Introduction

The genus Mimulus (Phrymaceae) comprises more than 120 species, the majority (75%) of which occur in western North America, and the remaining having a world-wide distribution including Eastern North America, South America, Australia, the Himalayas, Japan and Madagascar (Grant 1924, Beardsley and Olmstead 2002, Wu et al. 2007). Species of Mimulus have been spread outside their native range due to deliberate and accidental introductions. For example, Mimulus guttatus, a native of western North America, is now found in New Zealand and more than 16 European countries (Truscott et al. 2008, van Kleunen and Fischer 2008, Tokarska-Guzik and Dajdok 2010). In some of these areas of introduction, Mimulus guttatus has become naturalized and widely distributed, forming a nontrivial component of the local flora (e.g. Wales, Northern England, Scotland, Poland, Germany and New Zealand; Roberts 1964, Stace 2010, Tokarska-Guzik and Dajdok 2010, Vallejo-Marin unpublished). In the United Kingdom (UK), naturalized populations of Mimulus are widespread (Preston et al. 2002), and the genus is represented here by three currently extant species (Mimulus guttatus, Mimulus luteus and Mimulus moschatus), and a complex array of interspecific hybrids, some of which are locally invasive (Stace 2010).

One of the most conspicuous hybridization complexes in the UK involves closely related taxa, of isolated geographic origin: the North American Mimulus guttatus DC. (2n = 28, 30, 56, with most North American and British plants 2n = 2x = 28, Mukherjee and Vickery 1962, Vickery 1995, Stace 2010), and the South American taxa Mimulus luteus L. (2n = 4x = 60, 61, 62) and Mimulus cupreus Dombrain (2n = 4x = 62) (Stace 2010). These taxa belong to Mimulus Section Simiolus Greene (= Erythranthe Section Simiola (Green) Nesom & Fraga; Barker et al. 2012). Crosses between Mimulus guttatus and Mimulus luteus/Mimulus cupreus yield sexually sterile triploids (2n = 3x = 44, 45, 46) that are nevertheless vegetatively vigorous (Roberts 1964, 1968, Stace 2010). In the UK, hybrids between Mimulus guttatus and Mimulus luteus/Mimulus cupreus have been grown since the 1800’s and some of them have become well established throughout the country. For instance, the hybrid between Mimulus guttatus and Mimulus luteus (= Mimulus × robertsii; Silverside 1990) escaped cultivation at least by 1872 (Preston et al. 2002), and currently forms numerous naturalized populations with a scattered distribution in the British Isles (Preston et al. 2002, Stace 2010, BSBI 2011).

Despite being widely distributed and having persisted in the UK for 140 years, the evolutionary fate of Mimulus guttatus × Mimulus luteus/Mimulus cupreus triploid hybrids has been thwarted by their high pollen- and seed-sterility (Mukherjee and Vickery 1962, Roberts 1964, 1968, McArthur 1974, Parker 1975). Sterility is common in hybrids produced by the merging of genetically differentiated genomes (Mallet 2007), including cases when parents have different chromosome numbers (Stebbins 1950, Stebbins 1958). When hybridization gives rise to viable triploids, these tend to generate high proportions of unbalanced, aneuploid, and usually non-functional gametes (Ramsey and Schemske 1998). However, sterile plant hybrids often recover fertility after genome duplication (Stebbins 1958). Polyploidization in interspecific hybrids — allopolyploidization — has been linked to the restoration of sexual fertility in some natural triploid hybrids (e.g., Senecio, Abbott and Lowe 2004).

Polyploidization plays a particularly important role in species formation, as chromosome doubling results in immediate and strong reproductive isolation between the derivative and parental species (Rieseberg and Willis 2007, Köhler et al. 2010). It is therefore not surprising that polyploidization is often thought to be fundamental to angiosperm diversification (Stebbins 1950, Grant 1971, Ramsey and Schemske 2002, Soltis 2005, Wood et al. 2009). In Mimulus, speciation by hybridization and polyploidization may have played an important role during the diversification of this group (Vickery 1995, Beardsley et al. 2004). For instance, allopolyploidization between diploid Mimulus guttatus and Mimulus nasutus has given rise to a widespread North American tetraploid taxon that is strongly reproductively isolated from its progenitors (Sweigart et al. 2008). Despite the importance of hybridization and polyploidization for plants in general, the opportunity to study early events in speciation via this route is limited by the small number of angiosperm species known to have originated via allopolyploidization in the last 150 years (e.g. Spartina anglica (Ayres and Strong 2001), Tragopogon mirus, T. miscellus (Soltis et al. 2004, 2012), Senecio cambrensis and Senecio eboracensis (Abbott and Lowe 2004)). The discovery of a recently formed polyploid hybrid species in the wild therefore would provide a window of opportunity to study the evolution and speciation of polyploid taxa.

In this note, I describe a new, fertile, polyploid (2n = 6x = 92) species of Mimulus (Phrymaceae), Mimulus peregrinus, which has currently been found in a single locality in the Lowther Hills, Scotland. A comparison of vegetative and reproductive morphology, DNA content, and chromosome number of this new polyploid species against other British Mimulus, strongly suggests a hybrid origin for Mimulus peregrinus and a close affinity with the sterile triploid hybrid Mimulus × robertsii. I speculate that Mimulus peregrinus may represent the hexaploid derivative of a hybrid between Mimulus guttatus and Mimulus luteus, although a careful examination of additional populations of both parental and hybrid taxa is required to elucidate the genetic origin, extent and distribution of this new polyploid species. If an allopolyploid origin is demonstrated, Mimulus peregrinus has the potential to serve as a study system to understand the evolutionary processes associated with the origin of species through hybridization and polyploidization following the breakdown of geographic barriers caused by human-assisted dispersal.

Methods

Field surveys in August 2011 uncovered the existence of fertile individuals in a large population of Mimulus × robertsii in South Lanarkshire, Scotland. To further investigate these unusual plants, open-pollinated seeds were collected on 27 August 2011 from multiple seed-bearing fruits in a single patch at Shortcleuch Waters, near Leadhills, South Lanarkshire, Scotland (NS 9029 1578; 55.4237°N, 3.7349°W). Field-collected seeds—accession number 11-LED-seed—were germinated and grown in a controlled environment cabinet (Microclima 1750E; Snijders Scientific, Tilburg, the Netherlands) at the University of Stirling under 16 light-hours at 24°C and 8 dark-hours at 16°C, and 70% constant humidity. Individual plants were grown in 0.37 l round pots, filled with general purpose peat-sand compost (Sinclair, Lincoln, Lincolnshire, UK), and kept on plastic trays with abundant water. Plants were sporadically treated with SB Plant Invigorator (Fargro Ltd, Littlehampton, West Sussex, UK) to control for fungal infections. Seven plants were brought to flowering (F₁ generation; 11-LED-seed-1 to 11-LED-seed-7), and each individual plant was used to generate F₂ offspring via manual self-fertilization of emasculated flowers kept inside the pollinator-free growth cabinet. A representative individual of this F₂ generation (11-LED-seed-2-14) was chosen as the holotype for the type description presented here (deposited at the Herbarium of the Royal Botanic Garden Edinburgh; E).

Pollen measurements were conducted using fresh pollen fixed in 1 ml of 70% ethanol and dyed with 50 μl of lactophenol-aniline blue (Kearns and Inouye 1993). Darkly stained grains were considered viable (Sweigart et al. 2006). Pollen diameter was measured at the widest point in expanded pollen grains using image analysis software (analySIS, Olympus Soft Imaging Solutions, Münster, Germany) at 200× magnification in an Olympus BX50 light microscope.

Stomata size was measured in casts obtained from the adaxiall side of healthy leaves. A negative cast was first obtained with polysiloxane precision impression material (Xantoprene VL Plus, Heraeus Kulzer Gmbh, Hanau, Germany), and a positive cast was then generated with quick-drying nail polish. Measurements of stomata length and width were done using a light microscope at 400×.

Chromosome counts were conducted by John Bailey (University of Leicester) in mitotic cells from root tips of two F₂ individuals (11-LED-seed-3-21 and 11-LED-seed-5-8). Genome size was measured using DAPI-stained nuclei analysed in a CyFlow ML flowcytometer (Partec GmbH, Münster, Germany) in a commercial facility (Plant Cytometry Services, Schijndel, The Netherlands) in six F₁ individuals (11-LED-seed-1 to 11-LED-seed-4, 11-LED-seed-6, 11-LED-seed-7). Vinca major was used as internal standard (2n = 92, 2C = 3.80 pg; Obermayer and Greihulber 2006). Because DAPI preferentially binds to AT-rich regions, the flow cytometry results presented here must not be treated as absolute measurements of DNA content.

Data resources

The data underpinning the analysis reported in this paper are deposited at GBIF, the Global Biodiversity Information Facility, http://ipt.pensoft.net/ipt/resource.do?r=mimulus_peregrinus

Taxonomic treatment Discussion

Mimulus peregrinus can be distinguished from closely related Mimulus species and their hybrids in the UK based on a number of morphological and functional characters (Table 1, Fig 2). Its chromosome number, DNA content, larger stomata and pollen grain size, clearly indicate that Mimulus peregrinus is a polyploid species. Although the parentage of this new polyploid has not been firmly established yet, its close affinity with Mimulus × robertsii suggest that Mimulus peregrinus has been derived from hybridization between Mimulus guttatus and Mimulus luteus and thus it might have arisen through a recent (<140 years) allopolyploidization event. Below I contrast Mimulus peregrinus with related Mimulus taxa in the UK, and end with a brief discussion on its putative origin.

Comparison with related Mimulus in Britain

1. Mimulus guttatus DC. (Section Simiolus Green) (yellow monkeyflower). Mimulus peregrinus has a more open corolla throat, in contrast to the nearly closed corolla throat of Mimulus guttatus. The 2–5 mm red spot in the central lower lip of Mimulus peregrinus, is absent in most British populations of Mimulus guttatus. The margins of the lower leaves of Mimulus peregrinus are more triangular and regular than those of most Mimulus guttatus, although leaf traits are highly variable in the genus. Field and herbarium specimens could potentially be distinguished by the much larger size of the pollen grains in Mimulus peregrinus. Chromosome number and genome content as measured in flow cytometry are also diagnostic characters to distinguish these two species (Table 1, Fig. 3).

2. Mimulus luteus L. (Section Simiolus Green) (blood-drop emlets). Mimulus luteus, is a group of polymorphic perennial herbs comprising several interfertile varieties that are distinguished based on the presence, size and colour of markings on the corolla lobes. Taxa in this group include Mimulus luteus var. rivularis Lindl. 1826, with a single large red spot on the middle lower lip; Mimulus luteus var. variegatus (Lodd.) Hook 1834, with pale yellow corollas tinted with pink at the lobe margins; and Mimulus luteus var. youngana Hook 1834 (= Mimulus smithii Lindl 1835, not Paxton), with deep yellow corollas and lobes with large red spots at the margins (Grant 1924). In the UK, many extant populations of Mimulus luteus likely represent crosses between taxa in this interfertile group (e.g. Mimulus luteus var. rivularis × Mimulus luteus var. variegatus) (MVM pers. obs.), and present highly variable patterns of spots and blotches in the corolla lobes. Mimulus peregrinus can be distinguished from most species and hybrids in the Mimulus luteus aggregate by its more robust habit, elliptical leaves with dentate and slightly irregular margins, and the presence of only a small, faint, elongated central spot in the lower lip. Most importantly, Mimulus peregrinus possesses simple hairs in the calyx, which are always absent from all varieties of Mimulus luteus. Other diagnostic characters of Mimulus peregrinus are pollen grain size, stomata size, DNA content as measured in flow cytometry and chromosome number (Table 1, Fig. 3).

3. Mimulus cupreus Dombrain (Section Simiolus Green) (copper monkeyflower). Mimulus cupreus with orange to yellow corollas, and which is closely related to Mimulus luteus, has been reported in the UK but most likely in error for the hybrid between Mimulus guttatus and Mimulus cupreus (Mimulus × burnetii S. Arn.) (Stace 2010). In contrast with Mimulus peregrinus, the copper monkeyflower usually has orange corollas, more open corolla throat, lacks simple hairs in the calyx, and has a smaller chromosome complement (2n = 62).

(3) Mimulus moschatus Douglas ex Lindl. (Section Paradanthus Grant) (musk). Mimulus moschatus is easily distinguished from other British Mimulus including Mimulus peregrinus by its smaller yellow corollas (1–2.5 cm), glandular-hairy pubescence throughout the plant, and chromosome number (2n = 4x = 32 × = 8, 9, 10, Vickery 1995). Mimulus moschatus does not hybridize with other British Mimulus.

(4) Mimulus × robertsii Silverside (Mimulus guttatus × Mimulus luteus). A highly pollen- and seed-sterile, perennial herb rooting at the nodes, its yellow flowers are marked with orange to red to brown spots of various sizes in the petal lobes (Roberts 1964, Silverside 1990, Silverside 1998, Stace 2010). The corolla is 2.5-4.5cm in length and the throat is partially open (Stace 2010). This is a taxon of variable pubescence, but is usually hairy in the upper parts of the plant (Stace 2010) including the inflorescences which present simple hairs in the base of the calyx (MVM pers. obs.). Of garden origin Mimulus × robertsii can occasionally arise in the wild; this hybrid is produced by crosses of Mimulus guttatus with Mimulus luteus var. rivularis, M. luteus var. variegatus or Mimulus × smithii Paxton (the latter a hybrid between Mimulus luteus var. rivularis and Mimulus luteus var. variegatus, which is phenotypically very similar to Mimulus luteus var. youngana) (Stace 2010). In the UK it can be found up to 610 m (Ochil Hills, Scotland), and is suggested to be the commonest taxon of high ground (Preston et al. 2002, Stace 2010).

Mimulus peregrinus resembles Mimulus × robertsii rather closely in habit, size and general vegetative and floral morphology, suggesting a close affinity between these two taxa (Table 1). Mimulus × robertsii and Mimulus peregrinus can be distinguished by their differences in chromosome number, pollen and seed fertility, pollen grain size, and stomata size (Table 1). Mimulus peregrinus presents consistently high levels of pollen fertility (0.86 ± 0.04) and is capable of producing abundant seed set following artificial pollination. In contrast, both natural and artificial specimens of Mimulus × robertsii present very low levels of pollen viability (proportion of viable pollen = 0.05 ± 0.01, for both naturalized (N = 7) and synthetic hybrids (N = 15)), and do not set seed following artificial pollination (Roberts 1964) (see also Table 1). In addition, the two taxa differ markedly in chromosome number: Mimulus × robertsii is a triploid (e.g. 2n = 45), while Mimulus peregrinus has twice as many chromosomes (2n = 92), and this difference in genome size is clearly seen in flow cytometry analysis of DAPI-stained nuclei (Fig. 3). Finally, associated with the different genome size of the two taxa, Mimulus peregrinus has larger pollen grains, larger seeds, and larger stomata than Mimulus robertsii (Table 1).

(5) Other hybrids. Mimulus × burnetii S. Arn. (Mimulus guttatus × Mimulus cupreus) is a sterile triploid (2n = 45) with copper-coloured corolla, and often presenting a petaloid calyx (Stace 2010). Mimulus × polymaculus Silverside nom. nud. (Mimulus guttatus × (Mimulus luteus × Mimulus cupreus)) is also a sterile triploid that differs from Mimulus × burnetii in having dark blotches in the corolla lobes. Both can be easily distinguished from Mimulus peregrinus based on corolla colour, calyx morphology, fertility, and chromosome number. Mimulus × maculosus W. Bull ex T. Moore (Mimulus cupreus × Mimulus luteus) and Mimulus × hybridus Siebert & Voss (Mimulus cupreus × Mimulus × smithii) are fertile hybrids with variably-coloured corollas, often copper-coloured or with blotches on the petal lobes. They can both be easily distinguished from Mimulus peregrinus by their corolla colours, lack of abundant simple hairs in the keels of the calyx, and evenly triangular, flat teeth in the leaf margins. Chromosome numbers for these latter two hybrids are not yet available, but it is to be expected that they are similar to their parental species (2n = 60-62).

Putative origin and distribution of Mimulus peregrinus

The intermediate floral and vegetative characteristics of Mimulus peregrinus between Mimulus guttatus and Mimulus luteus, as well as its close morphological similarity to Mimulus × robertsii clearly suggest a hybrid origin for this new taxon associated with a polyploidization event. The alternative, that Mimulus peregrinus is an autopolyploid derivative of a pure Mimulus guttatus or Mimulus luteus seems highly unlikely based on vegetative and floral characteristics of the different taxa (Table 1). Moreover, both chromosome counts and genome size data are inconsistent with the expectations of an early generation autopolyploid of either Mimulus guttatus or Mimulus luteus or a backcross between Mimulus × robertsii and either parent (Fig. 3). The fact that Mimulus peregrinus presents approximately twice the number of chromosomes and has double the amount of DAPI-staining DNA than a common cytotype of Mimulus × robertsii (Fig. 3), immediately suggests that the most parsimonious explanation for the origin of Mimulus peregrinus is through hybridization between Mimulus guttatus and Mimulus luteus linked to a polyploidization event. Given that Mimulus peregrinus was indentified amongst a large population of Mimulus × robertsii, a possible origin of this new taxon is via genome doubling of the triploid hybrid.

The known distribution of Mimulus peregrinus iscurrently restricted to a single locality in Scotland. A preliminary examination of herbarium specimens at the Royal Botanic Gardens in Edinburgh did not uncover any hybrid specimens that were obviously fertile. However, the widespread distribution of Mimulus × robertsii in the UK suggests, along with anecdotal records of fertility in hybrids (Silverside 1998), may suggest that Mimulus peregrinus could be significantly under recorded, and future studies are required to determine its actual distribution.

It is well known that polyploidization can act as a mechanism restoring fertility even in highly sterile triploid hybrids (Dobzhansky 1937, Stebbins 1950, Grant 1971, Ramsey and Schemske 1998, Briggs and Walter 2000), and polyploidization has resulted in the evolution of other non-native allohexaploid species from highly sterile triploids in the UK (e.g. Senecio cambrensis, 2n = 6x; Abbott and Lowe 2004). While firmly establishing the origin and distribution of Mimulus peregrinus must await further ecological and genetic work, the discovery of this taxon provides an exciting opportunity to study the recent evolution of a new allopolyploid British species.