Richard I. Milne

The origin and evolution of tertiary relict floras

Abstract Tertiary relict floras contain survivors from plant communities that were distributed throughout a large part of the Northern Hemisphere during much of the Tertiary (i.e. 65 – 15 million years ago (Ma)). They are now mainly restricted to warm humid areas (refugia) in southeastern and western North America, East Asia and southwest Eurasia. Recent molecular phylogenetic studies show that within East Asia the Tertiary relict flora is best divided into two distinct refugial groups, with geographical distributions centred on the Japan/Korea/northeast China and southeast China/Himalayas regions respectively. Recognition of this division leads to a significant improvement in our understanding of the origins and evolution of Tertiary relict floras in East Asia and elsewhere. Molecular studies also indicate two putative clusters of divergence times for East Asian-North American Tertiary relict disjuncts occurring at 5 and 10 Ma. These clusters might reflect a break in the continuity of the Tertiary flora between East Asia and North America across Beringia during a cold period 6–8 Ma, i.e. before the Bering Land Bridge was severed approximately 5 Ma. In addition, there is some evidence that evergreen disjuncts diverged earlier than their deciduous counterparts, possibly due to the high latitude of Beringia. Molecular studies further suggest that divergence times for transatlantic Tertiary relict disjuncts generally fall between 10 and 40 Ma, even though most geological evidence shows that the North Atlantic Land Bridge (NALB), which connected the floras of Europe and North America, was severed around 50 Ma. This raises the issue of whether a partial NALB allowed migration of floras between Eurasia and North America throughout much of the Tertiary. Tertiary relict floras are notable for exhibiting slow morphological evolution (stasis). This might result from large-scale allopatric speciation, together with stabilising selection.

Origin and evolution of invasive naturalized material of Rhododendron ponticum L. in the British Isles

Information concerning the area of origin, genetic diversity and possible acquisition of germplasm through hybridization is fundamental to understanding the evolution, ecology and possible control measures for an introduced invasive plant species. Rhododendron ponticum is extensively naturalized in the British Isles, but it is not known whether native material in Turkey, Spain or Portugal gave rise to the naturalized material, or to what extent introgression has affected this material. Chloroplast (cp) and nuclear ribosomal DNA (rDNA) restriction fragment length polymorphisms (RFLPs) were sought which could distinguish between native material of R. ponticum, and between 15 other Rhododendron species including R. ponticums closest relatives. Thereafter, a total of 260 naturalized accessions of R. ponticum from throughout the British Isles was examined with respect to informative polymorphisms. It was found that 89% of these accessions possessed a cpDNA haplotype that occurred in native material of R. ponticum derived almost entirely from Spain, while 10% of accessions had a haplotype unique to Portuguese material. These results therefore indicated an Iberian origin for British material. rDNA or cpDNA evidence of introgression from R. catawbiense was found in 27 British accessions of R. ponticum, and such accessions were significantly more abundant in Britains coldest region, eastern Scotland, than elsewhere. This could indicate that introgression from R. catawbiense confers improved cold tolerance. Introgression from R. maximum and an unidentified species was also detected.

Diversification and biogeography of Juniperus (Cupressaceae): variable diversification rates and multiple intercontinental dispersals

• A central aim of biogeography is to understand when and how modern patterns of species diversity and distribution developed. Many plant groups have disjunct distributions within the Northern Hemisphere, but among these very few have been studied that prefer warm semi-arid habitats. • Here we examine the biogeography and diversification history of Juniperus, which occurs in semi-arid habitats through much of the Northern Hemisphere. A phylogeny was generated based on > 10,000 bp of cpDNA for 51 Juniperus species plus many outgroups. Phylogenies based on fewer species were also constructed based on nuclear internal transcribed spacer (nrITS) and combined nrITS/cpDNA data sets to check for congruence. Divergence time-scales and ancestral distributions were further inferred. • Both long dispersal and migration across land bridges probably contributed to the modern range of Juniperus, while long-term climatic changes and the uplift of the Qinghai-Tibetan plateau probably drove its diversification. Diversification apparently slowed down during climate-stable period of the Oligocene, and then speeded up from the Miocene onwards. • Juniperus probably originated in Eurasia, and was a part of the south Eurasian Tethyan vegetation of the Eocene to Oligocene. It reached America once at this time, once in the Miocene and once more recently.

Distribution of living Cupressaceae reflects the breakup of Pangea

Most extant genus-level radiations in gymnosperms are of Oligocene age or younger, reflecting widespread extinction during climate cooling at the Oligocene/Miocene boundary [∼23 million years ago (Ma)]. Recent biogeographic studies have revealed many instances of long-distance dispersal in gymnosperms as well as in angiosperms. Acting together, extinction and long-distance dispersal are likely to erase historical biogeographic signals. Notwithstanding this problem, we show that phylogenetic relationships in the gymnosperm family Cupressaceae (162 species, 32 genera) exhibit patterns expected from the Jurassic/Cretaceous breakup of Pangea. A phylogeny was generated for 122 representatives covering all genera, using up to 10,000 nucleotides of plastid, mitochondrial, and nuclear sequence per species. Relying on 16 fossil calibration points and three molecular dating methods, we show that Cupressaceae originated during the Triassic, when Pangea was intact. Vicariance between the two subfamilies, the Laurasian Cupressoideae and the Gondwanan Callitroideae, occurred around 153 Ma (124–183 Ma), when Gondwana and Laurasia were separating. Three further intercontinental disjunctions involving the Northern and Southern Hemisphere are coincidental with or immediately followed the breakup of Pangea.

Phylogeography of Pinus tabulaeformis Carr. (Pinaceae), a dominant species of coniferous forest in northern China

How coniferous trees in northern China changed their distribution ranges in response to Quaternary climatic oscillations remains largely unknown. Here we report a study of the phylogeography of Pinus tabulaeformis, an endemic and dominant species of coniferous forest in northern China. We examined sequence variation of maternally inherited, seed‐dispersed mitochondrial DNA (mtDNA) (nad5 intron 1 and nad4/3–4) and paternally inherited, pollen‐ and seed‐dispersed chloroplast DNA (cpDNA) (rpl16 and trnS‐trnG) within and among 30 natural populations across the entire range of the species. Six mitotypes and five chlorotypes were recovered among 291 trees surveyed. Population divergence was high for mtDNA variation (GST = 0.738, NST = 0.771) indicating low levels of seed‐based gene flow and significant phylogeographical structure (NST > GST, P < 0.05). The spatial distribution of mitotypes suggests that five distinct population groups exist in the species: one in the west comprising seven populations, a second with a north–central distribution comprising 15 populations, a third with a southern and easterly distribution comprising five populations, a fourth comprising one central and one western population, and a fifth comprising a single population located in the north‐central part of the species’ range. Each group apart from the fourth group is characterized by a distinct mitotype, with other mitotypes, if present, occurring at low frequency. It is suggested, therefore, that most members of each group apart from Group 4 are derived from ancestors that occupied different isolated refugia in a previous period of range fragmentation of the species, possibly at the time of the Last Glacial Maximum. Possible locations for these refugia are suggested. A comparison of mitotype diversity between northern and southern subgroups within the north‐central group of populations (Group 2) showed much greater uniformity in the northern part of the range both within and between populations. This could indicate a northward migration of the species from a southern refugium in this region during the postglacial period, although alternative explanations cannot be ruled out. Two chlorotypes were distributed across the geographical range of the species, resulting in lower levels of among‐population chlorotype variation. The geographical pattern of variation for all five chlorotypes provided some indication of the species surviving past glaciations in more than one refugium, although differentiation was much less marked, presumably due to the greater dispersal of cpDNA via pollen.

A hybrid zone dominated by fertile F1s: maintenance of species barriers in Rhododendron.

Isolating barriers between interbreeding sympatric or parapatric interfertile species are maintained by processes that occur within their hybrid zones. Although the effects of intrinsic selection on hybrid fitness are well known, less is understood about extrinsic fitness variation. At Tiryal Dag, northeast Turkey, Rhododendron×sochadzeae (R. ponticum×caucasicum) forms large populations in which neither segregation nor backcrossing occur, in habitats intermediate between those of its parents. Using single‐copy species‐specific random amplified polymorphic DNA and inter simple sequence repeat markers, it was determined that most or all R.×sochadzeae plants are F1s, and that there are many separate genets present. Hand pollination and germination experiments showed that R.×sochadzeae plants can produce viable seed of F2s or backcrosses in either direction. Furthermore, adult backcrosses have been observed in habitats atypical for R.×sochadzeae. From this, all non‐F1 hybrid derivatives appear to be eliminated in the hybrid zone at Tiryal Dag as a result of postgermination selection. This absence of post‐F1 hybrid derivatives apparently prevents introgression. This type of hybrid population is here termed an F1‐dominated hybrid zone (F1DZ), and also occurs in Encelia. The observed dominance of F1s within a narrow habitat range is best explained by habitat‐mediated superiority of F1s over all other genotype classes. Therefore, habitat‐mediated selection against the second hybrid generation might be preventing interspecific gene flow in R.×sochadzeae. F1DZ formation is postulated to require the formation of F1s in quantity, habitat‐mediated superiority in F1s, and highly specific habitat conditions.

Reproductive isolation among two interfertile Rhododendron species: low frequency of post-F1 hybrid genotypes in alpine hybrid zones

Hybrids between the acid‐loving species Rhododendron ferrugineum and the basic soil species Rhododendron hirsutum occur on soils of intermediate pH in the European Alps. Material from two hybrid zones ~500 m apart, and also nearby populations of each parent species, was surveyed for presence/absence of 31 random amplified polymorphic DNA markers that distinguish parents. Based on morphological assessment, the material comprised 51 putative hybrids, 18 putative R. ferrugineum individuals and 26 putative R. hirsutum plants. RAPD data were analysed using a Bayesian approach implemented by the program newhybrids, and also by principal coordinates analysis. The identity of all R. ferrugineum plants examined was confirmed; however, of the putative R. hirsutum individuals examined, two were certainly and 11 possibly hybrid derivatives. Among all hybrid derivatives examined, about half were designated as F1s or a similar class, otherwise backcrosses to R. hirsutum appeared to be common whereas other hybrid classes were rare and backcrosses to R. ferrugineum possibly absent. Despite this, artificially generated seed of F2 class and backcrosses in each direction showed greater viability than one parent (R. hirsutum). Introgression from R. ferrugineum was also detected in a population that from morphology appeared to contain only R. hirsutum. Hence, the direction of backcrossing might be highly asymmetric within hybrid zones, causing unidirectional gene flow from R. ferrugineum into R. hirsutum. Conversely, the rarity of backcrosses to R. ferrugineum, F2s and later hybrid generations, which might be due to phenology effects and habitat‐mediated selection, could play a part in restricting gene flow towards R. ferrugineum.

Gene flow and species delimitation: a case study of two pine species with overlapping distributions in Southeast China.

Species delimitation detected by molecular markers is complicated by introgression and incomplete lineage sorting between species. Recent modeling suggests that fixed genetic differences between species are highly related to rates of intraspecific gene flow. However, it remains unclear whether such differences are due to high levels of intraspecific gene flow overriding the spread of introgressed alleles or favoring rapid lineage sorting between species. In pines, chloroplast (cp) and mitochondrial (mt) DNAs are normally paternally and maternally inherited, respectively, and thus their relative rates of intraspecific gene flow are expected to be high and low, respectively. In this study, we used two pine species with overlapping geographical distributions in southeast China, P. massoniana and P. hwangshanensis, as a model system to examine the association between organelle gene flow and variation within and between species. We found that cpDNA variation across these two pine species is more species specific than mtDNA variation and almost delimits taxonomic boundaries. The shared mt/cp DNA genetic variation between species shows no bias in regard to parapatric versus allopatric species’ distributions. Our results therefore support the hypothesis that high intraspecific gene flow has accelerated cpDNA lineage sorting between these two pine species.

The willow genome and divergent evolution from poplar after the common genome duplication

Willows (Salix) and poplars (Populus) are known worldwide as woody species with diverse uses. Although these two genera diverged from each other around the early Eocene, they share numerous traits, including the same chromosome number of 2n = 38 and the common ‘Salicoid’ genome duplication with a high macrosynteny. However, most willow species flower early in their lives with short, small and sometimes indistinct stems, and thus differ from poplars in their life histories and habits. In addition, multiple inter- and intrachromosomal rearrangements have been detected involving chromosomal regions present in both lineages, suggestive of the likely genomic divergence after the common genome duplication.

Hybridization among sympatric species of Rhododendron (Ericaceae) in Turkey: morphological and molecular evidence

Rhododendron (Ericaceae) is a large genus in which barriers to hybridization are especially weak, but many species are maintained in sympatry. Hybridization among four species of Rhododendron subsect. Pontica, which occur in sympatry in Turkey, was investigated. Material of R. ponticum, R. smirnovii, R. ungernii, and R. caucasicum and their putative hybrids was collected from the wild. Based on morphology, chloroplast DNA and nuclear ribosomal DNA restriction fragment length polymorphism (RFLP) profiles, each accession was identified as a species or hybrid combination. Five of the six possible hybrid combinations among the four species were detected. Rhododendron ponticum × R. smirnovii was represented by a single individual and R. caucasicum × R. smirnovii by one small group of hybrid plants. The combinations R. ponticum × R. ungernii and R. ungernii × R. smirnovii showed evidence of frequent backcrossing, while R. ponticum × R. caucasicum appeared unusual in that an intermediate hybrid type was abundant, whereas hybrids with phenotypes approaching either parent were rare. Possible explanations of this latter situation are discussed. The results suggest that natural hybridization among Rhododendron species is common and that ecological factors are important in maintaining integrity when species occur in sympatry.