Karyological variation, reproductive isolation, morphological differentiation and geographic distribution of the cytotypes of Centaurea phrygia were investigated in Central Europe. Occurrence of two dominant cytotypes, diploid (2n = 22) and tetraploid (2n = 44), was confirmed and additionally triploid, pentaploid and hexaploid ploidy levels identified using flow cytometry. Allozyme variation as well as morphological and genome size data suggest an autopolyploid origin of the tetraploids. Crossing experiments and flow cytometric screening of mixed populations revealed strong reproductive isolation of the cytotypes. Multivariate morphometric analysis revealed significant differentiation between the cytotypes in several morphological characters (pappus length, length and colour of appendages on involucral bracts, involucre width). The cytotypes have a parapatric distribution with only a small contact zone: diploids occupy the whole of the Central and North European geographic range of the species except for the major part of the Western Carpathians, whereas tetraploids are confined to the Western Carpathians and adjacent areas, both cytotypes co-occurring only in a limited area of intra-montane basins of the Western Carpathians. Based on this array of data, taxonomic treatment of the cytotypes as autonomous species is proposed. The name Centaurea phrygia is applied to the diploids and the name C. erdneri belongs to the tetraploids; nomenclature of hybrids with C. jacea is also resolved.
Laboratory and field tests support the "voltinism-suitability hypothesis" of host selection at various latitudes as well as in local "cold pockets": The best hosts for rapid development will be selected by herbivorous insects under severe thermal constraints for completion of the generation before winter. Papilio canadensis and P. glaucus females do select the best hosts for rapid larval growth in Alaska and in southern Michigan, but not in northern Michigan and southern Ohio. In addition to latitudinal patterns, local host preferences of P. canadensis are described in relation to "phenological twisting" of leaf suitability for larval growth in cold pockets with "thermally constrained" growing season lengths. White ash leaves (Fraxinus americana) have the highest nutritional quality (relative to cherry, aspen, birch, and other local trees) throughout June and July for P. canadensis populations inside the cold pocket, but not outside. In all areas outside the cold pockets, even with bud-break occurring much later than other tree species, ash leaves rapidly decline in quality after mid-June and become one of the worse tree host species for larvae. This temperature-driven phenology difference creates a geographic mosaic in host plant suitability for herbivores. However, we also report here that the cold pocket of northern Michigan has basically disappeared since 1998. Implications of these changes are discussed in relation to voltinism and host plant selection.
Various physiological and ecological adaptations to thermal unit constraints are evident with both species of naturally hybridizing butterflies (P. canadensis to the North and P. glaucus to the South). The rapid regional climate warming since 1998 in the Great Lakes region has allowed us to document some critical aspects of gene flow via analysis of independent "species-diagnostic" trait clines (including morphological, biochemical, and physiological characters). Narrower black bands on hind-wings, larger fore-wings, larval detoxification abilities for tulip tree leaves, behavioral preference of tulip tree leaves, and species-diagnostic allozymes such as PDG (x-linked) and HK (autosomal) provide evidence of these genodynamics. Laboratory hybridization studies (providing known reference groups) and field observations along the hybrid zone show that gene flow between P. glaucus and P. canadensis has recently been extensive across the historical hybrid zone.
Pollen viability was analysed causally between and within Central European Cirsium species and their hybrids to determine (i) how frequently hybrids are fertile and produce viable pollen; (ii) how the pollen viability of hybrids and their parents are related and how this is affected by the genetic distance between parents; (iii) how species promiscuity relates to species pollen viability; (iv) to what extent the pollen viability of a hybrid may predetermine its frequency in nature; (v) how the pollen viability of a hybrid and sympatricity of its parental species are related; and (vii) how the frequency of females in populations of gynodioecious species may affect the observed pollen viability. Altogether, the viability of 656,363 pollen grains was analysed using Alexander’s staining (1185 flowers from 301 plants from 67 field populations of 13 pure species and 1693 flowers from 345 plants from 96 field populations of 16 natural hybrids). The particular characters potentially related with pollen viability were estimated using following methods: natural hybrid frequency and species interfertility (by herbarium data), genetic distance (by AFLP), sympatricity (in local scale based on herbaria and literature data; on a global scale using the similarity between digitized maps of natural ranges). The strengths of pre- or postzygotic isolation were estimated for hybridizing species pairs using geographical data and pollen viability analyses. All hermaphrodite plants of the Cirsium hybrids had viable pollen, generally at lower levels than those found in pure species. The pollen viability of a hybrid generally decreased with increasing genetic distance between the parents and when the parental species had lower pollen viability. The pollen viability was decreased in frequently hybridizing species where occasionally individuals of pure species morphology may show decreased pollen viability. In some instances these might represent some unrecognized hybrid backcrosses. In populations of gynodioecious species where females co-occurred, pollen viability (in hermaphrodites)was also lower, indicating some degree of inbreeding depression. Hybrids between sympatric species exhibited higher post-pollination isolation (decrease of pollen viability), which suggests that the reproductive isolation had been increased by natural selection (effect similar to the Wallace effect). The strength of the postzygotic barrier (based on pollen viability) was generally stronger than that of the prezygotic barrier (based on distribution overlap) in studied hybridizing species pairs.