The analysis of divergence patterns between species has been a major focus in systematics and molecular ecology [1–5]. Species descending from a common ancestor are expected to differentiate from each other and eventually attain reciprocal monophyly. Coalescence theory predicts that sister species are likely to be polyphyletic/paraphyletic in their genetic composition during the early stages after they split from their common ancestor [cf. ]. As sufficient time goes by, they become reciprocally monophyletic with the loss of complementary haplotypes. In contrast, lack of monophyly can be a result of several factors, such as incomplete lineage sorting, gene duplication, or hybridization [7–12]. Of them, hybridization leading to speciation has been known as one of the major forces for the diversification in plants . As many hybridizing species remain morphologically discrete , hybrids usually share traits with parental species, often resulting in taxonomic difficulties. Interspecific gene flow usually leads to a mixed genetic composition in hybrids, but may have low impacts on parental species' genomes [cf. ]. Although molecular tools provide sufficient power in detecting genetic variation, empirical data on species delimitation are often difficult to interpret, since both gene introgression after speciation and shared ancestral polymorphisms can result in species paraphyly . A complex of incipient species with different degrees of morphological or ecological differentiation provides an ideal model for studying species divergence.
Taiwan, an island of the island-arc system along the western edge of the Pacific Ocean, first emerged from the waters via collision between Eurasian and Pacific plates about 9 million years before present (Proto-Taiwan Stage), but attained its modern shape only 5-6 million years ago (MYA) [16, 17]. Its rugged topography is characterized by hundreds of steep mountains, which in turn provide diverse habitats along distinct vegetation zones, including tropical, coastal evergreen forests, subalpine shrubs and alpine tundra along the Central Mountain Range . Taiwan was connected to mainland SE Asia by a land bridge during lower sea levels of the last glacial maximum [19, 20]; some species may have reached Taiwan by this route and subsequently become endemic [2, 21].
Phylogeographical patterns and genetic characteristics of populations/species are shaped by the interaction between historical vicariance and recurrent genetic exchanges. These evolutionary events would leave evolutionary footprints in the genetic polymorphisms within and between populations across the distributional range. Of the geological events, regular historical glacial cycles in the Eurasia Continent had prevalent influences on survival and recolonization of populations/species . Postglacial expansion of alpine species may follow the phalanx model, which describes the effects of slower expansions from refugia due to habitat constraints [2, 22, 23]. That is, during interglacial periods, alpine species moved upward to peaks and became fragmented. In contrast, down-slope movement of alpine species during glacial periods could connect populations that were previously isolated and allow for genetic exchange among populations . On Taiwan island, only the peaks of high mountains over 3000 m in elevation were covered by ice sheets during the glacial maxima.
Rhododendron, one of the largest and most widespread woody plant genera, is distributed from the northern temperate zone, throughout tropical Southeast Asia, to northeastern Australia and consists of over 1000 species . Hymenanthes, one of eight subgenera comprising Rhododendron, consists of 225 species, most of them distributed in the Himalaya-Southwest China region [25–28]. In Taiwan, 13 taxa occur within Rhododendron . The R. pseudochrysanthum complex consists of R. pseudochrysanthum Hayata sensu stricto (s.s.), R. morii Hayata, and R. rubropunctatum Hayata, all in subgenus Hymenanthes subsection Maculifera, and all are phylogenetically related based on molecular analyses [30–32]. The remaining taxa of subsection Maculifera occur in west and central China, disjunctly from those in Taiwan . R. pseudochrysanthum s.s. is an alpine species (3000-3900 m a.s.l.), while R. morii has a wider range from 1650-3600 m a.s.l., with some overlap in distribution. In contrast, R. rubropunctatum is restricted to elevations of 600-1200 m a.s.l. in northern Taiwan. The three species of R. pseudochrysanthum complex, all distributed along the central mountain range (CMR) and other mountain ranges west of the CMR, share morphological polymorphisms in leaf shape, petiole and flower shape, thus making taxonomy difficult, despite the differentiation of ecological distributions . Possibly the closest relative of this species complex is R. hyperythrum Hayata, which is also endemic to Taiwan, occurring 1400-3700 m a.s.l. [25, 28, 29]. This species was previously placed in subsection Pontica [25, 34], but phylogenetic work indicates that it is closely related to R. pseudochrysanthum . These two might be sister species, but at present a phylogeny of subsection Maculifera is lacking, so this hypothesis cannot yet be tested. Rhododendron hyperythrum is also known to hybridise with members of the R. pseudochrysanthum complex . Closely related to R. hyperythrum, the three species of the R. pseudochrysanthum complex with low morphological differentiations may be recognized as a single species (R. pseudochrysanthum sensu lato [s.l.]) [25, 28, 29, 35].
The power of molecular markers in elucidating plant phylogeography has been detected in many studies [2, 36–41]. Comparative phylogenies between biparental (nuclear) and uniparental (chloroplast and mitochondrial) markers are useful for resolving phylogeographic patterns, origin of hybridization, and assessing the migratory routes of species [40–42]. Because one or both organelle genomes are often maternally inherited in plants , they are particularly suitable for investigating processes associated with seed dispersal, such as range expansions , contribution of seed movement to total gene flow [44–47], refugial isolation [48, 49], and origin of maternal or paternal lineage .
Previous studies have investigated the phylogeography of the R. pseudochrysanthum in Taiwan, revealing close relationships among these taxa [30, 32, 51] and a lack of geographical subdivision. However, little attention has been paid to the demographic dynamics of R. pseudochrysanthum across the mountain ranges. In contrast to previous studies, sampling in this study covered vaster areas, with the aid of the bureau of the Yushan National Park. The present study analyzed genetic variation of the R. pseudochrysanthum s.l. at atpB-rbcL intergenic spacer of cpDNA and the internal transcribed spacer regions (ITS) of nrDNA. A phylogeographical approach was used to infer past evolutionary history and processes and to examine species delimitation and population structure of the species complex. The objectives of this study were to investigate the following:
Do the lineages of cpDNA correspond with those of nrDNA?
Given geographical barriers, does significant genetic differentiation exist among populations of different mountain ranges?
Have the populations of different mountain ranges of R. pseudochrysanthum s.l. experienced different demographic dynamics?