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The flowers of primroses and relatives (family Primulaceae) have played a central role in our understanding of the form, function, genetics, and evolution of distyly (e.g. Darwin, 1877; Charlesworth & Charlesworth, 1979b; Ganders, 1979; Barrett, 1992; Richards, 2003). However, the common co-occurrence of distyly and monomorphy (the term is here used to describe populations or species fixed for a single floral morphology) in closely related groups within the family has made interpretation of the evolution of distyly difficult. Ninety-two per cent of the c. 430 species of Primula have distylous populations (Richards, 2003), as do members of the closely related genera Hottonia (one of two species), Dionysia (40 of 41 species), and Vitaliana (one of one species). The 45 species of Primula known to have monomorphic populations outside cultivation have been placed into 19 of the 38 sections (Richards, 2003; Table 1), and they share 18 of these 19 sections with distylous species. Here, we infer a chloroplast DNA (cpDNA) phylogeny for the family using the largest taxonomic and character sampling published to date and present the optimal states for key ancestral nodes, including the most recent common ancestor (MRCA) of Primula. This permits us to assess an important evolutionary hypothesis that some of the monomorphic species in Primula, particularly in Primula sections Proliferae and Sphondylia, represent a persistence of the monomorphy that existed before the evolution of distyly and thus perhaps reveal the suite of floral traits preceding that complex adaptation.
Table 1. Distribution of monomorphic taxa1 in Richards’ (2003) classification of Primula with presence (+) or absence (–) in sampling indicated
|Subgen. Sphondylia||+ P. eximia|
| Sect. Sphondylia||Sect. Cordifoliae|
| + P. verticillata||Sect. Fedtschenkoana|
| + P. simensis||Sect. Proliferae|
| + P. floribunda2||+ P. prolifera2|
|Subgen. Auriculastrum||+ P. chungensis2|
| Sect. Auricula||+ P. cockburniana|
| Sect. Cuneifolia||+ P. japonica|
| + P. cuneifolia ssp. saxifragifolia3||− P. miyabeana|
| Sect. Suffrutescens||+ P. prenantha|
| Sect. Amethystina||− P. polonensis|
| Sect. Parryi||Sect. Sikkimensis|
|Subgen. Primula||− P. morsheadiana|
| Sect. Primula||Sect. Oreophlomis|
| + P. vulgaris2||Sect. Armerina|
| Sect. Sredinskya||+ P. egaliksensis|
| + P. grandis||Sect. Glabra|
|Subgen. Auganthus||− P. macrocarpa|
| Sect. Auganthus||Sect. Yunnanensis|
| Sect. Monocarpicae||− P. homogama|
| Sect. Obconicolisteri||− P. clutterbuckii|
| + P. sinolisteri var. aspera3||Sect. Aleuritia|
| − P. filipes||− P. frondosa2|
| − P. dumicola||+ P. halleri2|
| − P. listeri||− P. scotica|
| Sect. Malvacea||+ P. scandinavica|
| Sect. Pycnoloba||− P. stricta|
| Sect. Reinii||+ P. incana|
| Sect. Cortusoides||+ P. laurentiana|
| + P. mollis||− P. magellanica|
| − P. septemloba||− P. yuparensis|
| Sect. Bullatae||Sect. Pulchella|
| Sect. Dryadifolia||Sect. Minutissimae|
|Subgen. Pinnatae||− P. annulata|
| Sect. Pinnatae||+ P. muscoides|
| + P. cicutariifolia||− P. subularia|
|Subgen. Carolinella||− P. praetermissa|
| Sect. Carolinella||Sect. Denticulata|
| − P. larsenii||Sect. Capitatae|
|Subgen. Aleuritia||Sect. Muscarioides|
| Sect. Chartacea||+ P. watsonii|
| Sect. Davidii||− P. concholoba|
| Sect. Petiolares||+ P. bellidifolia2|
| − P. hookeri||Sect. Soldanelloides|
| Sect. Crystallophlomis||− P. sherriffae|
In distylous plants, a genetic polymorphism produces two floral types (morphs) among individuals of a population. The two floral morphs have their anthers and stigma at reciprocal heights (reciprocal herkogamy; Webb & Lloyd, 1986). This structural difference is often accompanied by a sporophytically controlled, diallelic incompatibility system that makes intermorph crosses more successful than intramorph crosses. Reciprocal herkogamy reduces pollen wastage, and thus increases male fitness, whereas the diallelic incompatibility protects against self-fertilization and inbreeding depression, and thus increases female fitness (Barrett, 2002). Additional morphological features, such as pollen size and stigmatic papillae length, differ between floral morphs in Primula, Hottonia, and Dionysia (Darwin, 1877; Schaeppi, 1934; Wendelbo, 1961a,c; Richards, 2003), although not in Vitaliana (Schaeppi, 1934). These might serve to further promote intermorph pollination (reviewed in Dulberger, 1992). Distyly, and the functionally similar tristyly, are currently known from 28 families scattered throughout the angiosperm phylogeny, suggesting many independent origins (Barrett et al., 2000; Barrett, 2002).
Within Primula, distyly is thought to be controlled by at least three tightly linked genes (Dowrick, 1956), which are collectively referred to as the ‘heterostyly supergene’ (e.g. Ganders, 1979; Barrett, 2002). The style length, stigmatic papillae length, and female mating type are thought to be controlled by locus G, the pollen size and male mating type by locus P (or two separate loci Pp and Pm, respectively; Kurian & Richards, 1997), and the anther height by locus A. ‘Pin’ plants, with their stigma positioned high in the flower and their anthers low, are homozygous recessive for these loci (gpa/gpa); ‘thrum’ plants, with their stigma and anther positions reversed, are heterozygous (GPA/gpa; Bateson & Gregory, 1905; Lewis & Jones, 1992). The rarity of homozygote thrums might be attributable to the presence of recessive sublethal alleles linked to GPA (Mather & de Winton, 1941; Kurian & Richards, 1997; Richards, 1998). The loci are likely ordered GPA or GAP based on the frequency of observed recombinations (Dowrick, 1956; Lewis & Jones, 1992; Richards, 2003; cf. Charlesworth & Charlesworth, 1979a).
Monomorphy in members of Primula has been interpreted as either primitive (representing the condition before the origin of distyly in the genus; ‘primary homostyly’) or derived from distyly (‘secondary homostyly’). The most common route to monomorphy from distyly is thought to be via a recombination of the heterostyly supergene to produce the genotype gPA/Gpa with subsequent fixation of the gPA alleles in a population (Charlesworth & Charlesworth, 1979a). This produces a single, self-compatible morph with anthers at the high position of the thrum producing pollen that is the large size of the thrum and a stigma at the high position of the pin (‘long homostyle’). The term ‘homostyly’ has a long history in discussions of heterostyly (e.g. Darwin, 1877). Today, it is typically reserved for species or populations that have a single floral morphology, have their anthers and stigma at the same height in the flower, are thought to be derived from heterostylous ancestors, and/or are very closely related to extant heterostylous species (e.g. Ganders, 1979). Here, we use the term ‘monomorphy’ as a description of floral homogeneity in a population or species, as it does not imply a particular spatial relationship between anthers and stigma or a particular evolutionary scenario.
It has long been noted that there is differential reproductive success when interspecific crosses are made within and between morphs of two close distylous relatives (e.g. Darwin, 1877). It is also known that, upon crossing some monomorphic species with the two morphs of a close distylous relative, the monomorphic species will act as thrums when the pollen donor and pins when the pollen recipient in these crosses (e.g. Ernst, 1943), suggesting the persistence of a diallelic incompatibility system. This has been interpreted as evidence for recombination (Dowrick, 1956; Barrett & Shore, 1987; Wedderburn & Richards, 1992).
Ernst (1943, 1955) found that crosses between two monomorphic species (Primula cockburniana and Primula chungensis) and the two morphs of a close distylous relative (Primula pulverulenta) within Primula section Proliferae did not result in the differential success expected immediately after recombination. He concluded from this that these two species are primitively monomorphic. Although the predicted incompatibility responses are absent, these two monomorphic species are similar in morphology to what is expected if the typical recombinational route to monomorphy is taken (long style, anthers high in the corolla tube, and large pollen).
Examined species of Primula section Sphondylia (six of eight species) lack a diallelic incompatibility system (Al Wadi & Richards, 1993), and thus patterns in crossing success cannot be used to argue for the primitive or derived nature of monomorphy in the section. However, section Sphondylia displays a number of putatively primitive character states (e.g. inflorescences with superimposed whorls, and diploidy) that suggest an early divergence of the lineage (or lineages, if the section is not monophyletic) from other species in Primula before the origin of more derived character states (Al Wadi & Richards, 1993; Richards, 1993). Al Wadi & Richards (1993) assumed that two monomorphic species in the section, Primula simensis and Primula verticillata, are primitively so, ‘representing the original condition in the genus before distyly evolved, and that various distylous conditions in other species of the [section] can be considered to be representative of the evolutionary stages by which the distylous syndrome evolved’ (p. 337). In Al Wadi & Richards’ (1993) view, each species in the section is a ‘palaeo-endemic relict evolved from more widespread ancestors, which once ranged widely from eastern Africa to western India., [and] five species appear to represent isolates from a morphological and geographical continuum’ (p. 330). They asserted that interspecific comparisons involving these five species represent a complementary approach to previous theoretical work on the evolution of heterostyly (Charlesworth & Charlesworth, 1979b; Lloyd & Webb, 1992), and they constructed a scenario for it based upon what Richards (1993) called the ‘intermediate stages in the evolution of “full” heterostyly, “frozen” in evolutionary time’ (internal quotation marks his; p. 65). It is unclear whether Richards and Al Wadi preferred the view that the species of Sphondylia represent the intermediate steps in the origin of the distyly that is seen throughout Primula (e.g. last paragraph of Al Wadi & Richards, 1993), suggesting the paraphyly of the section with respect to the rest of the genus, or the view that they represent a build-up of distyly in section Sphondylia independent of the evolutionary origin(s) responsible for the distyly seen elsewhere in the genus (e.g. p. 65 in Richards, 1993). However, they clearly consider the monomorphic condition present in P. simensis and P. verticillata to represent the primitive combination of features for the genus, and thus a good starting point for a general scenario of the origin of distyly in Primula. Like P. cockburniana and P. chungensis from section Proliferae and monomorphic species that appear to have arisen following a recombinational route, P. simensis and P. verticillata have long styles, anthers high in the corolla tube, and large pollen.
In our interpretation of the Al Wadi & Richards (1993) scenario for the evolution of distyly, there are five major features that involve the introduction of new alleles to the heterostyly supergene.
The primitive condition for the genus is represented by P. simensis (in the Ethiopian highlands) and P. verticillata (in the south-west corner of the Arabian Peninsula). This involves a long style (g), large pollen size (P), anthers high in the corolla tube (A), and the absence of diallelic incompatibility.
The product of the first step, in which a dominant allele for short style length (G) arises in a population with genotype gPA, is represented by the thrum morph of Primula boveana (on the Sinai peninsula), Primula gaubeana (in west Iran), and Primula davisii (in south-east Turkey).
The product of the second step, in which a recessive allele for small pollen size (p) becomes linked to g in a population with genotypes of gPA and GPA, is represented by the other morph of P. boveana
[called the ‘pin’ morph by Al Wadi & Richards (1993
), although it has anthers high, rather than low, in the corolla tube].
The product of the third step, in which a recessive allele for low anther height (a) becomes linked to gp in a population with genotypes of GPA, gpA, and perhaps gPA, is represented by the pin morph of P. gaubeana and P. davisii.
The final step, the emergence of a diallelic incompatibility system, is represented by the remaining distylous species in the genus Primula.
Our focus here is on feature (1) of the scenario, and we will reserve detailed considerations of the remaining features of the scenario for elsewhere.
Recent DNA sampling in the Primulaceae has provided results critical to our understanding of the phylogeny and evolution of distyly and monomorphy in the family. Both cpDNA and nuclear DNA data suggest that Primulaceae, as traditionally circumscribed (e.g. Cronquist, 1981), is not monophyletic (Martins et al., 2003; Anderberg et al., 1998; Källersjöet al., 2000; Mast et al., 2001; Trift et al., 2002), and some genera – including Cyclamen, Lysimachia, and Samolus– are more closely related to Theophrastaceae and Mrysinaceae. With their extensive taxonomic sampling of cpDNA in the Primulaceae sensu stricto (s.str.), Mast et al. (2001) and Trift et al. (2002) inferred a bifurcation at the root of the family, with Androsace and genera nested in it (Vitaliana, Douglasia, and Pomatosase) sister to the remaining genera. Mast et al.'s (2001) sampling of the ribosomal protein L16 (rpl16) gene and tRNA-Leu (trnL) gene introns resolved a bifurcation in this latter clade that produced a clade of Primula and genera nested in it [Dionysia, Dodecatheon, Cortusa, and Sredinskya (treated as Primula grandis here)] sister to a clade formed by Hottonia, Omphalogramma, and Soldanella. Trift et al.'s (2002) sampling of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene resolved this latter clade as part of a polytomy with three clades of Primula and nested genera. The only extensive nuclear DNA sampling of the family to date (Martins et al., 2003; using the internal transcribed spacers of the nuclear ribosomal DNA) inferred poor support (≤ 62% bootstrap frequencies) for the relevant nodes, although it inferred strong support (100% bootstrap frequency) for the position of the previously unsampled monomorphic genus Bryocarpum, sister to the single sampled species of Omphalogramma.
The objectives of our study were (1) to infer the cpDNA phylogeny for an expanded taxonomic sampling within Primulaceae s.str. in order (2) to test by ancestral state inference the hypothesis that monomorphic members of Primula, particularly taxa in sections Sphondylia and Proliferae, are primitively monomorphic [i.e. represent lineages that diverged from the group before the origin(s) of distyly] and (3) to determine by ancestral state inference the number of origins of distyly in Primulaceae. We consider phylogenetic inference from cpDNA sequence variation to be a good starting point for estimating organismal relationships and character evolution in the family. However, wholesale acceptance of the cpDNA phylogeny as capturing all relevant evolutionary relationships is premature while fine-resolution taxonomic samplings of independent ‘linkage partitions’ (sensu Slowinski & Page, 1999) from the nuclear or mitochondrial genomes are unavailable. Comparison of independent linkage partitions can uncover biological processes (e.g. ancient hybridization and introgression) that would reduce the predictive utility of phylogenies inferred from any single linkage partition (Rieseberg & Soltis, 1991; Rieseberg & Brunsfeld, 1992).