Phylogenetic relationships and character evolution in Primula L.: The usefulness of ITS sequence data

ABSTRACT

The main goals of this research were to reconstruct the infrageneric phylogeny of the genus Primula based on both nuclear and chloroplast DNA sequences, and to use the resulting phylogenies to elucidate the evolution of breeding systems, morphological characters, chromosome number, and biogeographic distribution in the genus. In this paper, the results of a pilot study based on the nuclear ribosomal Internal Transcribed Spacer (ITS) region are described. ITS sequences from 21 taxa produced a number of variable characters sufficient to resolve relationships among sections. The resulting phylogeny confirmed the monophyly of sections Auricula and Aleuritia. Sections Armerina, Proliferae, Crystallophlomis, Parryi, and Auricula, with a base chromosome number of x = 11, and sect. Aleuritia, with a base chromosome number of x = 9, formed two well supported clades. The ITS topology also suggested that leaves with revolute vernation, previously believed to be a derived state, might represent the ancestral condition in Primula, with later reversals to the involute condition. Finally, this initial ITS tree provides preliminary support to the proposed role of the widespread, diploid and heterostylous P. mistassinica as having given origin to the polyploid, homostylous P. incana and P. laurentiana.     

Interspecific mating in the Piriqueta caroliniana (Turneraceae) complex: effects of pollen load size and composition

Two taxa of Piriqueta (P. caroliniana and P. viridis) form a broad hybrid zone that extends over much of the central Florida peninsula. We used genetic markers to examine the strength of the isolation barriers between these taxa and the patterns of mating at the initial stages of hybridization. Regression models were employed to analyze the effects of pollen load size and the proportions of intra- and interspecific pollen on the frequency of first-generation (F1) hybrid formation. Overall, the postpollination mating barriers between these two taxa were relatively weak. However, there were significant effects of pollen load composition and size on the patterns of hybridization in both taxa with frequency-dependent responses to composition in both taxa. The frequency of F1 hybrid formation was generally lower than expected based on the frequency of each pollen type on the stigma for P. caroliniana recipients. The lower frequencies of F1 seeds in this taxon were apparently due to a greater competitive ability for intraspecific pollen, since hybrid seed formation decreased with increasing pollen load size. Pollen from P. caroliniana donors was also competitively superior on P. viridis recipients, leading to higher than expected frequencies of hybrid seed formation. Pollen from P. caroliniana did suffer higher rates of pollen-tube attrition than intraspecific pollen on P. viridis recipients, so the frequency of hybrid seed formation would be lower when pellen load sizes were small. In general, reproductive isolation mechanisms were stronger in P. caroliniana, suggesting that introgression should occur into P. viridis when these taxa come into close contact. Comparison of these expected patterns of mating to the distribution of hybrid genotypes in Florida provides insights into the relative roles of mating and selection in the evolution of hybrid populations of Piriqueta.     

Breeding systems,hybridization and continuing evolution in Avon Gorge Sorbus

Background and Aims

Interspecific hybridization and polyploidy are key processes in plant evolution and are responsible for ongoing genetic diversification in the genus Sorbus (Rosaceae). The Avon Gorge, Bristol, UK, is a world ‘hotspot’ for Sorbus diversity and home to diploid sexual species and polyploid apomictic species. This research investigated how mating system variation, hybridization and polyploidy interact to generate this biological diversity.

Methods

Mating systems of diploid, triploid and tetraploid Sorbus taxa were analysed using pollen tube growth and seed set assays from controlled pollinations, and parent–offspring genotyping of progeny from open and manual pollinations.

Key Results

Diploid Sorbus are outcrossing and self-incompatible (SI). Triploid taxa are pseudogamous apomicts and genetically invariable, but because they also display self-incompatibility, apomictic seed set requires pollen from other Sorbus taxa – a phenomenon which offers direct opportunities for hybridization. In contrast tetraploid taxa are pseudogamous but self-compatible, so do not have the same obligate requirement for intertaxon pollination.

Conclusions

The mating inter-relationships among Avon Gorge Sorbus taxa are complex and are the driving force for hybridization and ongoing genetic diversification. In particular, the presence of self-incompatibility in triploid pseudogamous apomicts imposes a requirement for interspecific cross-pollination, thereby facilitating continuing diversification and evolution through rare sexual hybridization events. This is the first report of naturally occurring pseudogamous apomictic SI plant populations, and we suggest that interspecific pollination, in combination with a relaxed endosperm balance requirement, is the most likely route to the persistence of these populations. We propose that Avon Gorge Sorbus represents a model system for studying the establishment and persistence of SI apomicts in natural populations.     

Evolution of biogeographic patterns, ploidy levels, and breeding systems in a diploid-polyploid species complex of Primula

Primula sect. Aleuritia subsect. Aleuritia (Aleuritia) includes diploid, self-incompatible heterostyles and polyploid, self-compatible homostyles, the latter generally occurring at higher latitudes than the former. This study develops a phylogenetic hypothesis for Aleuritia to elucidate the interactions between Pleistocene glacial cycles, biogeographic patterns, ploidy levels and breeding systems. Sequences from five chloroplast DNA loci were analyzed with parsimony to reconstruct a phylogeny, haplotype network, and ancestral states for ploidy levels and breeding systems.The results supported the monophyly of Aleuritia and four major biogeographic lineages: an amphi-Pacific, a South American, an amphi-Atlantic and a European/North American lineage. At least four independent switches to homostyly and five to polyploidy were inferred. An Asian ancestor probably gave origin to an amphi-Pacific clade and to a lineage that diversified on the European and American continents. Switches to homostyly occurred exclusively in polyploid lineages, which mainly occupy previously glaciated areas. The higher success of the autogamous polyploid species at recolonizing habitats freed by glacial retreat might be explained in terms of selection for reproductive assurance.     

Intrapopulation variation in the breeding system ofVillarsia lasiosperma (Menyanthaceae), a distylous species

The restricted perennial diploid herb,Villarsia lasiosperma, of southwestern Western Australia has distylous flowers with dimorphism in style and stamen length, stigma morphology, and pollen size. In order to assess the presence and nature of an incompatibility system in this species, a crossing program was carried out using 17 plants grown from seeds collected in two field populations. Pollen stainabilities of these plants mostly exceeded 95%. Mean seed-set of Longs following intermorph pollinations was c. 24 seeds per pollination and for Shorts was c. 16 seeds per pollination. Approximately three-quarters of the Longs and Shorts produced no seeds after self-pollination; the remainder produced very few seeds following such pollinations. No Shorts produced seeds after intramorph cross-pollinations. However, about half the Longs exhibited various levels of weakened intramorph incompatibility, with mean seed-sets following intramorph cross-pollinations up to half the mean seed-sets following intermorph pollinations. In the behavior of Longs, the incompatibility system ofV. lasiosperma differs from the idealized distylous breeding system, but resembles that of a purported close relative,V. exaltata of eastern and southeastern Australia.     

Chromosome doubling inTurnera ulmifolia (Turneraceae) induced by regeneration of plants from in vitro cultured leaf explants

Plants were regenerated from cultured excised leaf segments ofTurnera ulmifolia (2n = 6x = 30). Cytological studies have demonstrated that chromosome doubling occurred in 100% of the regenerated plants. Probably it was produced by endomitosis, induced by excess of auxins in relation to cytokinins. High bivalent and low quadrivalent frequency, and univalents to octovalents were observed in metaphase I; lagging chromosomes were also found. Probably, the presence of multivalents may be due to the pairing among homoeologous chromosomes because the mother plant is a segmental allohexaploid. The high bivalent frequency may have been caused by preferential pairing of identical chromosomes against the homologous.     

Cytogeography of the South American Turnera sidoides L. complex (Turneraceae, Leiocarpae)

New chromosome number determinations are presented for 48 populations of five subspecies of Turnera sidoides. Chromosome counts in sspp. carnea, holosericea, integrifolia and sidoides confirm previous data while, in ssp. pinnatifida , the numbers 2n=2x = 14 and 2n = 6x = 42 are reported for the first time. The results show that polyploidy is a very frequent phenomenon. Populations are primarily tetraploids (2n = 4x = 28), while diploids (2n = 2x = 14), hexaploids (2n = 6x =42) and octoploids (2rc = 8x = 56) are less frequent. Diploid, tetraploid and hexaploid cytotypes occur in sspp. carnea and pinnatifida , whereas tetraploids and hexaploids are known in ssp. holosericea. Turnera sidoides ssp. integrifolia shows a polyploid series with ploidy levels from 2x to 8x. The ssp. sidoides appears to be uniformly tetraploid, with the exception of an isolated pentaploid (2rc = 5x = 35). On the basis of these counts and previous reports the geographical distribution of the cytotypes has been determined and related to climatic and ecological factors.     

Karyotype variation, evolution and phylogeny in Borago (Boraginaceae), with emphasis on subgenus Buglossites in the Corso-Sardinian system

BACKGROUND AND AIMS: Karyological variation in the Mediterranean genus Borago and cytogeography of subgenus Buglossites in Corsica, Sardinia and the Tuscan Archipelago were investigated in combination with a molecular phylogenetic analysis aimed at elucidating relationships between subgenera and taxa with different chromosome features. METHODS: Karyotype analysis was performed on population samples of B. pygmaea, B. morisiana, B. trabutii and B. officinalis. Phylogenetic analyses were based on ITS1 nrDNA and matK cpDNA sequences. KEY RESULTS: Four base numbers were found, x = 6, 8, 9 and 15, and three ploidy levels based on x = 8. In subgenus Buglossites the Sardinian endemic B. morisiana is diploid with 2n = 18, while B. pygmaea includes three allopatric cytotypes with 2n = 30 (Sardinia), 2n = 32 (southern Corsica) and 2n = 48 (central northern Corsica and Capraia). In subgenus Borago, the Moroccan endemic B. trabutii and the widespread B. officinalis have 2n = 12 and 2n = 16, respectively. Molecular data support the monophyly of Borago, while relationships in subgenus Borago remain unclear. Borago trabutii appears as the earliest divergent lineage and is sister to a clade with B. officinalis, B. morisiana and B. pygmaea. Subgenus Buglossites is also monophyletic, but no correspondence between ITS1 phylogeny and B. pygmaea cytotypes occurs. CONCLUSIONS: Chromosome variation in Borago is wider than previously known. Two base numbers may represent the ancestral condition in this small genus, x = 6 or x = 8. An increase in chromosome number and karyotype asymmetry, a decrease in chromosome size and heterochromatin content, and the appearance of polyploidy are the most significant karyological changes associated with the divergence of the Buglossites clade. High ITS1 variation in the tetra- and hypotetraploid races of B. pygmaea suggests a multiple origin, while the lower polymorphism of the hexaploid race and its allopatric distribution in the northernmost part of the range is better explained with a single origin via union of unreduced and reduced gametes.     

Heterostyly promotes compatible pollination in buckwheats: Comparisons of intraflower,intraplant, and interplant pollen flow in distylous and homostylous Fagopyrum

Premise of the Study

Heterostyly, the reciprocal positioning of stigmas and anthers in different floral morphs, has long been thought to promote intermorph pollination. However, extensive intramorph pollination occurs commonly in heterostylous species, leading to recurrent questions about the functional and evolutionary significance of heterostyly.

Methods

To identify the sources of stigmatic pollen (autogamous [intraflower], geitonogamous [intraplant], vs. interplant), we emasculated either one flower or entire plants in experimental populations of the two closely related buckwheat species, distylous Fagopyrum esculentum and homostylous F. tataricum. Differences in pollen size allowed unambiguous identification of pollen on stigmas.

Results

Only 2.4% of F. tataricum pollen and 1.5% of F. esculentum pollen arrived successfully on compatible stigmas of other plants. In the former (homostylous) species, 71.3% of the pollen load on stigmas was autogamous, 10.8% was geitonogamous, and 17.9% was interplant. In the latter (distylous) species, 37.45% of the pollen on stigmas was autogamous, 13.8% was geitonogamous, 17.0% was intramorph, and 31.75% was intermorph. The amount of incompatible pollen arriving on stigmas was greatly decreased by both one‐flower and whole‐plant emasculations, and thus, the proportion of compatible pollen deposited increased with one‐flower emasculation and increased even more with whole‐plant emasculation.

Conclusions

Our quantification of pollen‐donor sources in these two species indicated that heterostyly in Fagopyrum esculentum provided a nearly 2‐fold fitness advantage (in terms of compatible pollination) over expected (random) pollen transfers between morphs. Because of reduced herkogamy, the homostylous F. tataricum was highly autogamous.     

Heterostyly and the possibility of its breakdown by polyploidization

Heterostyly is a genetically controlled sexual polymorphism promoting outcrossing by animal pollinators. The occurrence of heterostyly in angiosperms was investigated, and 199 genera in 28 families in 15 orders were recognized as taxa that contain heterostylous species. A correlation between the occurrence of heterostyly and ploidy level was found in some limited groups in Primulaceae and Rubiaceae. In such taxa, individuals with a lower ploidy level tended to have heterostyly and individuals with a higher ploidy level tended to have monomorphic flowers. It appears reasonable that the first step of the breakdown of heterostyly was the recombination of the supergene in a diploid heterostylous plant, and then poyploidization incidentally occurred. Polyploidization itself does not necessarily appear to have direct effects on the breakdown of heterostyly.     

Male sterility, fitness gain curves and the evolution of gender specialization from distyly in Erythroxylum havanense

The evolution of dioecy from a monomorphic hermaphroditic condition requires two mutations, one producing females and one producing males. Conversely, a single mutation sterilizing one sexual function in one morph of distylous species would result in functional dioecy because such a mutation also affects the complementary function in the other morph. In this study, we tested these ideas with Erythroxylum havanense, a distylous species with morph-biased male sterility. Based on sex allocation theory we evaluated whether the invasion of thrum females is favoured over the maintenance of this morph cosexuals. Completely male sterile thrum plants obtained higher fitness returns than hermaphrodites or partial male sterile individuals of the same morph, thus favouring the invasion of female thrum plants. We concluded that because fruit production of pin individuals depends on the pollen produced by thrum plants, the invasion of thrum females would result on the evolution of functional dioecy.     

Breeding system of the threatened endemic Primula cusickiana var. maguirei (Primulaceae)

Maguire primrose (Primula cusickiana var. maguirei) is a threatened, narrow endemic found only along a 20‐km section within a single canyon in northern Utah, USA. Here we describe the breeding strategy of this perennial, distylous, primarily cliff‐dwelling species to test for reproductive constraints on the plants. Intermorph outcrossing treatments (i.e. between pin and thrum flowers) were the most successful hand pollinations performed, but those were usually inferior to natural pollination. Intramorph out‐crossing also was somewhat successful, but was consistently inferior to intermorph outcrossing. Seed‐set resulting from autogamy and geitonogamy treatments was least successful. Morph‐specific differences in seed set were observed for our hand pollinations, but not in natural pollination conditions. Our results suggest that pollinators play a crucial role by providing the pollen transfer necessary for maximal seed set in this rare and narrowly distributed distylous plant.     

Cytology and mating systems in the climbing cacti Hylocereus and Selenicereus

Chromosome numbers and meiotic behavior are reported for the climbing cacti species Hylocereus undatus, Hylocereus polyrhizus, and Selenicereus megalanthus. The Hylocereus spp. are diploid (2n = 22), while S. megalanthus is a tetraploid (2n = 44). Irregular chromosome disjunction at anaphase I in pollen mother cells of S. megalanthus is probably the major cause of its reduced pollen viability and may contribute to low seed set, low number of viable seeds and, consequently, low fruit mass. A pollination study confirmed self-incompatibility in H. polyrhizus and a weakened incompatibility reaction in H. undatus and S. megalanthus. Major crossability barriers do not exist between the Hylocereus spp. investigated. Reciprocal intergeneric crosses were successful between Hylocereus spp. and S. megalanthus, suggesting that an Hylocereus sp. might be one of the diploid progenitors of the tetraploid S. megalanthus. The implications of the results on cacti nomenclature and systematics are briefly discussed.