The palaeo-Jinshajiang flowing southward in the Oligocene
Our results show that the ancestors of the endemic East Asian cyprinid clade, which produces adhesive eggs, appeared in the late Oligocene (~24 Ma; 95% credibility interval (CI): 22.3–26.7 Ma), including metzins, aphyocyprins and opsariichthyins (Fig. 2a). Combined with the fossils of Ecocarpia ningmingensis in the Ningming Basin, Guangxi Province (30), and the primary distribution of extant species of these cyprinids, we inferred that their ancestors were distributed in southern East Asia largely within the palaeo-Pearl River and palaeo-Red River before ~24 Ma (Fig. 2b). This implies that the modern Yangtze River had not yet been formed and that the palaeo-Jinshajiang likely flowed towards the south, connecting with a stream similar to the modern Red River.
The cyprinid fossils Nanningocyprinus wui and Huashancyprinus robustispinus found in Oligocene formations of the Nanning and Ningming Basins are also consistent with our results (31). Other biological and geological evidence suggests that the palaeo-Jinshajiang once flowed southward and probably connected through the palaeo-Red River into the South China Sea (Supplementary Table S1). The age of the river capture at Laojunshan near the First Bend of the Yangtze River occurred prior to 24 Ma, maybe as early as the Early Oligocene as suggested by the change in sediment composition in the Red River delta, (32). Incision of the gorge in the First Bend area started between 20 and 30 Ma based on bedrock apatite (U-Th-Sm)/He thermochronology (33). Recently, studies using 40Ar/39Ar mica dating and zircon U-Pb dating methods coupled with statistical analysis suggest that a major Paleogene river probably originated in the southeastern Qinghai-Tibetan Plateau and flowed through the Jianchuan Basin, extending to northern Vietnam during the late Eocene-Oligocene period, but disappearing by the early Miocene (34,35). Biological evidence from a time-calibrated phylogeny of only one fish genus (36-38) showed that the dating of the south-flowing palaeo-Jinshajiang is younger than that predicted in this study by using the endemic East Asian Cyprinidae.
Schizothoracine fishes commonly live on the Qinghai-Tibetan Plateau and surrounding area at an elevation of 1250–4750 m a.s.l. (39,40). In this study, the time-calibrated phylogeny of Cyprinidae (Supplementary Fig. S3) reveals that the schizothoracine fish endemic to the Qinghai-Tibetan Plateau did not appear before ~20 Ma (Supplementary Fig. S6). Combined with palaeontological evidence (41), we infer that the palaeo-elevation of the central Qinghai-Tibetan Plateau was fairly low in the Oligocene, may not above 2000 m a.s.l. During the Eocene, reorganization of rivers did not occur because the southeastern part of the plateau was not uplifted significantly until the Oligocene (34). At the same time, southern East Asia was in a humid belt, while a broad arid belt stretched across central East Asia from west to east (1,42). These results indicate that the middle and lower reaches of the Yangtze River system had not yet been connected to the Jinshajiang. Our study provides new biological dating for the southern flow of the palaeo-Jinshajiang in the Oligocene.
The formation of the Yangtze River close to the Oligocene-Miocene boundary
Fishes with semi-buoyant eggs consisting of squaliobarbins and hypophthalmichthyins existed in the Yangtze during early Miocene (~19 Ma; 95% CI: 17.1–21.3 Ma) (Fig. 2a). The earliest fossils of Hypophthalmichthys, Ctenopharyngodon and Elopichthys were found from the Lower Miocene of the Sihong Basin, Jiangsu Province (25). These results indicate that the endemic East Asian cyprinids dispersed to the position of the current Yangtze River and evolved into fishes laying semi-buoyant eggs by approximately 19 Ma (Fig. 2c), suggesting that the Yangtze River had reversed its flow direction eastward and formed the present drainage system before that time, close to the Oligocene-Miocene boundary (~24–19 Ma). The hematite/goethite proxy from Ocean Drilling Program (ODP) Site 1148 in the South China Sea (43), which is positively correlated to the strength of East Asian monsoons, rapidly rose to a peak at approximately 19 Ma (Fig. 3d). This indicates that the climate in East Asia became humid at that time, and abundant rainfall was conducive to the formation of a major Yangtze River system with high discharge.
Geological studies constrain the age of formation of the present Yangtze River system to 23–36.5 Ma based on 40Ar/39Ar dating of basalts and U-Pb dating of zircon sand grains from the lower reaches of the Yangtze River and the appearance of evaporites and lacustrine sedimentation in the Jianghan Basin (9). Detrital zircon U-Pb geochronology and heavy mineral analysis from the Cenozoic sediments of the Jianghan Basin define their provenance and indicate that the age of incision of the Three Gorges must have postdated 32 Ma. The best date for initiation of the modern river is likely after the ~24.6 Ma unconformity (44). These results are close to the date of Yangtze River formation estimated from the timing of divergence of the semi-buoyant egg group in our study. Due to using different sampling locations, dating methods and proxies, the ages of the connection and formation of the Yangtze River system were incompatible with other geologically based models (Supplementary Table S2).
The formation of the current Yangtze River system hindered gene flow of some terrestrial species between the north and south sides of the mainstream, resulting in genetic diversification and speciation. The divergence dating of the primitively segmented spider genera Sinothela and Ganthela, which are distributed on the north and south sides of the Yangtze River, was estimated to be 13–30 Ma, which is consistent with the suggested formation the modern Yangtze River system before the Miocene (45). This divergence timing has a much broader range than we inferred, probably due to fewer species and the lack of fossil calibrations.
In addition, the specialized schizothoracine fishes mostly live in the Qinghai-Tibetan Plateau at an elevation of >2750 m a.s.l (39,40). Based on the results of the time-calibrated phylogeny of schizothoracine fishes (Supplementary Fig. S6), the timing of divergence between primitive and specialized grades was likely at ~18 Ma, indicating that the Qinghai-Tibetan Plateau had reached a high elevation in the early Miocene. The surface of the southeastern Qinghai-Tibetan plateau was uplifted when ductile lower crust beneath the central plateau flowed towards the plateau margin from the late Oligocene to the early Miocene (46,47). At the same time large-scale strike-slip faults linked to extrusion of crustal blocks from Tibet by the colliding Indian block resulted in the reversal or capture of river systems (6,9). Therefore, the Yangtze River diverted its flow from being towards the south to eastward and incised through the Three Gorges to form the modern river system at that time.
Formation of the potamo-lacustrine ecosystem in the Yangtze River in the middle Miocene
Formation of a potamo-lacustrine complex ecosystem greatly promoted fish diversification (48). Previous studies have mainly focused on the formation mechanisms and ages of several of the modern lakes in the Yangtze River basin (49,50), while the earliest formation of the potamo-lacustrine ecosystem remains unclear.
Based on the result of the diversification dynamic from Bayesian Analysis of Macroevolutionary Mixtures (BAMM) and RPANDA analyses (Fig. 3a-c), the net diversification rate of the endemic East Asian cyprinids peaked at approximately 13 Ma, together with a significant rate shift configuration in the maximum a posteriori probability. These results imply that the drainage network was rich in the Yangtze River basin, and provided a large number of niches, facilitating rapid radiation and dispersal of fishes. In the middle Miocene (~13 Ma; 95% CI: 11.4–14.6 Ma), fish laying adhesive eggs arose again, including xenocyprins and cultrins (Fig. 2a). This finding indicates that to adapt to the lake environment, endemic East Asian cyprinids evolved into fishes spawning adhesive eggs that attached to aquatic plants to develop. These results suggest that the potamo-lacustrine ecosystem of the Yangtze River had appeared by that time (Fig. 2d). Coincidentally, the hematite/goethite proxy of sediment at ODP Site 1148 in the South China Sea, which is related to humidity and temperature peaked at ~13 Ma (Fig. 3d) (43), indicates that the climate in East Asia was humid at that time. Strong precipitation would have sustained a potamo-lacustrine ecosystem in the Yangtze River, greatly increasing species diversification.
In summary, we used the spatiotemporal evolutionary pattern and diversification dynamics of endemic East Asian cyprinids from the largest molecular phylogenetic tree of Cyprinidae, fossil records and information on egg type evolution while adapting to varied hydrologic conditions to reconstruct the formation history of the Yangtze River system. Our results indicate that the ancestors of East Asian cyprinids were confined to the south of East Asia between the palaeo-Pearl and palaeo-Red rivers during the Oligocene, prior to formation of the Yangtze River system. At that time the palaeo-Jinshajiang flowed southward to the South China Sea roughly along the course of the modern Red River. Endemic East Asian cyprinids had dispersed to the Yangtze River basin and evolved into fishes laying semi-buoyant eggs by ~19 Ma, which suggests that the Yangtze River system had formed by that time in response to regional surface uplift, large strike-slip tectonism and climate change. The formation of the Yangtze River is constrained to be around the Oligocene-Miocene boundary (~24–19 Ma) when monsoon climate was strengthened. Notably, the endemic East Asian cyprinids evolved into fishes spawning adhesive eggs again by approximately 13 Ma, coinciding with a peak in the net diversification rate of this endemic clade and the intensity of the East Asian summer monsoon (43), indicating that the Yangtze River system probably had developed into a potamo-lacustrine ecosystem with high productivity by the middle Miocene. Our studies constrain the ages of important geological events during the evolution of the Yangtze River from a biological perspective, helping us to understand the evolutionary history of the Yangtze River system.