4.2 The role of rivers on evolutionary history of P. heterotricha
Our phylogenetic trees and dating analyses convincingly indicate that the closest phylogenetic relationship of P. heterotricha isP. pteropoda (mainly distributed in the South of mainland China), and the colonization event that gave rise to P. heterotrichaoccurred at c. 0.83 Myr, approximately coinciding with the formation of the Qiongzhou Strait (Zhao et al. , 2007).
The analyses of the ribotypes and chlorotypes converge in suggesting a widespread pristine distribution of P. heterotricha in the south-central mountain system of Hainan Island. At c . 0.83 Myr (Figure 3), two vicariance events gave rise to the genetic split of the species first in two clades (SW and the rest), and later in three clades (SW, NW and SE, Figure 2, 3, S3). Meanwhile, The barriers represented by the Changhua and Wanglou rivers are very old, as their origins date back to Hercynian-Indosinian ages (236-257 Myr) and Late Yanshanian ages (c. 99 Myr), respectively (Wang et al. , 2015), long before the estimated differentiation within P. heterotricha and in other Hainan endemic Gesneriaceae (Ling et al. , 2017b). Thus, gene flow restrictions induced by these rivers may have affected many species of the biota of Hainan Island throughout long geological periods.
Cogent with genetic isolation between clade SW and other two clades (atc. 0.83 Myr) and between clades NW and SW (starting at c.0.48 Myr), one possibility to explain the high differentiation of the three current lineages of P. heterotricha is limited gene flow after the colonization of the south-central mountain (feasibly triggered by the tropical monsoon, Ling et al. , 2017a, b). The small brown and fusiform shaped seeds intimate weak dispersal capabilities either by anemochory (i.e. wind-dispersal) or zoochory (i.e. animal-mediated dispersal), deep and wide rivers may enhance barriers of seeds dispersal. Furthermore, at the estimated time of isolation between clades the water discharge of Changhua River and Wanglou River was probably greater, consistent with the increased precipitation regime that prevailed in the area in the early Holocene (Zheng et al. , 2004). Both factors likely contributed to the strengthening of the earlier genetic barriers and shaped the observed inter-regional genetic differentiation.
This scenario is not incompatible, however, with the possibility of multiple colonization events from the mainland to Hainan (the distance from the mainland is quite low, some 20 km across the straits of Qiongzhou), followed by secondary contact and further dispersal and isolation to originate the three genetic lineages. This would also help explain the high genetic variation levels and structuring detected inP. heterotricha . Similar processes related to low distances to the mainland and the geological ontogeny of the islands have been suggested to be frequent in other islands and archipelagos with high levels of endemicity (Caujapé-Castells et al., 2017; Stuessy et al. , 2014). Finally, the possibility of independent colonization events to the three areas of current distribution of P. heterotricha is not supported by the mismatch distribution analysis. However, three clades of P. heterotricha had no observable morphological differentiation within south-central mountain system, which may indicate the species could be at an early diversification stage.