Morphometric Analysis and Synopsis of Pseudognaphalium (Gnaphalieae, Asteraceae) in North America

: Pseudognaphalium Kirp. (Asteraceae, Gnaphalieae) consist of about 60 species mainly distributed in South, Central, and North America. As a fi rst contribution toward a comprehensive taxonomic review of Pseudognaphalium, we perform here the fi rst morphometric analysis of North American species, using UPGMA method for the construction of the dendrogram. Based upon these results we present a synopsis including a key to identify species and their associated synonymy. Thirty-seven species are recognized, two taxa are newly synonymized, Pseudognaphalium microcephalum under the name P. canescens and Pseudognaphalium semilanatum under the name P. semiamplexicaule , and two other names are confi rmed as synonyms as previously proposed, Pseudognaphalium micradenium as a synonym of P. helleri and Pseudognaphalium crenatum as a synonym of P. viscosum . Lectotypes are newly designated for Gnaphalium beneolens, G. berlandieri (= Pseudognaphalium stramineum ) , Gnaphalium decurrens (= Pseudognaphalium macounii ) , G. leucocephalum, G. oxyphyllum, G. oxyphyllum var. semilanatum (= P. semiamplexicaule ) , G. semiamplexicaule, G. sulphurescens (= P. stramineum ) , G. thermale, and second-step lectotypifi cations are proposed for G. helleri and G. wrightii (= P. canescens ). In addition, the fi rst illustrations of Pseudognaphalium helleri and P. semiamplexicaule , and a colour fi gure of P. canescens and P. beneolens emphasizing the results of the morphometric analysis are provided.


INTRODUCTION
Ps e u d o g n a p h al i u m K i r p. ( A s te ra cea e, Gnaphalieae) was segregated from Gnaphalium L. by Kirpichnikov & Kuprijanova (1950). Estimates of the number of species ranges between about 90 , Bayer et al. 2007) and 150 (Dillon 2005), and they are mainly distributed in South, Central, and North America, but some species also occur in Asia and Africa. However, as treated in recent revisions of the South American species (Freire et al. 2014a(Freire et al. , b, 2018, the number of taxa accepted in Pseudognaphalium has been reduced to ca. 60. The genus is mainly characterized by its herbaceous habitat and the presence of disciform, heterogamous capitula in clusters arranged in corymbs or panicles, the possession of monochromous phyllaries with a divided stereome, truncate style branches with apical sweeping hairs, achenes either glabrous or with short oblong myxogenic duplex hairs, and pappus bristles free at the base. Studies of detailed morphological characters  and phylogenetic analysis inferred from morphology (Anderberg 1991) and molecular data (Bayer et al. 2007, Ward et al. 2009, Smissen et al. 2011, Galbany-Casals et al. 2010, 2014 have been interpreted as providing support for recognizing Pseudognaphalium as a distinct genus. However, the most recent molecular phylogenies (e.g. Nie et al. 2016, Acosta Maindo & Galbany-Casals 2018 recover Pseudognaphalium as polyphyletic in two independent clades: a clade containing mainly North American species (Canada, United States of America, and Mexico), and the other clade including mainly South American species. The generitype, Pseudognaphalium oxyphyllum (DC.) Kirp., was included in the North American clade with the other North American species. Both clades were nested within the southern African HAP clade (Helichrysum, Anaphalis, Pseudognaphalium, etc.) showing a southern African origin of Pseudognaphalium (Nie et al. 2016).
Taxonomic treatments and catalogues of North American species of Pseudognaphalium were carried out by Espinosa-García (1985, and Villaseñor (2016) for Mexican species, and by Nesom (2006) for North American North Mexican species. These treatments include several taxonomically critical groups of closely related species, as well as polymorphic species with complex intraspecific variation. As an example, the species Gnaphalium beneolens Davison, G. microcephalum Nutt., G. thermale E.E.Nelson, and G. wrightii A.Gray, were accepted by Ferris (1960), Munz & Keck (1959), and Munz (1968Munz ( , 1974. On the other hand, Cronquist (1950) recognized Gnaphalium thermale as a variety of G. microcephalum while  considered it as a subspecies. Stebbins & Keil (1992) and Stebbins (1993) merged all these species into a single polymorphic species, Gnaphalium canescens, recognizing G. beneolens, G. microcephalum, and G. thermale as subspecies, and placing G. wrightii into synonymy. Meanwhile, Nesom (2004) considered these four taxa as species under Pseudognaphalium, accepting Gnaphalium wrightii as a synonym of P. canescens. This taxonomic instability affects the identification of specimens of these and other species, and this prompted an investigation based on multivariate analysis to shed light on the delimiting morphologically close, infraspecific taxa or species classification. Cluster analysis and other multivariate techniques have been useful for solving taxonomic problems and circumscribing taxa from morphometric variability data. Several authors have used this type of study to identify entities based on morphology in diverse groups of plants (Owen et al. 2006, Lopez Laphitz et al. 2011, Grossi et al. 2011, Robbiati et al. 2014, Fernández et al. 2017.
The purpose of this study is to examine the current circumscription of the North American Pseudognaphalium taxa to define which species should be recognized based on the variation of morphological characters, including diagnostic characters, using cluster analysis for delimitation of species.

Sample collection
A total of 297 specimens (including type material), representing 41 of the 43 North American species of Pseudognaphalium recognized in previous studies (Nesom 2006, Villaseñor 2016, Villarreal-Quintanilla et al. 2020, were chosen for the morphometric analysis (Appendix 1). Only two species, Pseudognaphalium altamiranum and Gnaphalium oaxacanum could not be included in the analysis since no material was available from these species. Herbarium material was examined from G, GH, M, MEXU, MO, NY, S, and UC (acronyms following Thiers 2020). Type images at high resolution from BM, C, G-DC, GH, L, LINN, MEXU, MICH, NDG, OS, P, RM, TEX, UC, US, and WIS were also examined (http://plants.jstor.org/).

Morphological data and cluster analysis
We analyzed a total of 36 characters: 13 vegetative and 23 reproductive. A list of the characters and their states can be seen in the Table I. All characters used to separate the North American species of Pseudognaphalium by former authors were included in our analysis. The measurements were performed on mature specimens. All specimens were studied by direct observation and by using a WILD Heerbrugg M5-26799 stereoscope; measurements were taken using a calibrated ocular micrometer. The leafblade observations were limited to the midsection of flowering branches. Three florets per capitulum at same stage of anthesis from 2 or 3 capitula in each specimen were dissected. When the availability of specimens made it possible, replicates of three measurements for each character were obtained and the average was used in the ensuing statistical analyses.
We carried out a cluster analysis on all the OTUs. The similarity between two OTUs was calculated on the basis of Gower's general similarity coefficient (Gower 1971) and the UPGMA method was used for the construction of the dendrogram (Sneath & Sokal 1973).
The multivariate analyses with Gower's (1971) coefficient are suitable for the analysis of mixed characters, e.g., qualitative (binary and ordinal) and quantitative characters to generate a distance/dissimilarity matrix (Mapaya & Cron 2016). Likewise, UPGMA is an accurate and spread method to deduce similarity/dissimilarity among OTUs (Radford 1986, Ward 1993. The qualitative characters were coded accordingly as binary (presence/absence) or a code was assigned for each character state (see Table I for the list of characters and coding). Cluster analysis was performed on all the OTUs using all 36 characters, to obtain information about general relationships and similarities between them. The analyses were carried out using the software's PAST (Hammer & Harper 2006).

Concepts of species delimitation
The treatment here reported is based on the conservative and widely accepted morphological species concept. According to this concept, continuous variation of characters is allowed within the species, while discontinuous variation in more than one character define distinct species (Davies & Heywood 1967). Table I. Morphological characters and character states evaluated in the study.

RESULTS AND DISCUSSION
The cluster analysis (UPGMA) (Figure 1a) showed that most of the specimens of each species grouped together in separated clusters. A few specimens were intermingled in four main groups (Figure 1b), each with little metric distance suggesting high similarity between the species of each group.
T h e f i r s t g r o u p ( I ) i n c l u d e s Pseudognaphalium canescens (Figure 2a, b) and P. microcephalum. These species are morphologically similar with weakly discolorous leaves, which are oblanceolate, not decurrent and eglandular, and corymbiform capitulescences. Both are confined to United States of America and Mexico and are sympatric in part of their distributions, i.e. they are found together in Chihuahua (Mexico) and California (USA). Stebbins & Keil (1992), suggested that Pseudognaphalium beneolens (= G. beneolens), P. microcephalum (= G. microcephalum), and P. thermale (= G. thermale) could be treated as subspecies within a single polymorphic species, P. canescens (= G. canescens). Nesom (2004) considered these four taxa as separate species mainly by leaf base, shape of leaves, nature of tomentum, apex of phyllaries, capitulescence, number of florets, and size of capitula. Our results recovered P. beneolens and P. thermale in separate clusters. Pseudognaphalium beneolens (Figure 2c, d) is characterized by its linear to linear-oblanceolate leaves and paniculate capitulescences, and P. thermale characterized by its concolorous oblanceolate leaves, decurrent leaf base, and corymbiform capitulescences. However, the specimens of P. microcephalum and P. canescens appear intermingled. Consequently, based on this study, we consider Pseudognaphalium microcephalum as a synonym of P. canescens. T h e s e co n d g ro u p ( I I ) i n c l u d e s Pseudognaphalium semiamplexicaule (Figure 4a-g) from Mexico and Mesoamerica and P. semilanatum, endemic to Mexico. These two species have discolorous lanceolate leaves, clasping leaf bases, white shiny phyllaries and corymbiform to paniculate capitulescences. Villaseñor (2016) accepted both names as valid species. Espinosa-García (2005) considered Pseudognaphalium semiamplexicaule as a valid species and P. semilanatum as a dubious species. According to Pruski (2018) the identity of Pseudognaphalium semilanatum would be based on its adaxially stipitateglandular leaves (vs. arachnoid-tomentose in P. semiamplexicaule). The present analysis shows specimens with glandular and eglandular leaves of Pseudognaphalium semilanatum and P. semiamplexicaule intermingled. Consequently, based on this study, we consider Pseudognaphalium semilanatum as a synonym of P. semiamplexicaule.
T h e t h i rd g ro u p ( I I I ) i n c l u d e s Pseudognaphalium hellerii (Figure 3 a-h) and P. micradenium from East of United States of America, characterized by narrow oblonglanceolate leaves. Pseudognaphalium hellerii (= Gnaphalium helleri) was mainly described based on its densely glandular-pubescent stems and obtuse phyllaries. According to Weatherby (1923), the identity of Pseudognaphalium micradenium (= Gnaphalium obtusifolium var. micradenium) was based on its glandular-puberulent stems (vs. glandular-villous in P. helleri), linear or linear-lanceolate leaves (vs. leaves oblonglanceolate in P. helleri), and acute phyllaries (vs. obtuse phyllaries in P. helleri). Mahler (1975), considered Pseudognaphalium micradenium (= Gnaphalium obtusifolium var. micradenium) as a variety of P. hellerii (= G. helleri; i.e. G. helleri var. micradenium). Cronquist (1980) distinguished Pseudognaphalium obtusifolium (= Gnaphalium obtusifolium) from P. helleri (= G. helleri, including G. obtusifolium var. micradenium as its synonym) by its wooly pubescence (vs. principally glandular in P. helleri). Nesom (2006) considered Pseudognaphalium obtusifolium, P. helleri, and P. micradenium as separate species, using leaf shape, pubescence, and the number of florets as a way to distinguish them. The present cluster analysis shows the specimens of Pseudognaphalium obtusifolium in a separate cluster, but those of P. helleri and P. micradenium appear intermingled. Consequently, based on this study, we consider Pseudognaphalium micradenium as a synonym of P. helleri as was previously suggested by Cronquist (1980). T h e f o u r t h g ro u p ( I V ) i n c l u d e s Pseudognaphalium crenatum from Mexico and P. viscosum, from United States of America (Texas), Mexico, and Mesoamerica, characterized by its stipitate-glandular stems, linear-lanceolate leaves, white shiny phyllaries, and numerous pistillate florets. Villaseñor (2016) accepted Pseudognaphalium crenatum as a valid species, but McVaugh (1984) and Pruski (2018) placed P. crenatum into synonymy of P. viscosum. The present cluster analysis shows the type specimen of P. crenatum grouped together with the specimens of P. viscosum and confirms previously proposed synonymy.
Therefore, in agreement with our results Pseudognaphalium consists of 37 North American species (Figures 1a, b), and the corresponding synonyms for the three newly circumscribed species are indicated in the taxonomic treatment provided below.

Taxonomic Treatment of Pseudognaphalium in North America
Ps e u d o g n a p h a l i u m K i r p . (1 9 5 0 : 3 3 ) .
Worldwide distribution, mostly American, some African and Asian species. About 60 species of which 37 grow in North America.   2. According to the protologue, Gnaphalium microcephalum was based on the specimen 'St. Diego, Upper Californiaʼ. We found at BM, where the original herbarium of Nuttall is mainly deposited, the sheet Nuttall s.n. BM 001010942, which is in accordance with the protologue and matches the locality. Since Nuttall mentioned in the protologue '(I have seen but a single specimen)ʼ this specimen is almost surely the holotype. There are other two collections on the same sheet BM 001010942, i.e. Suksdorf s.n. and Macoun s.n. 3. Nesom (2004): 782 indicated the type of Gnaphalium wrightii A.Gray as 'Type. U.S.A. Texas [El Paso or Hudspeth Co.] valley between El Paso and the Guadalupe Mts., Oct [1849], C. Wright 394 (Lectotype, designated here: GH!; Isolectotypes: GH!, US!)ʼ. This constitutes a valid [first-step] lectotypification. Since we located two sheets of this collection at GH, 'secondstep' lectotypification is required (ICN, Art. 9.17. Shenzhen Code;Turland et al. 2018). The specimen GH 00008314 which presents the most complete plant is here designated as the lectotype of G. wrightii. Furthermore, this is the specimen that was labelled as the 'lectotype' by Kittredge in 2009, and shows that Nesom did not annotate either of the two sheets in GH. 4. The types of new species described by Osterhout were deposited in his private herbarium and, upon his death, all his sheets were bequeathed to the Rocky Mountain Herbarium in Laramie, Wyoming. Consequently, the only collection kept at RM (RM 0001102) is considered as the holotype of Gnaphalium albatum. Distribution: Mexico and United States of America. 11. Pseudognaphalium chartaceum (Greenm.) Anderb. (Anderberg 1991: 147). Gnaphalium chartaceum Greenm. (Greenman 1904: 95  which bears the number '2' written in pencil in the top right hand corner, as the lectotype of Gnaphalium semiamplexicaule, since this is the only one that bears the annotations provided by Candolle. 2. Candolle (1838: 225) mentioned 'β. semilanatum, foliis subtùs albo-lanatis, ̶ Cum var. α mixtum in pl. Mendezianis. (v.s.)ʼ. We located at G-DC, where the original herbarium of Candolle is deposited, the collection ' Villalpando, au Sud est de Guanajuato, J. Mendez s.n. ʼ G-DC G-00469505 of four sheets (see observation of Pseudognaphalium oxyphyllum). On sheet 1 and 2 the three plants on right have discolorous lanceolate leaves, with abaxial surface white lanate. We propose the sheet 1-G-DC G-00469505