Fossil climbing perch and associated plant megafossils indicate a warm and wet central Tibet during the late Oligocene

Understanding the Tibetan Plateau’s palaeogeography and palaeoenvironment is critical for reconstructing Asia’s climatic history; however, aspects of the plateau’s uplift history remain unclear. Here, we report a fossil biota that sheds new light on these issues. It comprises a fossil climbing perch (Anabantidae) and a diverse subtropical fossil flora from the Chattian (late Oligocene) of central Tibet. The fish, Eoanabas thibetana gen. et sp. nov., is inferred to be closely related to extant climbing perches from tropical lowlands in south Asia and sub-Saharan Africa. It has osteological correlates of a labyrinth organ, which in extant climbing perches gives them the ability to breathe air to survive warm, oxygen-poor stagnant waters or overland excursion under moist condition. This indicates that Eoanabas likewise lived in a warm and humid environment as suggested by the co-existing plant assemblage including palms and golden rain trees among others. As a palaeoaltimeter, this fossil biota suggests an elevation of ca. 1,000 m. These inferences conflict with conclusions of a high and dry Tibet claimed by some recent and influential palaeoaltimetry studies. Our discovery prompts critical re-evaluation of prevailing uplift models of the plateau and their temporal relationships with the Cenozoic climatic changes.


Tibet (
, and see Supplementary Information) is an important addition to the fossil record of the Tibetan Plateau. These fossils represent a biota that comprises ideal environmental indicators of warm and humid environment at low elevations.
The climbing perches (anabantid fishes) are closely related to kissing gouramies and fighting fishes [23][24][25] in the perciform suborder Anabantoidei (labyrinth fishes) 25 . They are common in freshwaters in tropical Asia and sub-Saharan Africa 23,24 . Their labyrinth organs are so large that they occupy a large portion of the gill chamber. In fact, their tissues are not sufficient to meet the respiratory needs, and they are therefore obligatory air-breathers 24 . The capability of accessary air breathing allows some of anabantids to move onto land under moist conditions, or even reputedly climb trees, thus earning the common name "climbing perch" 23 .
Diagnosis. A labyrinth fish displaying anabantid characteristics including a posterior notch of the opercle bounded by spines, a V-shaped strut on inner side of opercle and six to nine anal-fin spines. It shares with Asian anabantids the following derived characters: broad infraorbitals 3-5 completely covering the cheek, a sensory canal pore just behind sphenotic/pterotic junction and pelvic plate lying flat; and it shares with African anabantids some derived characters, e.g., sensory canal opening in between the infraorbitals, supraorbital commissure of the sensory canal absent and male postocular contact organ present.  The skull roof is smooth. The frontals carry paired coronal pores on the supraorbital sensory canals (Fig. 1a,b and Supplementary Fig. 3a,b) and, as in the species of African Microctenopoma 24 , lack the supraorbital commissure. The circumorbital bones include a ventrally serrated lacrimal and broad infraorbitals 3-5 covering the cheek completely (Figs 1 and 2a, Supplementary Fig. 5a,b). Similarly large infraorbitals are only seen in Anabas (Fig. 2g); however, the sensory pores are open between these elements, a character known only in African anabantids 24 . Just posterior to the sphenotic/pterotic junction, the pterotic bears a sensory opening ( Fig. 1 and Supplementary  Fig. 5a,b), which is present in Anabas but not in African forms 24 . More posteriorly, it has a depressed attachment facet for the levator operculi muscle (Fig. 1c,d and Supplementary Fig. 5a,b) as in living anabantids 24 . Immediately ventral to this facet is the suprabranchial chamber (Fig. 1c,d) housing the labyrinth organ referred to below. The parasphenoid shaft is straight without oral or orbital processes (Supplementary Figs 3a,b,g and 4j-m), and the transverse process is likely present (Supplementary Fig. 3h).
The opercle has a round posterior notch bounded by two large spines or spine patches ( The ventral postcleithrum directly contacts the flat lying pelvic plate (Fig. 1a,b and Supplementary Fig. 3g), which resembles that of Anabas, but differs from the dorsally angled one in African forms 24 .
The long-based dorsal and anal fins show relatively low numbers of anal spines (7-9) and soft rays (7-9) in both fins (Figs 1a,b and 2b,c, Supplementary Figs 3a,b,i,j, 4a-e, j-m and 5a,b,d-f, and Supplementary Table 1). The truncated caudal fin has a principal fin-ray formula of I-7/7-I (Figs 1a,b and 2b,c, Supplementary Figs 3a,b,i,j, 4j-m and 5d,e and Supplementary Table 1). The parhypural firmly attaches to the compound vertebra (Fig. 1a,b and Supplementary Fig. 3a,b,i,j), whereas in living anabantids, it is detached and widely separated from the vertebra 24 (Fig. 2g).
In one specimen of Eoanabas, layers of bony lamellae just above the branchial chamber ( Fig. 2c and Supplementary Fig. 3g,h) show a cribrate structure (Fig. 2d) almost identical to that of the expanded plates of the labyrinth organ (modified first epibranchial) 24 in living anabantids (Fig. 2e,f), suggesting the presence of a labyrinth organ also in the fossil fish (Fig. 2h). Sexual dimorphism is exemplified by the postocular contact organ located immediately posteroventral to the orbit, a patch of scales with large ctenii (Fig. 2a,b and Supplementary Figs 3a,b,d and 5c) that are much larger than those of flank scales (Supplementary Fig. 3a,c,e). This organ is rare among perciform fishes 24 and is seen in all African anabantids except for the species of Microctenopoma and Sandelia capensis 24, 27 . The Plant Megafossils Assemblage Associated with Eoanabas. Eoanabas was found in the lacustrine depositional environments with several aquatic herbs ( Fig. 3 and Supplementary Information) such as duckweed (Limnobiophyllum, Lemnoideae, Araceae) (Fig. 3k) and bulrush (Typha) (Fig. 3q), associated with a diverse lowland vegetation (Fig. 3 and Supplementary Information) including palms (Fig. 3a), golden rain trees (Koelreuteria) (Fig. 3b), Pistacia (Fig. 3c), Ulmaceae (Fig. 3d), Araliaceae (Fig. 3i), Handeliodendron (Fig. 3j), Exbucklandia (Fig. 3l) and aspens (Populus) (Fig. 3o). Among the 15 types of woody dicot leaves in this plant assemblage, only five types have a toothed leaf margin, whereas the remaining 10 types all have an entire margin (Supplementary Table 2). Regarding the leaf size 28 , microphyll leaves are dominant in the assemblage, with at least 10 morphotypes.

Discussion
A phylogenetic analysis based on morphological data resolves Eoanabas as the earliest diverging lineage within the family Anabantidae, with its African relatives being monophyletic and sister to the Asian single genus Anabas (Supplementary Fig. 6). Eoanabas is also the oldest anabantid known so far, extending the geological record (previously represented only by three opercular bones from Pleistocene Java) 29, 30 of this family for ca. 20 Ma and expanding the geographic distribution of this family to the middle Cenozoic central Tibet.
Living anabantids are confined mostly to the regions in Asia and Africa dominated by the tropical monsoon ( Supplementary Fig. 7), with the greatest diversity in the rain forests of western and central Africa 24,31 . They inhabit small bodies of water in lowlands in tropical Asia and sub-Saharan Africa 23,24,31 (Supplementary Table 3), rarely higher than 1,000 m asl 31 . With few exceptions in the rivers of west Africa 24 , they prefer warm and periodically oxygen-poor stagnant waters, under the optimal temperature range of 18 to 30 °C (Supplementary Table 3).
The key adaptations to such conditions are the labyrinth organ (Fig. 2f), a highly convoluted structure covered by richly vascularized epithelia 24 , and the resultant accessary air-breathing capability 24,32 . Their air-breathing behaviour is facilitated by several muscles, including the levator operculi muscle 32 that leaves a characteristic marked facet on the pterotics in living anabantids 24 . Interestingly, Eoanabas also has a labyrinth organ (Fig. 1d) and similar arrangement of the air-breathing muscle on the pterotics (Fig. 1c,d and Supplementray Fig. 5a,b). These strikingly similar characters appear physiologically specific and environmentally diagnostic 31 , and thus may indicate similar habitat conditions.
Eoanabas possesses a postocular contact organ in the males, which is unique to certain African anabantids 24,27 . This structure indicates a typical anabantid spawning behavior. When the male embraces the female, it thrusts the spines of this organ into her abdomen to stimulate her to release eggs and ensure fertilization 27 . The similar reproductive behaviour strengthens the ecological similarities between Eoanabas and its living confamilial relatives.
The plant megafossil assemblage found with Eoanabas contains some taxa with living analogues (e.g., the palms and duckweed) commonly associated with the extant climbing perches (Supplementary Information), and hence reinforces the above palaeoenvironmental inference. Although the available material is insufficient to conduct a quantitative climatic reconstruction, the relatively high percentage (>60%) of the entire-margined types among the woody dicot leaves suggests a warm and humid climate (Supplementary Information). The dominance of microphyll leaves is consistent with this climatic inference, and appears to further suggest that the late Oligocene central Tibetan vegetation is more comparable to modern subtropical vegetation in Zhejiang, East China 33 . Based on the co-existing range of the living analogues of these fossil plants, this assemblage is assumed to grow most likely at an elevation of ca. 1,000 m asl ( Supplementary Information).
Collectively, the ecological signal of Eoanabas and associated plant megafossils, as well as the co-existing fossil barbel fish Tchunglinius 21 , suggests that warm and humid lowland habitats were present in the Tibetan interior ca. 26 Ma, probably at an elevation ~1,000 m asl, just like the environmental settings for modern climbing perches ( Supplementary Fig. 8). Such a vibrant ecosystem represented by a spiny-rayed fish, barbel carp, and a diverse subtropical vegetation is in sharp contrast to that in the central Tibet today 15,17,18 . Yet the general lowland landscape might exhibit some topographic variation within the catchment area according to the contemporaneous pollen data of a mixed coniferous-broadleaved forest 26 .
Such palaeoenvironmental and palaeotopographic inferences will help us reevaluate our current understanding of the rise of the plateau and its environmental and climatic consequences. A warm and humid central Tibet requires a palaeogeographic setting allowing sufficient warm moisture to reach as far as the plateau interior at least up to the late Oligocene. As such, the current high-standing topographic features in southern Tibet might not exist then to block the southerly moisture from the ocean. This is in conflict with the notions that high Gangdese range has already been in place since the Palaeocene 10 or the plateau had undergone northward stepwise uplift from late Eocene time onward 12 , but is compatible with the predictions of the rise of the Himalaya until the Neogene 34 and newly proposed hypothesis for the oceanic Greater Indian Basin that would have maintained a deep tropical water mass between Asia and India throughout most of the Palaeogene 35 . Given the intrinsic environmental signals of the fossil climbing perch and the highly diversified sub-tropical plants, the establishment of a central Tibet with near-present elevation and climatic condition since early to late Eocene 5, 7-9 or late Oligocene 6 also seems highly problematic. Therefore, we caution against simple links between the rather early uplift of the plateau and the global climatic cooling during the early Cenozoic era 9 . The generally low elevation in plateau interior and probably more southern areas around the Palaeogene/Neogene transition is an important condition to test the existing tectonic models of plateau uplift. It is compatible with the inferences of either a period of un-roofing 2 or the cease of the uplift process and a Tibet no higher than 1,000 m during ca. 30-20 Ma 11 , but contradicts the models for the constantly thickened crust in central Tibet since at least as early as the late Oligocene 6 or even earlier 8,9 . Our estimation of a fairly low, warm and wet central Tibet implies that this area has been elevated ~4,000 m to attain its current conditions since latest Palaeogene. In light of our fossil discovery, the geological history of the Tibetan Plateau is in need of critical re-evaluation.

Methods
Fossils were prepared mechanically with sharp needles. The Recent material for comparison was cleared and stained according to the method of Bai 36 . Photographs of fossils were taken with Nikon except Fig. 2d and Supplementary Fig. 3f,h that were images produced using Hitachi S-3700N Scanning Electron Microscope (SEM) (working condition: Fig. 2d, 3 kV, 98 µA, Working Distance = 25.4 mm; Supplementary Fig. 3f, 5 kV, 99 µA, WD = 16.7 mm; Supplementary Fig. 3h, 3 kV, 101 µA, WD = 25.5 mm); and Fig. 2e that is an X-ray micro-computerized tomography. For the latter, the scanning was carried out at the Key Laboratory of Vertebrate Evolution and Human Origins, Chinese Academy of Sciences (CAS) using the 225 kV micro-computerized tomography (developed by the Institute of High Energy Physics, CAS. The specimen (partial labyrinth organ of Anabas) was scanned with beam energy of 90 kV and a flux of 100 μA at a detector resolution of 6.27 μm per pixel using a 360° rotation with a step size of 0.5° and an unfiltered aluminium reflection target. A total of 720 transmission images were reconstructed in a 2,048*2,048 matrix of 1,563 slices using a two-dimensional reconstruction software developed by the Institute of High Energy Physics, CAS. The three dimensional reconstructions were created in VG Studio Max (v2.0), and images of the reconstructions were exported from VG Studio and finalized in Adobe Photoshop and Adobe Illustrator. Line drawings were done under a Leica MZ8 microscope with a camera lucida attachment and further prepared using Adobe Photoshop CS4.
To conduct a phylogenetic analysis to assess the systematic position of Eoanabas within the family Anabantidae, we developed a data matrix including 165 soft tissue, behavioural, and skeletal morphological characters (Wu et al. 2016.nex, Wu et al. 2016.tnt). These morphological characters are updated from ref. 24 and include 33 newly defined and other characters adopted from references elsewhere (for details see Appendix section below). Of the total 21 taxa, Badis and kissing gouramy Helostoma were designated as outgroups. The ingroup comprises fossil Eoanabas thibetana and 18 living anabantid species and species assemblages according to ref. 24. The data matrix was edited in WinClada 1.00.08 37 and saved in the NEXUS format (Wu et al. 2016.nex) and TNT format (Wu et al. 2016.tnt). Additionally, to further test the phylogenetic position of the fossil taxon, we also tried the backbone-constrained search with the relationships based on Rüber et al. 's gene matrix 25 of the extant taxa involved in this study used as backbone (datamatix: Wu et al. 2016 backboned.tnt). Parsimony analysis was undertaken using TNT, Tree analysis using New Technology, a parsimony analysis program supported by the Willi Hennig Society 38 . The analyses were conducted using a traditional search strategy, with default settings except for the following: 10,000 maximum trees in memory and 100 replications. Bremer support values were generated in TNT by applying the 'New Traditional Search' using TBR and collecting suboptimal topologies with 100 replicates. All characters were treated as unordered, and weighted equally.