Polyploidy and new chromosome counts in Pseudognaphalium Kirp. (Compositae: Gnaphalieae)

ABSTRACT

Pseudognaphalium Kirp. (Compositae: Gnaphalieae) comprises approximately 90 species and it has a worldwide but fragmented distribution. This study provides a total of 20 chromosome counts, nine of which are the first report for a species, and the rest are confirmation of previous reports. With the addition of these data to the previously published reports, the chromosome numbers for a total of 32 species of the genus Pseudognaphalium are now available. Considering all chromosome data available at present, c.22% of the species are diploids and c.78% are polyploids, mainly tetraploids. African and Asian species are predominantly diploid whereas the vast majority of the American species are tetraploid, suggesting that polyploidy played a key role in the colonization of the American continent by the genus Pseudognaphalium.

KEYWORDS:

• America
• Asteraceae
• chromosome number
• Compositae
• Gnaphalieae
• polyploidy
• Pseudognaphalium

Introduction

The genus Pseudognaphalium Kirp. (Compositae: Gnaphalieae) is one of the most species-rich genera of the tribe Gnaphalieae and it is constituted by annual, biennial or perennial herbs with clusters of capitula usually arranged in corymbiform or paniculiform synflorescences and with monochromous involucral bracts (Anderberg 1991; Hilliard and Burtt 1981; Hilliard 1983). It comprises approximately 90 species and has a worldwide but fragmented distribution. It includes African, Asian and American taxa, including some species endemic to the Caribbean and the Hawaiian archipelagos, and the subcosmopolitan Pseudognaphalium luteoalbum (L.) Hilliard & B.L. Burtt. However, the vast majority of the species are distributed in America (Anderberg 1991; Hilliard and Burtt 1981; Freire et al. 2014).

Existing chromosome data show that base chromosome number of Pseudognaphalium is x = 7, and the species mainly present 2n = 14 chromosomes (diploids) or 2n = 28 chromosomes (tetraploids). Additionally, the numbers 2n = 16, 2n = 20 and 2n = 40 have occasionally been reported (Table 1). Smissen et al. (2011) and Galbany-Casals et al. (2014), based on molecular phylogenies, hypothesized that Pseudognaphalium could have one or multiple allopolyploid origins. However, these authors highlighted the need for obtaining more chromosome counts and also more complete and well-resolved phylogenies to corroborate this hypothesis. Allopolyploidy is considered a significant mechanism in the evolutionary success of many species, in particular by enabling the colonization of new niches and the adaptation of polyploids to new habitats (Ramsey 2011; Mota et al. 2016). Also, a pattern of higher probability of long distance dispersal events and colonization of new areas has been observed in polyploid groups (Linder and Barker 2014), and several adaptive radiations in islands have been preceded by allopolyploidization (Barrier et al. 1999; Mandáková et al. 2010; Joly et al. 2009). The recurrent presence of polyploids in habitats or geographic areas different from those of their diploid progenitors suggests the ability of polyploids to colonize new environmental niches (Hegarty and Hiscock 2008). However, it may show also their difficulty in succeeding in areas in which the parental species occur (Thompson 2005; Ramsey 2011).

Table 1. Available chromosome counts for the genus Pseudognaphalium, with indication of the relevant publication references and of the geographic area of the species. North America here refers to the American part of the Boreal Kingdom. Central America here refers to the area from Guatemala and Belize to Panama.

Species	Geographic area	2n	References
Pseudognaphalium affine (D. Don) Anderb.	Asia	14	Arano 1956 [sub Gnaphalium affine D. Don]; King 1965^a [sub Gnaphalium luteoalbum subsp. affine (D. Don) Koster]; Shetty 1967 ^a [sub G. luteoalbum subsp. affine]; Hsu 1970 ^a [sub G. affine]; Karna Mallick et al. 2011 [sub G. affine]; Gupta et al. 2017 [sub G. affine]
Pseudognaphalium arizonicum (A. Gray) Anderb.	North America and Mexico	28*	This work
Pseudognaphalium attenuatum (DC.) Anderb.	Mexico and Central America	28	Turner and King 1964 ^a [sub Gnaphalium cf. attenuatum DC.]; Keil and Stuessy 1977 ^a [sub G. cf. attenuatum]; Lane and Li 1993 ^a [P. attenuatum var. sylvicola Hinojosa & Villaseñor, sub Gnaphalium schraderi DC.]^b
Pseudognaphalium beneolens (Davidson) Anderb.	North America	14	Keil and Pinkava 1976 ^a [sub Gnaphalium beneolens DC.]
Pseudognaphalium californicum (DC.) Anderb.	North America	28	Keil et al. 1988 ^a [sub Gnaphalium californicum DC.]
Pseudognaphalium canescens (DC.) Anderb.	North America and Mexico	28	Keil and Pinkava 1976 ^a [sub Gnaphalium canescens DC.]; Keil et al. 1988 ^a [sub G. canescens]; Carr et al. 1999 ^a [sub G. canescens]
		28*	This work
Pseudognaphalium chartaceum (Greenm.) Anderb.	Mexico	28	Keil and Stuessy 1977 ^a [sub Gnaphalium cf. chartaceum Greenm.]; Turner and Zhao 1992 [sub G. chartaceum]
		28*	This work
Pseudognaphalium cheiranthifolium (Lam.) Hilliard & B.L. Burtt	South America	28	Hunziker et al. 1990 ^a [sub Gnaphalium cheiranthifolium Lam.]; Carr et al. 1999 ^a [sub G. cheiranthifolium]
		28*	This work
Pseudognaphalium conoideum Anderb.	Mexico	28*	This work
Pseudognaphalium cymatoides (Kuntze ex DC.) Anderb.	South America	14*	This work
Pseudognaphalium dombeyanum (DC.) Anderb.	South America	28	Carr et al. 1999 ^a [sub Gnaphalium dombeyana DC.]
Pseudognaphalium elegans (Kunth) Kartesz	Mexico, Central and South America	28	Powell and King 1969 ^a [sub Gnaphalium cf. elegans Kunth]; Jansen et al. 1984 ^a [sub G. elegans]; Carr et al. 1999 ^a [sub G. elegans]
		28*	This work
Pseudognaphalium gaudichaudianum (DC.) Anderb.	South America	28	Hunziker et al. 1990 ^a [sub Gnaphalium gaudichaudianum DC.]
Pseudognaphalium graveolens (Kunth) Anderb.	South America	28	Powell and Cuatrecasas 1970 ^a [sub Gnaphalium graveolens Kunth]
Pseudognaphalium greenmanii (S.F. Blake) Anderb.	Mexico	28*	This work
Pseudognaphalium heterotrichum (Phil.) Anderb.	South America	28*	This work
Pseudognaphalium hypoleucum (DC.) Hilliard & B.L. Burtt	Asia	14	Arano 1963 [sub Gnaphalium hypoleucum DC.]; Bala and Gupta 2013 ^a [sub G. hypoleucum]
		28	Hsu 1970 ^a [sub G. hypoleucum]
Pseudognaphalium jaliscense (Greenm.) Anderb.	Mexico	28*	This work
Pseudognaphalium leucocephalum (A. Gray) Anderb.	North America, Mexico	28	Keil et al. 1988 ^a [sub Gnaphalium leucocephalum A. Gray]
Pseudognaphalium liebmannii (Sch. Bip. ex Klatt) Anderb.	Mexico and Central America	28	Beaman et al. 1962 [sub Gnaphalium vulcanicum I.M. Johnst.]
		28*	This work [P. liebmannii var. liebmannii]
		28*	This work [P. liebmannii var. monticola (McVaugh) Hinojosa & Villaseñor
Pseudognaphalium luteoalbum (L.) Hilliard & B.L. Burtt	Sub-cosmopolitan	14	Mehra et al. 1965 ^a [sub Gnaphalium luteoalbum L.]; Raj 1965 ^a [sub G. luteoalbum]; Gadella and Kliphuis 1966a [sub G. luteoalbum]; Turner 1970 ^a [sub G. luteoalbum]; Fernandes and Queiros 1971 [sub G. luteoalbum]; Love and Kjellqvist 1974 [sub G. luteoalbum]; Groves 1977 [sub G. luteoalbum]; Keil and Pinkava 1981 ^a [sub G. luteoalbum]: Kockx-Van Roon and Wieffering 1982^A [sub G. luteoalbum]; Ruíz De Clavijo 1990 ^a [sub G. luteoalbum]; Morton 1993 [sub G. luteoalbum]; Vogt and Oberprieler 1993; Dempsey et al. 1994 [sub G. luteoalbum]; Vogt and Oberprieler 1994; Razaq et al. 1994 ^a [sub G. luteoalbum]; Carr et al. 1999 ^a [sub G. luteoalbum]; Dawson and Beuzenberg 2000; De Lange et al. 2004 ^a; Gosavi et al. 2012 ^a [sub G. luteoalbum]; Bala and Gupta 2013 [sub G. luteoalbum]
		14*	This work
		16	Gill and Omoigui 1987 ^a [sub G. luteoalbum]
		20	Subramanian 1987 [sub G. luteoalbum]
		28	Vogt and Aparicio 1999
Pseudognaphalium melanosphaeroides (Sch. Bip. ex Wedd.) Anderb.	South America	28	Fernández-Casas and Fernández-Piqueras 1981 ^a [sub Gnaphalium melanosphaeroides Sch. Bip. ex Wedd.]
Pseudognaphalium microcephalum (Nutt.) Anderb.	North America	28	Powell et al. 1974 [sub Gnaphalium microcephalum Nutt.]; Morton 1981 [sub G. microcephalum]
Pseudognaphalium obtusifolium (L.) Hilliard & B.L. Burt	North America	28	Smith 1969 [sub Gnaphalium obtusifolium L.]
Pseudognaphalium oxyphyllum (DC.) Kirp. var. oxyphyllum	Mexico and Central America	28*	This work
Pseudognaphalium purpurascens (DC.) Anderb.	Mexico	40	Carr et al. 1999 ^a [sub Gnaphalium purpurascens DC.]
Pseudognaphalium ramosissimum (Nutt.) Anderb.	North America	28	Keil and Pinkava 1976 ^a [sub Gnaphalium ramosissimum Nutt.]
Pseudognaphalium semilanatum (DC.) Anderb.	Mexico	28*	This work
Pseudognaphalium stramineum (Kunth) Anderb.	North America, Mexico and Central America	28	Sundberg and Dillon 1986 ^a [sub Gnaphalium stramineum Kunth]; Keil and Stuessy 1977 ^a [sub G. stramineum]
		28*	This work
Pseudognaphalium undulatum (L.) Hilliard & B.L. Burtt	Southern Africa and Madagascar	14*	This work
Pseudognaphalium viravira (Molina) Anderb.	South America	28	Spooner et al. 1987 ^a [sub Gnaphalium viravira Molina]
		28*	This work
Pseudognaphalium viscosum (Kunth) Anderb.	North America, Mexico and Central America	14	Turner et al. 1962 ^a [sub Gnaphalium leptophyllum DC.]
		14*	This work

*Newly obtained chromosome counts reported in the present work.

^aValue inferred from gametophytic count.

^bThis count was based on the herbarium specimen TEX 141,442, collected in Mexico. After examining it, we conclude it belongs to P. attenuatum var. sylvicola and not to G. schraderi, which does not occur in Mexico (Villaseñor 2016).

Given the reported variation in chromosome numbers in Pseudognaphalium and their probable significance in its origin and biogeographical history, it is essential to have a more complete picture of chromosome data for the genus, as chromosome number is only available for 22 Pseudognaphalium species, which represent c.24% of the species of the genus. In this context, the aims of this work are to provide new chromosome counts for Pseudognaphalium, to compile all chromosome number reports available, and to examine the distribution patterns of ploidy levels in the genus. These data will be essential for the interpretation of future phylogenetic works focused on the origin and biogeographic history of the genus.

Material and methods

Chromosome counts were made on mitotic metaphases using standard squash techniques. Seeds collected in the wild were used, and voucher specimens were deposited in the herbaria of the Botanical Institute of Barcelona (BC) and in the National Herbarium of Mexico (MEXU). For one species, seeds were taken from a specimen deposited in the herbarium of the University of Salamanca (SALA). Root-tip meristems were obtained by germinating seeds on wet filter paper in Petri dishes at approximately 20°C. Samples were pretreated with 0.05% colchicine for 2 h 15 min at room temperature. The material was fixed in 3: 1 v/v absolute ethanol: glacial acetic acid at 4°C. Meristems were hydrolysed in 5 M hydrogen chloride (HCl) for 50 min to 1 h at room temperature. They were then stained in 2% acetic orcein for a minimum of 3 h at room temperature. Squashes were made in 45% acetic acid. Photographs were taken through a Zeiss Axioscope (Oberkochen, Germany) compound microscope with a Jenoptik ProGres C3 (Jena, Germany) digital camera using the program ProGres Capture 7 (Jenoptik, Jena, Germany).

Published chromosome data for the genus Pseudognaphalium were retrieved from the “Index to chromosome numbers in Compositae” website (http://www.lib.kobe-u.ac.jp/infolib/meta_pub/G0000003asteraceae_e), “Index to plant Chromosome Numbers” website (http://www.tropicos.org/Project/IPCN) and Nesom (2006). Most of the original references cited in these online sources were later searched and consulted to cover one or more references for each of the chromosome numbers reported for every species.

Results

The newly obtained chromosome numbers for each species are given below along with some relevant notes. The species are listed in alphabetical order. The data on species habitat and geographic distribution were taken from Hilliard (1983), McVaugh (1984), Anderberg (1991), Espinosa-García (2005), Nesom (2006), Chen and Bayer (2011), Freire et al. (2014) and Hinojosa-Espinosa and Villaseñor (2014), and from our own field observations. The new chromosome counts obtained in this work are also included in Table 1, where all gathered information of previously published chromosome counts of the genus is compiled. We only cite the references that have been directly checked by us, which are representative of all the chromosome numbers documented for each species up to the present.

Pseudognaphalium arizonicum (A. Gray) Anderb.

This taxon is an annual herb distributed from southern USA to central Mexico. It grows in open woodlands, chaparral and in pine-oak forest clearings (Espinosa-Garcia 2005; Nesom 2006).

Mexico: Veracruz, road 140, between Las Vigas de Ramírez and La Joya, 19°37ʹ35.2ʺ N, 97°4ʹ20.8ʺ W, 2412 m, 1 September 2014, Galbany GC-2470–2 & Arrabal (BC).

2n = 28 (Figure 1). This is the first chromosome count for this species, which is shown to be tetraploid.

Figures 1–6. Somatic metaphases in Pseudognaphalium. (1) P. arizonicum, 2n = 28. (2) P. canescens, 2n = 28. (3) P. chartaceum, 2n = 28. (4) P. cheiranthifolium, 2n = 28. (5) P. conoideum, 2n = 28. (6) P. cymatoides, 2n = 14. Scale bars = 20 μm.

Pseudognaphalium canescens (DC.) Anderb.

This species is an annual herb, distributed from the south of the USA to central Mexico. It occurs in lava beds, rocky sites, grasslands, scrublands, oak, pine-oak, and pine woodlands, often on slopes (McVaugh 1984; Espinosa-García 2005; Nesom 2006).

Mexico: Mexico City, Tlalpan, Ajusco Medio, ca. Km 5 of the Picacho-Ajusco road, about 150 meters south of the park Ecoguardas and the Tlalpan Police Subdelegation, 19°16ʹ16ʺ N, 99°12ʹ21ʺ W, 2610 m, 13 December 2014, Hinojosa-Espinosa 590 (MEXU).

2n = 28 (Figure 2). Our result agrees with previous gametophytic counts for this tetraploid species (Keil and Pinkava 1976; Keil et al. 1988; Carr et al. 1999), but this is the first mitotic count.

Pseudognaphalium chartaceum (Greenm.) Anderb.

This species is an annual or biennial herb endemic to Mexico (Espinosa-García 2005; Villaseñor 2016). It occurs on forested slopes, hills, ravines, forest clearings, and roadsides, mostly in tropical deciduous forests and oak or oak-pine forests (McVaugh 1984).

Mexico: Mexico City, “Bosque de Tlalpan”, Las Antenas road, at 500 m to the north of the entrance located in De las Torres Avenue, Colonia Miguel Hidalgo (2nd Section), 19°17ʹ21ʺ N, 99°11ʹ44ʺ W, 2410 m, 14 December 2014, Hinojosa-Espinosa 592 (BC, MEXU).

2n = 28 (Figure 3). Our mitotic chromosome count of this tetraploid species agrees with that of Turner and Zhao (1992) and with the gametophytic count of Keil and Stuessy (1977).

Pseudognaphalium cheiranthifolium (Lam.) Hilliard & B.L. Burtt

This taxon is a perennial herb distributed in southern South America, where it grows on rocky and sandy soils (Freire et al. 2014).

Chile: Valparaíso Region, Valparaiso, Quintero, Ritoque beach, rocky spur at the north end of the beach, 32°49ʹ36.7ʺ S, 71°31ʹ45.5ʺ W, 10 m, 18 November 2015, Galbany GC-2513 & Arrabal (BC).

2n = 28 (Figure 4). Our mitotic count for this tetraploid species is the first one from Chilean material and is consistent with previously published gametophytic counts based on material from Argentina (Hunziker et al. 1990; Carr et al. 1999).

Pseudognaphalium conoideum Anderb.

This taxon is an annual herb distributed in Mexico, where it appears in disturbed areas, meadows and crops (Espinosa-García 2005).

Mexico: State of Mexico, around Lagunas de Zempoala, 19°2ʹ52.7ʺ N, 99°19ʹ25.5ʺ W, 2887 m, 17 August 2014, Galbany GC-2443, Magallón & Arrabal (BC).

2n = 28 (Figure 5). This is the first chromosome count for this tetraploid species.

Pseudognaphalium cymatoides (Kuntze ex DC.) Anderb.

This species is an annual or biennial herb distributed in South America, particularly in Bolivia, Argentina and Chile (Freire et al. 2014).

Chile: Región IV, Coquimbo, arriving to Sotaquí from Monte Patria or Combarbalá, Quebrada Seca, 30°39ʹ29.1ʺ S, 71°05ʹ15.8ʺ W, 98 m, 20 November 2015, Galbany GC-2526 & Arrabal (BC).

2n = 14 (Figure 6). Our chromosome count is the first one for the species. It is diploid, which seems to be rather exceptional in the American species of the genus Pseudognaphalium.

Pseudognaphalium elegans (Kunth) Kartesz.

This taxon is distributed in Mexico, Central America and South America. It grows in volcanic beds, disturbed areas and forest clearings.

Mexico: Veracruz, road 140, between Las Vigas de Ramírez and La Joya, 19°37ʹ35.2ʺ N, 97°4ʹ20.8ʺ W, 2412 m, 1 September 2014, Galbany GC-2471–2 & Arrabal (BC).

2n = 28 (Figure 7). Our report is the first mitotic count for this tetraploid species, and the first count from Mexican material. It is consistent with previous gametophytic counts based on material from Colombia (Powell and King 1969; Jansen et al. 1984) and Venezuela (Carr et al. 1999).

Figures 7–12. Somatic metaphases in Pseudognaphalium. (7) P. elegans, 2n = 28. (8) P. greenmanii, 2n = 28. (9) P. heterotrichum, 2n = 28. (10) P. jaliscense, 2n = 28. (11) P. liebmannii var. liebmannii, 2n = 28. (12) P. luteoalbum, 2n = 14. Scale bars = 20 μm.

Pseudognaphalium greenmanii (S.F. Blake) Anderb.

This annual species is endemic to Mexico and occurs in oak-pine forests in steep grassy or rocky hills (McVaugh 1984).

Mexico: Jalisco, Zapopan, way to La Gotera, Pinar de la Venta, 20°42ʹ56.4ʺ N, 103°31ʹ29.0ʺ W, 1725 m, 20 August 2014, Galbany GC-2449–1, Arrabal & Juárez (BC).

2n = 28 (Figure 8). This is the first chromosome count for this tetraploid species.

Pseudognaphalium heterotrichum (Phil.) Anderb.

This taxon is a perennial herb endemic to Chile (Reiche 1903), where it grows in open shrublands with Cactaceae, in herbaceous communities of disturbed areas or in road margins.

Chile: IV Coquimbo Region, Coquimbo, Canela Baja, next to a chapel, 31°24ʹ29.3ʺ S, 71°28ʹ14.8ʺ W, 286 m, 19 November 2015, Galbany GC-2522 & Arrabal (BC).

2n = 28 (Figure 9). This is the first report for this tetraploid species. Freire et al. (2014) consider this species a synonym of Pseudognaphalium gayanum (J. Rémy) Anderb., for which there are no available chromosome counts.

Pseudognaphalium jaliscense (Greenm.) Anderb.

This annual plant is endemic to western and north-western Mexico, where it grows in meadows or grasslands, openings in oak-pine forests, often on roadsides or in disturbed places (McVaugh 1984).

Mexico: Jalisco, Zapopan, surroundings of the “Centro Universitario de Ciencias Biológicas y Agropecuarias”, 20°44ʹ22.5ʺ N, 103°30ʹ51.1ʺ W, 1647 m, 20 August 2014, Galbany GC-2446–1, Arrabal & Juárez (BC).

2n = 28 (Figure 10). This is the first chromosome count for this tetraploid species.

Pseudognaphalium liebmannii (Sch. Bip. ex Klatt) Anderb. var. liebmannii

This perennial herb is distributed in Mexico and Central America. It grows mostly at the timberline and above, occupying openings in pine forests, on grassy, rocky or gravelly slopes, in alpine grasslands and disturbed sites such as in volcanic debris (McVaugh 1984; Espinosa-García 2005).

Mexico: State of Mexico, Iztaccihuatl volcano, between Paso de Cortés and the parking area called “La Joya”, 19°5ʹ53.6ʺ N, 98°39ʹ7.2ʺ W, 3753 m, 6 September 2014, Galbany GC-2479 & Arrabal (BC) (Figure 11).

2n = 28 (Figure 11). Beaman et al. (1962) indicated Gnaphalium vulcanicum I.M. Johnst., which is a synonym of P. liebmannii var. liebmannii (McVaugh 1984), presents 2n = 28. Our chromosome count of P. liebmannii var. liebmannii is consistent with this previous count and confirms this species is tetraploid.

Pseudognaphalium liebmannii (Sch. Bip. ex Klatt) Anderb. var. monticola (McVaugh) Hinojosa & Villaseñor

This taxon is an annual or perennial herb and it is distributed in Mexico and Central America. It grows in clearings of fir forests, humid pine forests, and pine-oak forests, on rocky slopes and summits, and on roadsides and in disturbed areas (McVaugh 1984; Espinosa-García 2005; Hinojosa-Espinosa and Villaseñor 2014).

Mexico: State of Mexico, Iztaccihuatl volcano, between Paso de Cortés and the parking area called “La Joya”, 19°5ʹ38.3ʺ N, 98°39ʹ4.1ʺ W, 3710 m, 6 September 2014, Galbany GC-2476–3 & Arrabal (BC).

2n = 28. As in the previous case, our chromosome count agrees with that of Beaman et al. (1962); however, this is the first report for this variety.

Pseudognaphalium luteoalbum (L.) Hilliard & B.L.Burtt

This is an annual herb with a subcosmopolitan distribution, known from Europe, Asia, America, Australia, New Zealand and other Pacific Islands (Nesom 2006; Chen and Bayer 2011). It is considered introduced in most part of these areas and its original distribution is not known with certainty. It grows along roadsides, in fields and pastures, humid grasslands near streams and marshes, in gardens, river beds and disturbed areas (Hilliard 1983; Nesom 2006).

Mexico: Mexico City, “Ciudad Universitaria”, “Jardín Botánico”, 19°19ʹ5ʺ N, 99°11ʹ39.1ʺ W, 2327 m, 5 August 2014, Galbany GC-2411 & Arrabal (BC).

2n = 14 (Figure 12). Many studies have reported chromosome counts of this subcosmopolitan species (see Table 1). The most commonly found chromosome number is 2n = 14, which indicates it is diploid. Vogt and Aparicio (1999) reported the number 2n = 28, but to our knowledge this is the only report of a tetraploid level. This may indicate that the species can experience intraspecific polyploidy, or that the chromosome count might be an error. Unfortunately, the authors did not include an image of the chromosome count. Additional and exceptional chromosome numbers have been reported for P. luteoalbum: Gill and Omoigui (1987) reported n = 8, and Subramanian (1987) 2n = 20. None of these works contained an image of the chromosome count cited. These odd numbers have been found only once each. Thus, they could be considered as anomalous counts within the species, or perhaps errors. Finally, in the “Index to chromosome numbers in Compositae” database is a report of 2n = 18 (Fernandes and Queiros 1971). Actually, Fernandes and Queiros (1971) reported several counts of 2n = 14 for P. luteoalbum, but none of 2n = 18.

Pseudognaphalium oxyphyllum (DC.) Kirp. var. oxyphyllum

This annual herb is distributed in Central America and Mexico (Espinosa-García 2005). This species grows in clearings of oak or pine forests or in disturbed areas (Espinosa-García 2005).

Mexico: Mexico City, “Ciudad Universitaria”, close to “Ciudad Universitaria” metrobus station, between the station and the Pumabus stop, 19°19ʹ25.6ʺ N, 99°11ʹ19.8ʺ W, 2304 m, 8 September 2014, Galbany GC-2480 & Arrabal (BC).

2n = 28 (Figure 13). Our chromosome count is the first one for this tetraploid species.

Figures 13–18. Somatic metaphases in Pseudognaphalium. (13) P. oxyphyllum var. oxyphyllum, 2n = 28. (14) P. semilanatum, 2n = 28. (15) P. stramineum, 2n = 28. (16) P. undulatum, 2n = 14. (17) P. viravira, 2n = 28. (18) P. viscosum, 2n = 14. Scale bars = 20 μm.

Pseudognaphalium semilanatum (DC.) Anderb.

This taxon is an annual plant endemic to Mexico (Villaseñor 2016). It grows in rocky or steep mountainsides and summits, grasslands, open oak woodlands, pine or pine-oak forests, often in disturbed ground (McVaugh 1984).

Mexico: Veracruz, road 140, between Perote and Las Vigas de Ramírez, a little after passing Cruz Blanca, 19°38ʹ10.6ʺ N, 97°9ʹ9.1ʺ W, 2440 m, 1 September 2014, Galbany GC-2469 & Arrabal (BC).

2n = 28 (Figure 14). This is the first chromosome count for this tetraploid species. García-Espinosa (2005) stated that this species may be synonym of Pseudognaphalium semiamplexicaule (DC.) Anderb. or that both could be part of a complex of closely related species. McVaugh (1984) recognized it as a separate species closely related to P. oxyphyllum, P. semiamplexicaule, P. canescens and Pseudognaphalium roseum (Kunth) Anderb.

Pseudognaphalium stramineum (Kunth) Anderb.

This species is an annual or biennial plant, widely distributed in North America, Mexico and Central America. It occurs in a wide variety of habitats, such as humid fields and stream banks, dunes, chaparral slopes, roadsides or disturbed places (Espinosa-García 2005; Nesom 2006).

Mexico: State of Mexico, around Xalatlaco, 19°9ʹ29.2ʺ N, 99°24ʹ6.8ʺ W, 2850 m, 9 August 2014, Galbany GC-2430 & Arrabal (BC).

2n = 28 (Figure 15). Our report is the first mitotic count for this tetraploid species, which is consistent with the previous gametophytic counts (Keil and Stuessy 1977; Sundberg and Dillon 1986).

Pseudognaphalium undulatum (L.) Hilliard & B.L. Burtt

This species is an annual herb, naturally distributed in southern Africa and Madagascar, and naturalized in several parts of Europe and North Africa. It grows on damp places, particularly around rock outcrops, on stream- and river-banks, or near forest margins or in open fynbos (Hilliard 1983).

South Africa: Matroosberg Private Reserve, Matroosberg, Ski Hut., 33°20ʹ46.7ʺ S, 19°37ʹ26.5ʺ E, 1219 m, 21 January 2014, Andrés-Sánchez SA827 & Smuts (SALA 156,613).

2n = 14 (Figure 16). Our report is the first one for this African diploid species.

Pseudognaphalium viravira (Molina) Anderb.

This species is a perennial herb distributed in Chile and Argentina (Freire et al. 2014), where it grows on dry soils in open shrublands, along disturbed roadsides with other herbaceous species, or in forest clearings.

Chile: IV Coquimbo Region, from Quilimarí (Los Vilos) inland by D-875 road, 32°07ʹ17.4ʺ S, 71°27ʹ47.4ʺ W, 40 m, 19 November 2015, Galbany GC-2517 & Arrabal (BC).

2n = 28 (Figure 17). Our result is consistent with the gametophytic count of Spooner et al. (1987), but this is the first mitotic chromosome count for this species.

Pseudognaphalium viscosum (Kunth) Anderb.

This annual herb is distributed from the southern USA to Honduras (Espinosa-García 2005; Nesom 2006). It grows in pine or oak forest clearings, grasslands, fields, shrublands, often in rocky sites, disturbed areas or roadsides (Espinosa-García 2005; Nesom 2006).

Mexico: Mexico City, “Ciudad Universitaria”, “Jardín Botánico”, 19°19ʹ5ʺ N, 99°11ʹ39.1ʺ W, 2327 m, 5 August 2014, Galbany GC-2412–1 & Arrabal (BC); “Ciudad Universitaria”, next to the metrobus station called “Ciudad Universitaria”, 19°19ʹ25.6ʺ N, 99°11ʹ19.8ʺ W, 2304 m, 31 July 2014, Galbany GC-2408–1 & Arrabal (BC).

2n = 14 (Figure 18). We obtained the same chromosome diploid number for two different specimens. Our results are consistent with the gametophytic count of Turner et al. (1962) and are the first mitotic counts of 2n = 14. However, two counts of 2n = 28 also exist. The first one is from Canada (Chinnappa and Chmielewski 1987) and is clearly an error, given that P. viscosum is not present in that country. Nesom (2006) noted that reports of P. viscosum in states other than Texas are based on plants of Pseudognaphalium macounii (Greene) Kartesz, a very similar species that is widely distributed in the USA and Canada. In particular, P. macounii is present in British Columbia, where the material for that count was taken (Chinnappa and Chmielewski 1987). The other published tetraploid report is a gametophytic count based on material from Chihuahua, Mexico (Carr et al. 1999). We examined the voucher corresponding to this count [Chihuahua, 33.7 miles west of La Junta, 30 September 1989, R. M. King 9863 & P. M. Peterson (US 3,140,891)], and concluded it does not belong to P. viscosum, but to P. stramineum. This is consistent with the chromosome number obtained by us for P. stramineum in the present work, 2n = 28. Therefore, we note that the two reports of 2n = 28 for P. viscosum are errors and that this species is diploid with 2n = 14.

Discussion

This study includes a total of 20 chromosome counts, nine of which are the first report for a species, and the rest are confirmation of previous reports. With these data, the chromosome number is known for a total of 32 species of the genus Pseudognaphalium, c.35% of the species of the genus.

It is already known that Pseudognaphalium includes diploids and tetraploids (Smissen et al. 2011; Galbany-Casals et al. 2014). With chromosome data available at present, only seven species are diploids with 2n = 14 (c.22% of the species with available data) and 24 species are tetraploids with 2n = 28 (c.78% of the species with available data). Two of the diploid species, P. luteoalbum and P. hypoleucum (DC.) Hilliard & B.L. Burtt, are actually reported to present both ploidy levels, 2n = 14 and 2n = 28. Indeed, intraspecific polyploidy has been reported for many plant species (e.g. Gültepe et al. 2015; Meng et al. 2016). However, for the two mentioned species, the chromosome number 2n = 28 has only been reported once for each. Thus, additional counts of 2n = 28 would be desirable to confirm the tetraploid level in these two species. Finally, Carr et al. (1999) reported Pseudognaphalium purpurascens (DC.) Anderb. as 2n = 40. These authors interpreted this odd number as a dysploid derivate of the hexaploid based on x = 7 or alternatively a tetraploid based on x = 10. Hexaploids with base x = 7 (2n = 42) occur within Helichrysum (Febles 1989; Galbany-Casals and Romo 2008), a genus that is closely related to Pseudognaphalium. These data, and the fact that all New World Pseudognaphalium species with available chromosome data present the base number of x = 7, makes the hypothesis of a dysploid derivate of an hexaploid based on x = 7 more probable.

The chromosome data available in this study show that the African and Asian species are predominantly diploid, whereas the vast majority of the New World species are tetraploid. Considering Galbany-Casals et al. (2014) placed the origin of Pseudognaphalium in Africa, chromosome data available may indicate that the colonization of the American continent by this genus could have been stimulated or facilitated by one or more polyploidization events, the colonization routes being still unknown. Chromosome data presented here will help to disentangle the role of allopolyploidy in the colonization of new territories when a more complete phylogeny of Pseudognaphalium is provided.

Acknowledgements

M. G.-C. greatly appreciates Sebastián Arrabal’s help, enthusiastic support, dedication and joy in all the shared field expeditions. David Juárez and Sebastián Teillier provided very valuable help and advice for field trips and plant collections, and Santiago Andrés provided material from his field collections; all of them are gratefully acknowledged. We thank Susana Magallón for organizing the stay of M. G.-C. at the “Universidad Nacional Autónoma de México”. We thank the staff of the herbaria of “Institut Botànic de Barcelona” (BC, Neus Nualart), “Universidad de Concepción” (CONC, Alicia Marticorena), “Universidad Nacional Autónoma de México” (MEXU, David Gernandt and José Luis Villaseñor), Missouri Botanical Garden (MO, John Pruski), “Universidad de Salamanca” (SALA, Francisco Javier Hernández), “Museo Nacional de Historia Natural de Santiago de Chile” (SGO, Gloria Rojas), University of Texas at Austin (TEX, George Yatskievych), and Smithsonian Institution (US, Ingrid P. Lin) for kindly attending M. G.-C. and for providing specimens and/or institutional support for plant collections. Laia Guàrdia provided the microscope and camera, and the technical support necessary for using them. Joan Prunera provided technical support for some of the chromosome counts. Samuel Pyke is acknowledged for the English correction. Two anonymous reviewers contributed to improve the paper.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

M. G.-C. benefited from a “Systematics Research fund” from the Systematics Association and the Linnean Society of London and a “Beca Iberoamérica Jóvenes Profesores Investigadores” from “Banco Santander”, which partly financed the stays and field work in Chile and Mexico, respectively. The Catalan government (“Ajuts a grups consolidats” 2014-SGR 514) partly financed this work.