade of Stellaria irrigua is the morphological differences detailed in Table 1 (Fig. 4). Al- the ecologically and morphologically distinctive lineage of though populations of A. polygonoides have been previously S. sanjuanensis nom. nov. that phylogenetic data resolved as attributed to S. irrigua (Weber, 1961), taxonomic, morpholog- monophyletic with strong support (Fig. 5). Plants of this line- ical, and ecological data in combination with molecular results age are found from southwestern Colorado to northern portions presented here constitute strong evidence for a separately of the Sangre de Cristo Mountains in New Mexico (Fig. 7). De- evolving lineage in the southern Rockies. Below, we propose spite extensive fieldwork in similar biomes and habitats in the the nomen novum Stellaria sanjuanensis (≡ Alsine Altai Mountains and elsewhere in central, southern, and east- polygonoides Greene ex Rydb.) to accommodate this lineage. ern Asia, no representatives of S. sanjuanensis were found in Molecular data. — RADseq data used in this study that hemisphere. Likewise, new populations of this species resolved phylogenetic relationships within the Stellaria have not been discovered during either authors’ various field irrigua complex as well as among samples of Stellaria with trips throughout alpine areas of the mountainous western very high ML bootstrap support (Fig. 5). Our final alignment United States and Mexico, nor have specimens been seen at (that utilized nearly the maximum amount of phylogenomic or from any institution outside of the distribution shown in data generated; suppl. Appendix S1) comprised 37 tips and contained 72,941 concatenated RAD loci with a total length of 6,128,319 nucleotides, 455,184 variable (SNP) sites, and the percent of missing data (undetermined N sites and gaps) in this alignment was 76%. Alignments of shorter lengths (ranging from 88,715 to 2,330,025 bases, and anywhere from ~15% to 52% missing data; Fig. 6; suppl. Appendices S2– S5) as well as alignments composed only of SNP data almost always resolved the same topologies as those inferred from our final concatenated locus alignment (Fig. 5), and these smaller alignments generally had comparable albeit slightly lower bootstrap support at some nodes (Fig. 6; SNP-inferred phylogenies not shown). All but two nodes were resolved at or near (two other nodes supported with values of 99% and 97%) 100% bootstrap support in the 76% missing data tree; these two nodes involved short branches and very closely related populations within species (Fig. 5). Our data recovered two Asian clades plus one southern Rocky Mountains clade of S. irrigua, with the North American populations embedded within the broader Asian lineages (Fig. 5). Geographic structure within the Asian clades was lim- ited: neither populations from the Altai Mountains nor the Sayan Mountains formed monophyletic groups (Fig. 5). Within the southern Rockies, S. irrigua as sampled occurs from the Park Range of northern Colorado to the South San Juan Mountains of southern Colorado; two accessions from the South San Juan Mountains formed a strongly supported clade (100% BS) whereas five populations from northern Colorado showed little geographic structure, although intrapopulation samples were recovered as monophyletic (Fig. 5).

Fig. 1. Major differences in morphology of mature capsules help distin- guish Stellaria sanjuanensis nom. nov. from S. irrigua. A, Stellaria sanjuanensis (M. Sharples 1367, Colorado). B, Early material of S. umbellata herein recognized as a synonym of S. irrigua (Turczaninow s.n., alps of Nuchu Daban). C, Type material of S. irrigua (A. v. Bunge s.n., alps of the Chuya River). In B and C, the mature capsules (solid arrows) far exceed the length of the sepals (hollow arrows). The southern Rocky Mountains endemic S. sanjuanensis is characterized in fruit by having mature capsules that are less than or equaling the length of the sepals (shown in A). — Photos: Mat Sharples.

4 TAXON 2019 Sharples and Tripp • RADseq rejects disjunction in Stellaria

Table 1. Salient habitat and morphological differences between the southern Rocky Mountains endemic Stellaria sanjuanensis nom. nov. and the widespread eastern Asian–western North American S. irrigua. Character Stellaria sanjuanensis Stellaria irrigua Habitat Well-drained alpine screes and tuffs Moist boreal and arctic-alpine substrata Habit Fleshy shoots; long-sprawling Not fleshy; plants solitary or densely clumped Leaves Purple/dark green Light green/not conspicuously anthocyanic Bracts Indistinguishable from other leaves Reduced, scarious Solitary or 2 flowers arising from same axil Terminal, subumbellate cymes Perianth ~2 mm, ~2/3 length to subequaling sepals Petals absent to rarely minute, <1mm Capsules Subequaling sepals at maturity Exceeding sepals at maturity Seeds Testa cells interleaved and ridged Testa smooth

Fig. 2. Differences in habitat between Stellaria sanjuanensis nom. nov. and S. irrigua. A, Dry, exposed alpine screes representing the sole habitat in which the southern Rocky Mountains endemic S. sanjuanensis occurs. Several individuals are encompassed by each of the ellipses. B, Stellaria irrigua on damp, mossy screes in the Kazakh Altai Mountains fed by persistent snowmelt. Ellipse indicates a tuft of individuals. C, Stellaria irrigua on late-snowmelt portions of tundra, holding moisture throughout a typical growing season in northern Colorado (Niwot Ridge, September 2017). Note subumbellate cymes. D, Stellaria irrigua in damp, grassy areas at the base of rock faces in alps of the Chuya River valley, Altai Mountains, Russia. Ellipse indicates umbellate of several individuals. — Photos: Mat Sharples.

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Fig. 7. The distinctiveness of S. sanjuanensis is further (M. Sharples & E. Tripp, in revision). In the present study, pet- highlighted by its geographical co-occurrence with plants of iolate members of Stellaria formed a clade with strong support: S. irrigua: the “SSJ” populations of S. sanjuanensis sampled Stellaria media (L.) Vill. (Colorado)+Stellaria nemorum L. here occur less than 20 air kilometers from our sampled (Europe)+Stellaria bungeana Fenzl (Altai) (Figs. 5, 6). “SSJ” populations of S. irrigua—both of these from the South Our results additionally suggest that Stellaria longipes Goldie San Juan Mountains—but are clearly not sister taxa (Figs. 5–7). (Colorado) and Stellaria longifolia Muhl. ex Willd. (Colorado) Furthermore, the “SSJ” S. irrigua populations are embedded are close relatives, and that Stellaria brachypetala Bunge within a clade of S. irrigua populations from throughout north- (Altai) is also related to the former two. The Californian coastal ern Colorado (Figs. 5–7). endemic Stellaria littoralis Torr. is here placed phylogeneti- Other phylogenetic relationships among Stellaria and cally for the first time as a close relative to the circumpolar, outgroups recovered in this study largely support the results shoreline-inhabiting Stellaria humifusa Rottb. (Norway). of Greenberg & Donoghue (2011). Although our outgroup sampling was very limited, Cerastium beeringianum was sister to core Stellaria when trees were rooted using Pseudostellaria ■ DISCUSSION jamesiana, both of these sampled from Colorado. This result is similarly supported by our ongoing work in Caryophyllaceae, Across the range of Stellaria irrigua, there exists a clear which includes a much broader sampling of outgroups phylogenetic distinction between Asian and North American

Fig. 3. Habitat and morphology of Stellaria sanjuanensis nom. nov. A, Many tiny individuals of S. sanjuanensis (these ca. 4 cm tall; one indicated by red ellipse) are found amongst a “grove” of comparatively colossal Rhodiola integrifolia individuals (these ca. 15 cm tall; one indicated by yellow ellipse). B, Close-up photograph showing two ostensibly solitary individuals linked through an extensive sub-surface system. C, A cluster of several rhizomatously branched individuals. D, A cluster of individuals that are lighter in color, perhaps as a function of less UV exposure attributable to shelter provided by rocks. — Photos: Mat Sharples.

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populations sampled here. This geographical separation of recovered no phylogenetic evidence for a unique relationship North American from Asian populations may be driving evolu- between plants of the southern Rocky Mountains and the Altai tionary divergence between populations on the two continents Mountains: Altai plants were not specifically sister to any of despite morphological and ecological cohesion. Nevertheless, the southern Rocky Mountains plants (but rather, were sister our phylogenetic results in combination with morphological to either Sayan or Himalaya individuals sampled), and while and ecological features shared among plants from these two the southern Rockies plants are indeed embedded within other- distant localities support a broad eastern Asian–western North wise eastern Asian lineages, these eastern Asian plants include American connection in this Stellaria species complex. While individuals from Altai, Himalaya, Sayan, and Kamchatka, with Weber’s (2003) hypothesis posits such a connection, he also little phylogenetic-geographical structure recovered across put forward the notion of subsequent extinction of intervening these areas. The expanded geographical concept of S. irrigua populations in oroboreal portions of a formerly much broader presented here requires treatment of a later and more familiar geographical range. In contrast to the latter, our results clearly name, S. umbellata, as a synonym of S. irrigua, which is sup- establish the presence of a widespread and relatively abundant ported by our study of type material, study of collections from (rather than very rare) species. This species, S. irrigua, tra- across much of the geographical range of these plants, and mo- verses a broad, amphi-Beringian oroboreal arc ranging more lecular data (Figs. 1, 4, 5; see Revised Taxonomic Concepts). or less contiguously from cold elevations and latitudes This name, S. umbellata, has been in common usage for over throughout western North America to cold elevations and lati- a century to represent the widespread eastern Asian–western tudes of eastern Asia, to as far west as the western Himalaya North American species, but our results support the earlier and Altai (Fig. 7). Himalayan plants have previously been re- name S. irrigua as applying to this taxon. ferred to as S. subumbellata, a taxon that is not distinguishable Our best-estimate RADseq phylogeny supports a sce- from S. irrigua (see Revised Taxonomic Concepts). We nario of in situ speciation in southern ranges of the southern

Fig. 4. A, Early material (1899) collected by Baker (NY barcode 00342332) and determined as Alsine polygonoides. B, Early material collected by Turczaninow and determined as Stellaria umbellata: left, LE barcode LE 01026093 (1836); right, K barcode 000723678 (without year). C, All ma- terial on Bunge’s holotype of Stellaria irrigua. This young material of S. irrigua on the holotype is identical to young material of S. umbellata (B) collected by Turczaninow. — Scale bars: 1 cm. Photos: Mat Sharples.

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Rocky Mountains of Stellaria sanjuanensis from a more two lineages are evolving under distinct trajectories. Central widespread ancestor, S. irrigua. Mechanisms of speciation to this evidence is the narrow ecology of, and hence dis- of the former from the latter are likely to have included eco- persal limitations to, S. sanjuanensis (i.e., restricted to logical isolation of S. sanjuanensis when plants reached al- mostly volcanic substrata above treeline), as well as likely pine scree slopes of volcanic southern Colorado, after reproductive isolation of species of Stellaria that have diver- having migrated eastward from eastern Asia and southward gent floral morphologies (i.e., one petalous and the other into southern portions of western North America. We hy- apetalous; M. Sharples, unpub. data). Our recognition of pothesize that this migration occurred via Beringia given S. sanjuanensis as a distinct species is further vindicated the contemporary presence of S. irrigua there, and that this by the fact that both Porsild and Hultén could not identify likely happened during the Pleistocene given the lack of arc- the endemic southern Rocky Mountains scree-restricted tic tundra habitat at present-day arctic latitudes during the taxon as anything they had seen in Stellaria anywhere be- Tertiary (Fig. 7) (Basinger & al., 1994). Future molecular fore, including S. umbellata (Weber, 1961). This narrow en- dating work across all of Stellaria will help address this demic was first described as Alsine polygonoides (by Greene hypothesis (M. Sharples & E. Tripp, in prep.). Despite the in Rydberg, 1906), but this name has rarely appeared in sub- close phylogenetic affinity of S. sanjuanensis and S. irrigua, sequent literature, perhaps in part because it is an illegitimate the southern Rockies endemic is sufficiently distinctive from name (Jessen, 1879; Prantl, 1884; Sprague, 1920). S. irrigua to warrant recognition at the species level. Paraphyletic taxa above the rank of species are common- Though this recognition renders S. irrigua paraphyletic, place and are often, implicitly accepted by scientists who S. sanjuanensis has never been confused in the literature teach such examples in the classroom. At a broad scale, a with what has widely been known as S. umbellata for well few common examples include prokaryotes (without inclu- over a hundred years, and we provide evidence that these sion of eukaryotes), reptiles (rendered paraphyletic by lack

Fig. 5. Maximum likelihood phylogeny of the Stellaria irrigua complex and additional relatives as inferred from RAD loci. The new name S. sanjuanensis is here represented by five individuals from the South San Juan Mountains and nearby Sangre de Cristo Mountains that form a well-supported, monophyletic group nested within a paraphyletic assemblage of Asian and North American populations of S. irrigua. All branches have 97% or higher bootstrap support (32 out of 36 nodes resolved with 100% support) except for two nodes marked by circles (lowermost: 82% in the S. sanjuanensis clade; uppermost: 87% in one Asian S. irrigua clade). Boxed populations of SSJ S. irrigua are sympatric with S. sanjuanensis, further supporting the monophyly and distinctiveness of S. sanjuanensis. Abbreviations: ALT, Altai Mountains; HM, Himalaya; ID, Idaho; KM, Kamchatka; NCO, northern Colorado Mountains; SoRo, southern Rocky Mountains; SSJ, South San Juan Mountains; SY, Sayan Mountains. Inset, differences between S. sanjuanensis (A) and S. irrigua (B) represent one of several morphological features distinguishing these two taxa (Table 1). Yellow lines in A run parallel with and indicate extent of a single bifid petal. B shows a entirely absent of petals at a similar de- velopmental stage to that in A. Well-developed petals most likely reappeared in the most recent common ancestor of the clade containing all acces- sions of S. sanjuanensis, as depicted. — Photos: Mat Sharples.

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Fig. 6. Cladograms inferred from alignments with lower missing data thresholds than those presented in Fig. 5. Percent missing data per alignment is as follows. A, 15.86%; B, 25.36%; C, 44.38%; D, 52.31%. Topologies and topological layouts are identical to those in Fig. 5 except in these few examples of incongru- encies: A and B infer a relationship of Stellaria longifolia as sister to S. brachypetala, as opposed to S. longifolia as sister to S. brachy- petala+S. peduncularis+S. longipes; in A, one specimen of S. sanjuan- ensis is more closely related to a different specimen of that species from the San Juan Mountains; and the exact position of the S. irrigua specimen from Krasnoyarsk Krai (= Stellaria irrigua SY1) slightly differs in both the 15% and 25% alignments. The relatively poor support in the S. sanjuanensis clade in A is likely solely attributable to generally lower data amounts for those samples (see SRA data for comparison of file sizes). That is, when we raised the minimum num- ber of taxa sharing a locus in the final alignment used to infer this phylogeny, these samples lost a sig- nificant number of their locus repre- sentatives in the alignment. As the most significant topological incon- gruency involves S. longifolia and close relatives, hybridization or other phenomena may be at work in the evolutionary history of this group (see Discussion).

9 Sharples and Tripp • RADseq rejects disjunction in Stellaria TAXON 2019

of inclusion of avians), bony fishes (without inclusion of tet- rapods), algae, the ANITA Grade in flowering plants, bryo- phytes, and lichens (Raven & al., 2005; Urry & al., 2016). On a more narrow scale, and within flowering plants, exam- ples include genera of Hawaiian lobeliads (Givnish & al., 2009), Ranunculus subg. Auricomus (Hörandl & Emadzade, 2012), Hawaiian mints (Welch & al., 2016), and numerous other higher taxonomic ranks within angiosperms (Willner & al., 2014). However, despite advocation for monophyly of lineages at the rank of species by numerous practicing systematists, paraphyletic species oftentimes reflect evolu- tionary processes (Crisp & Chandler, 1996; Hörandl, 2006). In such instances, sophisticated methods of phylogenetic in- ference such as coalescence-based approaches aid in the rec- ognition and understanding of paraphyletic lineages (Harrington & Near, 2011), and such an approach may be appropriate in the future within certain lineages of starworts. Lineages that can readily hybridize—thereby obscuring true species trees—are also regularly still recognized at the rank of species (Hörandl, 2006); many species shown to have ex- perienced recent introgression are customarily still recog- nized with binomials (e.g., within Vaccinium: Beeler & al., in press). Although we did not explicitly test for introgres- sion in this study, if hybridization between S. irrigua and S. sanjuanensis were prevalent, we would expect the sympat- ric populations of S. irrigua in the South San Juan Moun- tains to be more closely related to S. sanjuanensis than they are, and we would expect intermediate taxa between the two lineages to be represented in herbarium collections. Species endemic to islands recently derived via dispersal from mainland populations should recover the same phyloge- netic patterns as those recovered here: a monophyletic descen- dant embedded within an ancestral lineage that is more widespread, yet rendered paraphyletic, by the island dispersal event, which may be followed by reproductive isolation and peripatric speciation (e.g., Valtueña & al., 2017). In the case of Stellaria sanjuanensis, present-day geographical isolation seems less at work in maintaining species boundaries than do edaphic and other forms of ecological isolation; this system is thus consistent with models of parapatric rather than peripatric speciation, though the southern Rocky Mountains may be

Fig. 7. Geographical distributions of Stellaria irrigua (widespread) and S. sanjuanensis nom. nov. (endemic to southern Rocky Mountains). Tri- angles represent digitized as well as newly collected populations of S. irrigua, while stars represent all currently known populations of S. sanjuanensis (SEINet, 2013–; GBIF Secretariat, 2017). Blue triangles (Asia), green triangles (North America), or orange triangles (southern Colorado) represent individuals of S. irrigua included in phylogenetic analyses; the orange triangles represent sampled S. irrigua individuals that are broadly sympatric with S. sanjuanensis. Inset, distribution of both species in Colorado and far northern New Mexico. Red stars repre- sent individuals of S. sanjuanensis included in phylogenetic analyses. Note sympatry of the sampled populations of the two species in the South San Juan Mountains of southern Colorado. The worldwide map was constructed with ArcGIS v.10.5; the inset map was constructed with D-Maps (https://d-maps.com/carte.php?num_car=19882).

10 TAXON 2019 Sharples and Tripp • RADseq rejects disjunction in Stellaria

considered its own sort of terrestrial “sky island”. Related and evidence demonstrating progenitor-derivative speciation in not necessarily mutually exclusive modes of speciation that Stellaria using RADseq data. This mode of speciation may yield paraphyletic species include progenitor-derivative (i.e., be very common across other systems characterized by nar- budding) speciation, centrifugal speciation, and catastrophic rowly endemic taxa: signatures of progenitor-derivative speci- speciation (Anacker & Strauss, 2014). In an empirical study ation were phylogenetically widespread in a meta-analysis on in the Helianthus petiolaris complex, Rieseberg & Brouillet plants from the California Floristic Province (Anacker & (1994) speculated that progenitor-derivative speciation is a Strauss, 2014). We hypothesize that further work exploring or- rather important process in plants. They and others have further igins of range-restricted, high-elevation species in the southern hypothesized that a very high percentage of plant species Rocky Mountains may reveal these mountains as an under- (upwards of 25%–50%) may be expected to have paraphyletic recognized region of phylogenetically widespread progenitor- origins through a wide array of speciation processes; conse- derivative speciation. quently, these authors argue that strictly monophyletic plant The purported disjunction of Stellaria irrigua was the sole, species are untenable (Crisp & Chandler, 1996; Hörandl, improbable example of a plant species found only in Altai and 2006). Other examples of paraphyletic species-rank taxa, some- in the southern Rocky Mountains and was hypothesized to be times referred to as “paraspecies”, can be found outside of particularly representative of a deep Tertiary-Quaternary vicar- plants, such as in Anas, Bufo, Nesticus, Peromyscus, and Ursus iance event affecting multiple members of the floras from both (Avise, 2000; Crandall & al., 2000). Rejecting paraphyletic regions (Weber, 2003). Our results indicate that previous skep- species can furthermore have negative conservation implica- ticism towards the hypothesized long-distance disjunction be- tions (Crandall & al., 2000). tween the southern Rockies and Altai was not unwarranted Indeed, the case of Stellaria sanjuanensis closely (Kozhevnikov, 1994; Czerepanov, 1995; Morton, 2005). In- resembles that of Pozoa volcanica Mathias & Constance deed, the likelihood of a range-restricted, narrowly adapted, (Apiaceae) in relation to P. coriacea Lag. in the southern and narrowly distributed species in the southern Rockies is in- Andes, with the former comprising a monophyletic, volcanic, herently greater than that of a singularly peculiar intercontinen- edaphic endemic lineage embedded within a paraphyletic tal disjunction between this region and elsewhere. The alpine assemblage of P. coriacea, a characteristic phylogenetic life zone of the southern Rocky Mountains alone is already re- pattern of progenitor-derivative speciation (López & al., ported to harbor some 25 to 26 endemic taxa (Fowler & al., 2012). The California Floristic Province genus Layia similarly 2014), likely an underestimate and perhaps a reflection of the reflects progenitor-derivative speciation: the widespread highly circumscribed niches of species adapted to this region. L. glandulosa (Hook.) Hook. & Arn. is rendered paraphyletic Thus, in light of results herein presented in combination with by evolution of the monophyletic edaphic endemic L. discoidea prior knowledge of a relatively high number of endemic spe- D.D.Keck nested within it (Baldwin, 2005). Such speciation cies reported from an ecosystem inherently inhospitable to patterns have been found to be extremely widespread in angio- many other lineages, it may be expected that future phyloge- sperm evolution (e.g., Crisp & Chandler, 1996; Perron & al., netic studies on alpine plants in western North America may 2000; Jaramillo-Correa & Bousquet, 2003; Grossenbacher & similarly reveal evidence of overlooked speciation in such al., 2014; Chung & al., 2015). Here, we present strong ecosystems.

Fig. 8. A, Seeds of Stellaria sanjuanensis nom. nov. (M. Sharples 1367, Colorado) have testae that are distinctly interleaved and ridged. B, Seeds of S. irrigua lack visible testa boundaries (M. Sharples & S. Smirnov 1250, Altai).

11 Sharples and Tripp • RADseq rejects disjunction in Stellaria TAXON 2019

Despite evidence here presented, there exists a number of further derives from salient macromorphological and other plant species that serve as candidates for long-distance ecological differences, the geographical patterns inferred, range disjunctions between central or eastern Asia and the and the generally high level of congruence with the primary southern Rocky Mountains. These study systems, which re- extant phylogenetic hypothesis of Stellaria (Greenberg & quire expanded investigation that in part incorporates high- Donoghue, 2011). resolution phylogenetic information, include Artemisia Our RAD sequencing analyses across a diversity of laciniata Willd., Eutrema edwardsii R.Br., E. salsugineum subclades within Stellaria suggest that these data are likely to (Pall.) Al-Shehbaz & Warwick, and Gentiana algida Pall. resolve a more extensive set of relationships across the genus. (Weber, 2003; Wang & al., 2015). In some of these instances, As mentioned, the petiolate S. bungeana, S. media, and immense stretches of inhospitable continental interiors separate S. nemorum form a strongly supported clade, in agreement putatively conspecific populations (though present in the Arc- with results from prior investigation (Greenberg & Donoghue, tic) between central Asia and the southern Rockies; in other in- 2011). Our results also place the S. longipes group with some stances, a number of northern and temperate stations exist increased resolution. We demonstrate for the first time that between the two geographical extremes. However, none of S. longifolia may be phylogenetically distinct from, yet closely the hypothesized disjunctions is as dramatic as that proposed related to, S. longipes, a species with which the former is for Stellaria irrigua, which our investigation has rejected. Phy- thought to hybridize over evolutionary time scales (Chinnappa logenetic knowledge combined with field studies will help to & al., 2005). Previously recognized closer relatives of more broadly characterize floristic histories within and S. longipes, such as S. peduncularis Bunge (Altai), may ulti- amongst these two regions as well as to provide more robust mately be supported as regional variants of S. longipes pending tests of Weber’s (2003) as yet still underexplored hypothesis. further sampling of S. longipes across its distribution; as To our knowledge, the present study represents the first S. longipes is circumboreal, widespread, morphologically plas- use of RAD loci to resolve phylogenetic relationships within tic, and replete with localized synonyms, much expanded sam- Caryophyllaceae (here, specifically in tribe Alsineae cf. pling of this species across its range is needed. Stellaria Harbaugh & al., 2010). Our results confirm the utility of brachypetala, although a member of the putatively hybridizing these data for phylogenetic inference among closely related S. longipes–S. longifolia species complex, is supported by species of flowering plants (e.g., Hipp & al., 2014; Hou & morphology and phylogeny as a distinctive species rather than al., 2015, 2016; Massatti & al., 2016; Eaton & al., 2017; as a regional variant or synonym of either entity. We also dem- Tripp & al., 2017). Specifically, RAD loci utilized in our onstrate that the circumboreal S. borealis, S. humifusa, and the study resolved population- to species-level relationships, al- California endemic S. littoralis share a more recent common though the latter was not an explicit goal of our investigation ancestor with core ingroup taxa sampled here than with and thus outgroup inclusion was minimal—sampling was S. longipes and relatives, which conflicts with previous results geared primarily towards ingroup representation. Further- suggesting that the former three are part of the same clade as more, samples of different individuals from the same popu- the S. longipes complex (Greenberg & Donoghue, 2011). In lations of Stellaria irrigua were unambiguously always contrast to suggestions by Morton (2005), S. littoralis of Cali- recovered as monophyletic (i.e., Stellaria_irrigua_ALT1 & fornia is not at all closely related to S. dichotoma L. from the 2, Stellaria_irrigua_ALT-Chuya1 & 2, Stellaria_irrigua_ steppes of Asia, a taxon that most likely should be transferred NCO1 & 2, and Stellaria_irrigua_NCO4 & 5). In view of to a different genus based on phylogenetic and morphological these results, RADseq represents a powerful tool for investi- characteristics (M. Sharples & E. Tripp, unpub. data). Further gating questions across multiple phylogenetic scales in work will more clearly elucidate phylogenetic relationships Caryophyllaceae. Given numerous groups within the family among these and other species of Stellaria using a greatly have been challenged with robust phylogenetic resolution expanded RAD locus database. and taxonomic placement as a function of convergent or parallel evolution of non-sister taxa upon very similar morphologies, including within Minuartia and relatives (Dillenberger & Kadereit, 2014) as well as within Stellaria ■ REVISED TAXONOMIC CONCEPTS itself (Greenberg & Donoghue, 2011), RAD sequencing may prove a fruitful approach to forthcoming phylogenetic Stellaria sanjuanensis M.T.Sharples & E.Tripp, nom. nov. ≡ reconstruction and taxonomic classification in the family, Alsine polygonoides Greene ex Rydb., Fl. Colorado: which have heretofore involved morphology or DNA align- 127–128. 1906, nom. illeg., non Alsine polygonoides ments of relatively few loci (e.g., Scheen & al., 2004; (Wulfen) Prantl 1884, nec Stellaria polygonoides Harbaugh & al., 2010; Greenberg & Donoghue, 2011). (Wulfen) Jess. 1879 – Holotype: U.S.A., Colorado, La Although prior studies have expressed some concern over Plata Mountains, Little Kate Basin, 11,500 ft. alt., 14 Jul inflated bootstrap values and phylogenetic incongruence as- 1898, C.F. Baker & al. 515 (NDG barcode NDG16510!; sociated with data concatenation (e.g., Lambert & al., isotype: MO No. 1756694!). 2015; Fernández-Mazuecos & al., 2018), our confidence in Note. – See below regarding Tiehm’s (1989) erroneous the phylogenetic relationships recovered with these data lectotypification of this name.

12 TAXON 2019 Sharples and Tripp • RADseq rejects disjunction in Stellaria

Geographical and ecological notes. – As currently under- screes, 27 Aug 2016, M. Sharples & E. Tripp 1367 (COLO stood, Stellaria sanjuanensis is narrowly restricted to dry, barcode 02170504); Hinsdale County, Gunnison National exposed alpine scree slopes of usually volcanic origin in the Forest, La Garita Wilderness, upper portion of basin which San Juan, Elk, and Sangre de Cristo Mountains of Colorado, feeds West Fork Mineral Creek, ca. 0.8–1.1 air mi. ESE of extending into more southerly portions of the Sangre de unnamed peak 13,510′, ca 5 air mi. SSW of Mineral Moun- Cristo Mountains in far northern New Mexico (Figs. 3, 7). tain, 12,680–12,800 ft., alpine tundra, 11 Aug 1999, M. Arnett Oddly, it is not known from the Sawatch Mountains, with 6878 (COLO barcode 00895789); La Plata County, La Plata ostensibly suitable habitat less than 10 air kilometers from Mts., San Juan N.F., Tomahawk Basin (Little Kate Basin of vouchered localities in the Elk Mountains across the Taylor Rydb.’s Flora), SS-facing 67% slope, 11,950 ft., in sparsely River valley. vegetated, gravelly soil “island” in middle of steep large-rock Taxonomic and nomenclatural notes. – The holotype and talus slope, almost hidden by gravel, 23 Jun 1977, isotype cited above (originally identified by M.E. Jones as B. Johnston 1252 (COLO barcode 00003046); San Juan “Arenaria saxosa Gray var.”) represent the earliest known col- County, Rio Grande National Forest, basin SE of Stony Pass, lections of Stellaria sanjuanensis. The locality information on ca. 8 air miles ESE of Silverton, headwaters of the Rio these specimens (C.F. Baker & al. 515) matches verbatim that Grande, NW aspect of Sheep Mt., ca. 12,400 ft., loose scree referenced in the protologue of Alsine polygonoides. Digitized w/ Claytonia megarhiza, Ligularia soldanella, 8 Jul 2007, herbarium specimens (viewable on https://jstor.plants.org) T. Hogan & L. Tembrock 4692 (COLO barcode 00917229); of a different collection, C.F. Baker 307 (K barcode San Miguel County, near Trout Lake, 12,000 ft., on dirt K000742072!, MO barcode MO-216575!, NDG barcode slides, 21 Aug 1924, E. Payson & L. Payson 4202 (COLO NDG16455!, NY barcode 00342332!, US barcode barcode 00002956); San Miguel County, Uncompahgre Na- 00610728!), represent material used by Tiehm (1989: 153) tional Forest, Lone Cone, 11,850 ft., alpine meadow S of in attempt to lectotypify the name A. polygonoides. However, “Devil’s Chair”, loose schist colluvium, 12 Aug 1982, C.F. Baker 307 was collected “Near Pagosa Peak”—dozens V. Siplivinsky 4739 (COLO barcode 00002972). U.S.A., of kilometers to the east of Little Kate Basin—and thus can- New Mexico, Sangre de Cristo Mountains. Colfax County, not be considered original material. Tiehm’s lectotypification Philmont Scout Ranch, near Cimarron, Baldy Mountain, is thus in conflict with the protologue and must therefore be 12,300 ft., occasional among rocks on north exposure, revised following Article 9.19 in Turland & al. (2018). Be- 30 Jul 1968, Hartman 2529c (COLO barcode 01866169); cause the epithet “polygonoides” is occupied in Stellaria Taos County, Culebra Range, Vermejo Park Ranch, slope (Jessen, 1879), a new name is proposed here. on south side of an unnamed ridge 0.4 air mi. SW of Lake Selected additional specimens examined. – U.S.A., No. 2, 12,208 ft., steep south facing slope of fine scree, Colorado, Elk Mountains. Gunnison County, Gunnison 17 Jul 2008, Legler 9855 (COLO barcode 01866151). N.F., just SS of false summit between E and W peaks of Mt. Belleview, 12,040 ft., 60% SS-facing talus slope with Stellaria irrigua Bunge in Mém. Acad. Imp. Sci. St.- gravel on surface and hard-packed soil 1–3 in below surface, Pétersbourg Divers Savans 2: 548. 1835 – Holotype: 12 Jul 1977, B. Johnston & P. Lucas 1314 (COLO barcode Russia, Altai Mountains, alps of the Chuya River, A. v. 00003079); Gunnison N.F., steep talus slope below SSE of Bunge s.n. (LE barcode LE 01009386!). Italian Mtn., 12,200–12,300 ft., orange talus with gray = Stellaria umbellata Turcz. ex Kar. & Kir. in Bull. Soc. Imp. marbleized limestone boulders on surface, 17 Jul 1980, Naturalistes Moscou 15: 173–174. 1842 ≡ Alsine B. Johnston & al. 2347 (COLO barcode 00003087); trail baicalensis Coville in Contr. U.S. Natl. Herb. 4: 70. along Copper Creek to summit of Conundrum Pass, ca. 1893, nom. illeg. superfl. – Lectotype (designated 10 mi. NE of Gothic, ca. 12,000 ft., in trail and on fine scree, here): Russia, alps of Nuchu-Daban (Nukhu-Daban in 4–5 Aug 1955, W. Weber & S. Shushan 9388 (COLO Russian, near Baikal), years various, N.S. Turczaninow barcode 00003053). U.S.A., Colorado, Sangre de Cristo s.n. (K barcode K000723679!; isolectotypes: K barcodes Mountains. Costilla County, Culebra Range, southwestern K000723675!, K000723677! & K000723678!, LE ridge of Trinchera Peak, SE of Fort Garland, 12,800– barcodes LE 01026093!, LE 01026094!, LE 01026095!, 13,400 ft., alpine, 12 Aug 1998, B. Elliott 5394 (COLO LE 01026096!, LE 01026097!, LE 01026098!, LE barcode 00820902); Costilla County, Culebra Range, 01026099! & LE 01032100!). Vermejo Park Ranch, State Line Peak (12867), ca. 14 air Additional syntypes: Alatau ad fontes fluviorum Aksu mi. SE of San Luis, 12,800 ft., alpine, 19 Aug 1999, B. Elliott et Sarchan, 1841, G.S. Karelin & I.P. Kiriloff 1305 (K 11208 (COLO barcode 00931162); Saguache County, Sangre barcode K000723680! [Herb. Hookerianum], K! [pend- de Cristo Wilderness and environs, North Fork of North ing barcode, Herb. Benthamianum]). Crestone Creek, Eureka Mt. summit, ca. 13,500′, crestone = Stellaria subumbellata Edgew. in Hooker, Fl. Brit. India conglomerate, 11 Aug 2010, T. Hogan 5088 (COLO barcode 1(2): 233–234. 1874 – Lectotype (designated here): 00978080). U.S.A., Colorado, San Juan Mountains. India, interior of Sikkim, alt. 12–16,000 ft., J.D. Hooker Conejos County, Rio Grande National Forest, ~12 miles s.n. (K barcode K000723655!; isolectotype: K barcode SSW of Platoro, ~3.4 mile N of Trail Lake, 12,200 ft., alpine K000723651!).

13 Sharples and Tripp • RADseq rejects disjunction in Stellaria TAXON 2019

Additional syntype: Tibet, Nubra, alt. 11–15,000 ft., 1993, Ho & al. 792 (CAS barcode 546419, MO No. T. Thompson s.n. (K barcode K000723653!). 4648673); Xizang, Changdu Xian, road (highway 317) from = Stellaria gonomischa B.Boivin in Svensk Bot. Tidskr. 50: Changdu to Riwoqe at Zhuoga-La pass, 4540–4550 m, under 113–114. 1956 – Holotype: U.S.A., Colorado, Univer- shrubs of Salix in moss, 7 Aug 2004, Boufford & al. 31770 sity Camp, between Nederland and Ward, 9,500 ft. alt., (CAS barcode 546365). Russia. Altai Republic, Kosh- 14 Jul 1950, W.A. Weber 5658 (DAO barcode Agachsky District, high mountains of the Chuya River 000000031!; isotypes: BRIT barcode 24089!, CAS steppe, ~45 km WSW of Kosh-Agach, Elangash River Val- barcode 0005635!). ley, 2455 m, wet, mossy, north-facing base of rocks of alpine = Stellaria weberi B.Boivin in Svensk Bot. Tidskr. 50: 114. tundra-steppe, 12 Aug 2015, M. Sharples & S. Smirnov 1241 1956 – Holotype: U.S.A., Colorado, along trail from (COLO barcode 02170645); Amur, Tyndinsky District, upper Steven’s Mine to summit of Gray’s Peak, on rocky reaches of the Tas-Yuryakh river valley, 1700.5 m, 17 Aug tundra slopes, 13,000 ft. alt., 11 Jul 1950, W.A. Weber 1986, A. Kozhevnikov 2989 (VLA No. 119266); Khabarovsk 5621 (DAO barcode 000000045!; isotypes: BRIT Krai, Nanaysky District, Tordoki Yani mountain, Anyuy barcode 24090!, CAS barcode 0005631!, COLO river, 22 Jul 1983, S. Kharkevich & al. s.n. (VLA No. barcode 00408195!). 291019); Magadan Oblast, Srednekansky District, headwaters Geographical and ecological notes. – Stellaria irrigua is of the Namindykan, mosses by the stream, 6 Aug 1982, widespread in boreal and tundra habitats across the mountains O. Khokhryakova 2429 (VLA No. 119268); Sakhalin Oblast, of western North America and eastern Asia, including suitable East Sakhalin Mountains, Nabil’skiy Ridge, Mount Balagan, habitats at high northern latitudes (i.e., ≥71° N). Unlike 1471.9 m, podgoltsovy belt, 25 Jul 1988, I. Viyshin 3030 S. sanjuanensis, which occupies dry, well-drained alpine vol- (VLA No. 119262). U.S.A. Colorado, Conejos County, Rio canic and sedimentary tuffs and screes below 39° N, S. irrigua Grande National Forest, ~3 miles NW of Platoro, N side of is not found on substrata that lack consistent moisture through- Alamosa River, near Stunner CG, 9700′, 29 Jun 2016, out the majority of the growing season. M. Sharples & E. Tripp 1294 (COLO barcode 02170660); Taxonomic notes. – The type material of Stellaria irrigua Montana, Deerlodge County, Anaconda-Pintlar Range, Goat is immature and indistinguishable from early developmental Flats, 9250′, on moist, mossy bank, 4 Sep 1974, stages and type material of S. umbellata (Fig. 4). In comparing K. Lackschewitz 5782 (COLO barcode 01868165); Utah, the protologues of S. irrigua (from Altai) and S. umbellata Duchesne County, Uinta Mtns., West Fork Whiterocks drain- (from the geographically proximal Sayan Mountains near Lake age, 0.2 east of Lower of Rasmussen Lake, 10,480 ft., moist Baikal), the descriptions are for the most part identical where soils in engelmann spruce forest, quartzite subs., 4 Sep 1995, they overlap in characters (except for one feature pertaining A. Huber & J. Huber 3029 (COLO barcode 01868140). to minute to absent petals). Indeed, Turczaninow (1842) noted that his S. umbellata was very similar to Bunge’s S. irrigua. Key to the native montane Stellaria species of the Observations by the first author in North America and Asia in- southern Rocky Mountains dicate that plants of S. irrigua sometimes have, but more often lack, minute petals. Thus, this character appears to be variable 1. Leaves approximately or more than three times as long as across the full range of the species. wide, linear to very narrowly lanceolate or elliptic; petals Stellaria subumbellata Edgew. is here placed into synon- equaling or exceeding sepals; bracts scarious ...... 2 ymy with S. irrigua due to having overlapping morphological 1. Leaves less than three times as long as wide, lanceolate, and habitat characteristics with Stellaria irrigua, distinct only elliptic, or ovate; petals equaling sepals, shorter than se- in being geographically situated in the Himalaya. Our phylo- pals, or lacking; bracts leafy or scarious ...... 3 genetic results (Fig. 5: “Stellaria_irrigua_HM”) place the en- 2. Inflorescence terminal; flowers often solitary or sometimes tity from the Himalaya as embedded within S. irrigua, and few ...... Stellaria longipes the protologue (Edgeworth & Hooker, 1874) itself notes that 2. Inflorescence axillary; flowers in compound cymes...... the new entity was “very near the Baikal S. umbellata, ...... Stellaria longifolia Turcz.”. 3. Petals equaling sepals; bracts leafy ....Stellaria crassifolia Nomenclatural notes. – Although the name Stellaria 3. Petals shorter than sepals or absent; bracts leafy or umbellata has been used broadly for many decades and on scarious ...... 4 two different continents, the name S. irrigua is validly pub- 4. Leaves ± elliptic; bracts scarious...... 5 lished and has priority. A formal proposal to conserve the name 4. Leaves lanceolate, elliptic or ovate; bracts leafy...... 6 S. umbellata would be needed to allow retaining S. umbellata. 5. Inflorescence terminal, umbellate and often further Selected additional specimens examined. – Canada. Yu- branched (= subumbellate); flowers three to numerous; kon, St. Elias Mts., Observation Mt. and vic., at terminus petals absent to sporadically minute (<1 mm) ...... Kaskawulsh Glacier, 5500–7000 ft., mossy snowflush, ...... Stellaria irrigua 27 Jul 1966, D. Murray & B. Murray 645 (COLO barcode 5. Inflorescence axillary; flowers solitary or 2–5 in cymes, 01868215). China. Qinghai, Maqin Xian, Muchang, Dawu these cymes sometimes secondarily branched; petals pres- Xiang, SE of Maqin, 3980 m, growing on tussock, 5 Aug ent and up to 2/3 length of sepals ...... Stellaria alsine

14 TAXON 2019 Sharples and Tripp • RADseq rejects disjunction in Stellaria

6. Creeping/sprawling habit; leaves broadly elliptic to widely specimen images through public databases or personal request. We ovate, of similar width and length or slightly longer than thank former lab members Heather Stone, Erica Hsin-Tsai, and Yongbin Zhuang for assistance with RADseq library preparations and bioinfor- wide, often with tiny petioles; flowers solitary and axil- matics. Review of the manuscript by Richard Rabeler and others greatly lary; petals lacking...... Stellaria obtusa improved an earlier version of it. Stacey D. Smith also provided useful 6. Plants not creeping/sprawling; leaves lanceolate to feedback on an even earlier version of the manuscript. David Stock elliptic, longer than wide, epetiolate; flowers solitary and and the University of Colorado’s EBIO department gave us free access axillary or multiple in terminal cymes; petals present or to the SEM used for preparation of Fig. 8, which was much appreciated. This work utilized the JANUS supercomputer, which was supported by lacking...... 7 the National Science Foundation (award number CNS-0821794), the 7. Plants fleshy; leaves tinged to mostly deeply maroon; in- University of Colorado (Boulder), the University of Colorado (Denver), ternodes generally congested and less than length of and the National Center for Atmospheric Research. Funding for this re- leaves; flowers ± solitary and axillary; petals ca. 2/3 length search was graciously made possible by the Department of Ecology and of sepals, never absent...... Stellaria sanjuanensis Evolutionary Biology at the University of Colorado (Boulder), by the University of Colorado Museum, and the University of Colorado Her- 7. Plants not fleshy; leaves green; internodes generally barium (COLO). greater than length of leaves; flowers solitary and axillary or multiple in terminal cymes; petals up to 2/3 length of sepals or absent...... Stellaria borealis complex ■ LITERATURE CITED

– Ackerfield, J. 2015. Flora of Colorado. Fort Worth: BRIT Press. Note. Stellaria alsine Grimm has not been previously Anacker, B.L. & Strauss, S.Y. 2014. The geography and ecology of reported from Colorado, or indeed the Rocky Mountains. plant speciation: Range overlap and niche divergence in sister spe- Thus the report here is novel, with the closest known sta- cies. Proc. Roy. Soc. London, Ser. B, Biol. Sci. 281: 20132980. tions being in Minnesota or Washington state (Morton, https://doi.org/10.1098/rspb.2013.2980 2005). The only known occurrence of Stellaria alsine in Andrews, S. 2010. FastQC: A quality control tool for high throughput sequence data, version 0.11.5. http://www.bioinformatics.babr Colorado to date is from the following collection event. It aham.ac.uk/projects/fastqc is presumed native due to the high-quality habitat indicated Avise, J.C. 2000. Phylogeography: The history and formation of spe- on the collection label, but it possibly alternatively represents cies. Cambridge, MA: Harvard University Press. an anthropogenic introduction. Boulder County, 2 miles Baldwin, B.G. 2005. Origin of the serpentine-endemic herb Layia north of Nederland, peaty fen in broad gently sloping valley, discoidea from the widespread L. glandulosa (Compositae). Evolu- tion 59: 2473–2479. https://doi.org/10.1554/05-147.1 8400 ft., growing in dense clumps in rivulets, 29 Jun 1999, Basinger, J.F., Greenwood, D.R. & Sweda, T. 1994. Early Tertiary N. Lederer & Bill Jennings 99-60 (COLO barcode vegetation of Arctic Canada and its relevance to paleoclimatic inter- 00325092!; KHD barcode 00001368!) pretation. Pp. 175–198 in: Boulter, M.C. & Fisher, H.C. (eds.), Ce- nozoic plants and climates of the Arctic. NATO ASI Series (Series I: Global Environmental Change) 27. Berlin and Heidelberg: ■ AUTHOR CONTRIBUTIONS Springer. https://doi.org/10.1007/978-3-642-79378-3_13 Beeler, R.B., Sharples, M.T. & Tripp, E.A. In press. Introgression MTS conceived of the study and conducted field, herbarium, lab, and among three western North American bilberries (Vaccinium section bioinformatic work. EAT provided material/laboratory support and of- Myrtillus). Syst. Bot. fered phylogenetic and taxonomic guidance. Both investigators wrote Bunge, A.V. 1835. Verzeichniss der im Jahre 1832, im östlichen Theile the manuscript. — EAT, https://orcid.org/0000-0001-9340-8723 des Altai-Gebirges gesammelten Pflanzen: Ein Supplement zur Flora Altaica. Mém. Acad. Imp. Sci. St.-Pétersbourg Divers Savans 2(6): 548. ■ ACKNOWLEDGMENTS Catchen, J., Hohenlohe, P.A., Bassham, S., Amores, A. & Cresko, W. A. 2013. Stacks: An analysis tool set for population genomics. We wish to thank botanists of Russian Altai who hosted the first Molec. Ecol. 22: 3124–3140. https://doi.org/10.1111/mec.12354 author on field expeditions in search of S. irrigua and other taxa of cen- Chinnappa, C.C., Donald, G.M., Sasidharan, R. & Emery, R.J.N. tral Asia. We are particularly grateful to Alexander Shmakov (ALTB), 2005. The biology of Stellaria longipes (Caryophyllaceae). Canad. Sergei Smirnov (ALTB), and Andrey Erst (NSK) in this capacity. The J. Bot. 83: 1367–1383. https://doi.org/10.1139/b05-117 staff at LE, especially Irina Illarionova and Roman Ufimov, were also Chung, M.Y., López-Pujol, J. & Chung, M.G. 2015. Genetic evidence instrumental in the completion of this work by allowing the first author of a progenitor-derivative species pair in East Asian Lilium. Ann. access to starwort collections, including the type of S. irrigua. We wish Bot. Fenn. 52: 211–218. https://doi.org/10.5735/085.052.0313 also to thank Dr. William Weber (COLO), who laid the foundation for Crandall, K.A., Bininda-Emonds, O.R.P., Mace, G.M. & Wayne, R. addressing some of the questions in this paper and who personally sug- K. 2000. Considering evolutionary processes in conservation biol- gested to the first author that he should work on the Altai–southern ogy. Trends Ecol. Evol. 15(7): 290–295. https://doi.org/10.1016/ Rockies connection, as well as Collections Manager Tim Hogan S0169-5347(00)01876-0 (COLO), who identified our first collections of the southern Rockies en- Crisp, M.D. & Chandler, G.T. 1996. Paraphyletic species. Telopea 6: demic species and offered other useful feedback through his expert fa- 813–844. https://doi.org/10.7751/telopea19963037 miliarity with the southern Rockies flora. We appreciate feedback Czerepanov, S.K. 1995. Vascular plants of Russia and adjacent states from two other Collections Managers at COLO, Dina Clark and (the former USSR). New York: Cambridge University Press. J. Ryan Allen, and thank them also for other assistance related to this Dillenberger, M.S. & Kadereit, J.W. 2014. Maximum polyphyly: Mul- work. Besides those already mentioned, we thank other herbaria for tiple origins and delimitation with plesiomorphic characters require hosting visits by the first author: CAS, K, MO, NY, P, and VLA. Addi- a new circumscription of Minuartia (Caryophyllaceae). Taxon 63: tionally, BRIT, DAO, KHD, NDG, RSA, and US provided digital 64–88. https://doi.org/10.12705/631.5

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Appendix 1. Specimens sampled (tip labels bolded) with collector(s), herbarium and SRA (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA473254) acces- sion numbers, and geographical identities. Names in the appendix follow the new taxonomy proposed here. Cerastium beeringianum Cham. & Schltdl.: M. Sharples 912 (COLO 552998), SRX4178270, Colorado, U.S.A.; Pseudostellaria jamesiana (Torr.) W.A.Weber & R.L.Hartm.: M. Sharples 525 (COLO 553802), SRX4178271, Colorado, U.S.A.; Stellaria borealis Bigelow: S. Hill 17178 (NY 3317261), SRX4178268, New Hampshire, U.S.A.; Stellaria brachypetala Bunge 1: M. Sharples & S. Smirnov 1239 (COLO 553809), SRX4178269, Altai Republic, Russia; Stellaria brachypetala 2: G.I. Artemov 114 (NSK), SRX4178266, Altai Republic, Russia; Stellaria bungeana Fenzl: M. Sharples & S. Smirnov 1235 (COLO 553807), SRX4178267, Altai Republic, Russia; Stellaria graminea L.: M. Nee 52333 (NY 3327128), SRX4178264, Wisconsin, U.S.A.; Stellaria humifusa Rottbøll: M. Sharples 1190 (COLO 553805), SRX4178283, Finnmark, Norway; Stellaria irrigua Bunge ALT1: M. Sharples 1225, indi- vidual 1 (COLO 553793), SRX4178262, Altai Mountains, East Kazakhstan; Stellaria irrigua ALT2: M. Sharples 1225, individual 2 (COLO 553793), SRX4178263, Altai Mountains, East Kazakhstan; Stellaria irrigua ALT3: SRAE 2005057 (ALTB & COLO 553794), SRX4178251, Altai Mountains, Xinjiang, China; Stellaria irrigua ALT-Chuya1: M. Sharples & S. Smirnov 1250, individual 1 (COLO 553795), SRX4178252, Chuya Alps, Altai Republic, Russia; Stellaria irrigua ALT-Chuya2: M. Sharples & S. Smirnov 1250, individual 2 (COLO 553795), SRX4178253, Chuya Alps, Altai Republic, Russia; Stellaria irrigua HM: D. Boufford & al. 28756 (CAS 1009294), SRX4178254, Sichuan, China; Stellaria irrigua ID: J.F. Smith 7760 (NY 1191774), SRX4178247, Albion Mountains, Idaho, U.S.A.; Stellaria irrigua KM: S. Kharkevich/VLA Expedition 230 (NY), SRX4178248, Kamchatka Krai, Russia; Stellaria irrigua NCO1: M. Sharples & E. Tripp 1279, individual 1 (COLO 553797), SRX4178249, Never Summer Mountains, Colorado, U.S.A.; Stellaria irrigua NCO2: M. Sharples & E. Tripp 1279, individual 2 (COLO 553797), SRX4178250, Never Summer Mountains, Colorado, U.S.A.; Stellaria irrigua NCO3: M. Sharples & E. Tripp 1278 (COLO 553796), SRX4178258, Park Range, Colorado, U.S.A.; Stellaria irrigua NCO4: M. Sharples & E. Tripp 1277, individual 1 (COLO 553798), SRX4178259, Sawatch Range, Colorado, U.S.A.; Stellaria irrigua NCO5: M. Sharples & E. Tripp 1277, individual 2 (COLO 553798), SRX4178280, Sawatch Range, Colorado, U.S.A.; Stellaria irrigua SSJ1: M. Sharples 684 (COLO 553799), SRX4178279, South San Juan Mountains, Colorado, U.S.A.; Stellaria irrigua SSJ2: M. Sharples & E. Tripp 1301 (COLO 553792), SRX4178282, South San Juan Mountains, Colorado, U.S.A.; Stellaria irrigua SY1: D. Shaulo & I. Kovaleva 3427 (NSK), SRX4178281, western Sayan Mountains, Krasnoyarsk Krai, Russia; Stellaria irrigua SY2: D. Shaulo 4493 (NSK), SRX4178276, western Sayan Mountains, Tuva Republic, Russia; Stellaria irrigua SY3: D. Shaulo & N. Saaya 2019 (NSK), SRX4178275, western Sayan Mountains, Tuva Republic, Russia; Stellaria littoralis Torr.: M. Sharples 1158 (COLO 553804), SRX4178278, California, U.S.A.; Stellaria longifolia Muhl. ex Willd.: M. Sharples & E. Tripp 1318 (COLO 553800), SRX4178277, Colorado, U.S.A.; Stellaria longipes Goldie: M. Sharples & E. Tripp 1276 (COLO 553803), SRX4178274, Colorado, U.S.A.; Stellaria media (L.) Villars: M. Sharples 1280 (COLO 553801), SRX4178273, Colorado, U.S.A.; Stellaria nemorum L.: M. Sharples & R. Ufimov 1169 (COLO 553806), SRX4178260, Leningrad Oblast, Russia; Stellaria peduncularis Bunge: M. Sharples & S. Smirnov 1248 (COLO 553808), SRX4178261, Altai Republic, Russia; Stellaria sanjuanensis M.Sharples & E.Tripp Sangres: M. Sharples 897 (COLO 553788), SRX4178255, Sangre de Cristo Mountains, Colorado, U.S.A.; Stellaria sanjuanensis SSJ1: M. Sharples 969 (COLO 553832), SRX4178265, South San Juan Mountains, Colorado, U.S.A.; Stellaria sanjuanensis SSJ2: M. Sharples 1050 (COLO 553783), SRX4178272, South San Juan Mountains, Colorado, U.S.A.; Stellaria sanjuanensis SSJ3: M. Sharples 1070, individual 1 (COLO 553789), SRX4178256, South San Juan Mountains, Colorado, U.S.A.; Stellaria sanjuanensis SSJ4: M. Sharples 1070, individual 2 (COLO 553789), SRX4178257, South San Juan Mountains, Colorado, U.S.A.

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