Evidence of a Cooler Continental Climate in East China during the Warm Early Cenozoic

The early Cenozoic was characterized by a very warm climate especially during the Early Eocene. To understand climatic changes in eastern Asia, we reconstructed the Early Eocene vegetation and climate based on palynological data of a borehole from Wutu coal mine, East China and evaluated the climatic differences between eastern Asia and Central Europe. The Wutu palynological assemblages indicated a warm temperate vegetation succession comprising mixed needle- and broad-leaved forests. Three periods of vegetation succession over time were recognized. The changes of palynomorph relative abundance indicated that period 1 was warm and humid, period 2 was relatively warmer and wetter, and period 3 was cooler and drier again. The climatic parameters estimated by the coexistence approach (CA) suggested that the Early Eocene climate in Wutu was warmer and wetter. Mean annual temperature (MAT) was approximately 16°C and mean annual precipitation (MAP) was 800–1400 mm. Comparison of the Early Eocene climatic parameters of Wutu with those of 39 other fossil floras of different age in East China, reveals that 1) the climate became gradually cooler during the last 65 million years, with MAT dropping by 9.3°C. This cooling trend coincided with the ocean temperature changes but with weaker amplitude; 2) the Early Eocene climate was cooler in East China than in Central Europe; 3) the cooling trend in East China (MAT dropped by 6.9°C) was gentler than in Central Europe (MAT dropped by 13°C) during the last 45 million years.


Introduction
The early Cenozoic climate was characterized by a much warmer mean global temperature than today, the extreme case being the Early Eocene Climatic Optimum (EECO), 53-51 million years ago, when concentrations of greenhouse gases were high and global temperature reached a long-term maximum [1]. The Early Eocene is also marked by the rapid evolution and diversity of early modern plants and vertebrates with important intra-and intercontinental dispersals starting during the Paleocene-Eocene Thermal Maximum, about 55 million years ago [2,3]. The response of vegetation succession to the Early Eocene climatic changes in the Northern Hemisphere has been extensively studied in North America and Europe [4][5][6][7][8][9][10][11], but it is still poorly understood in Asia. Only a series of quantitative researches on Paleogene climate in East China were carried out in recent years [12][13][14][15][16]. In this study, China would thus provide important information for the third Northern continent.
The Early Eocene Wutu Formation in Shandong Province, Eastern China is one of the most important deposits in Asia for the understanding of early modern mammal evolution. Since the 1930s, this deposit has indeed yielded an intriguingly mixed fauna of archaic and early modern mammals [17,18]. More recently, early modern plants such as the oldest Asian Nymphaeaceae and earliest record of Prunus have been found in the Wutu deposit [19,20] that corresponds to one of the warmest time periods of the Cenozoic. Therefore, Wutu occupied a key position in the palaeogeography of Asia especially for biotic exchanges with the rest of Asia and with North America.
Previous investigations at Wutu have indicated a potentially wide palynostratigraphical distribution [21,22]. Therefore, we analyzed the palynological samples of a 300 m borehole drilled in the Wutu Formation in order to reconstruct the Early Eocene vegetation succession and climatic changes in Wutu by the coexistence approach method. Furthermore, in combination with the prior quantitative researches, we explored the Cenozoic climatic changes in East China.

Materials and Methods
The Wutu coal mine (36°39 0 N, 118°55 0 E) is located near the town of Wutu, Linqu County, Shandong Province, East China (Fig 1). The sediments in the coal mine belong to the Wutu Formation which is late Early Eocene in age based on the seed-eating Carpolestidae mammals [23] and confirmed by a diversified mammal association (e.g. Preonictis youngi, Zodiocyon zetesios, Pappomoropus taishanensis, Chowliia laoshanensis, Homogalax wutuensis, Wutuhyus primiveris) [18]. The Wutu Formation consists of four members [24], from bottom to top: the lower coal-bearing member, the oil shale member, the middle coal-bearing member, and the upper coal-bearing member. Twenty-five palynological samples were collected from the lower  coal-bearing and oil shale members (Fig 2). The borehole was taken several years ago from Wutu coal mine during a geology survey. After a series of geological researches, there were only 25 left samples for the palynological study. In this study, the specimen collection did not involve endangered or protected species, so specific permission was not required.
The palynological samples were treated by the method of Heavy Liquid Separation (density = 2.1 g/ml) [25,26]. Palynomorph grains were found in all samples. More than 100 grains (113-766) were found in 11 samples (samples number : 1, 2, 7, 10, 11, 18, 19, 21, 22, 24 and 25), while less than 100 grains (1-59) were found in the other 14 samples. The low amounts of palynomorph grains may be caused by taphonomy condition. More than 2,800 palynomorph grains were observed under a Leica DM 2500 light microscope and identified based on the classification of Song [27]. By applying the single-grain technique [28], palynomorph grains were photographed with a Zeiss Axioplan 2 light microscope and a FEI Quanta 200 environmental scanning electron microscope (Figs 3-5).
The palynomorph relative abundance (RA) of a taxon was calculated by the following equation: RA = N/Nt, where N is the pollen/spore number of a taxon and Nt is the total pollen/ spore number of all taxa combined in the pool of samples [29,30]. TILIA and TILIAGRAPH softwares were used to construct the pollen diagram (Fig 6).
In this work, according to the temperature requirements of their nearest living relatives (NLRs), the palynomorph taxa were divided into seven groups: megathermic, mega-mesothermic, mesothermic, meso-microthermic, microthermic, nonsignificant elements and herbs and/ or shrubs [29][30][31][32]. We used the CA [33][34][35] in the quantitative reconstruction of the palaeoclimate in Wutu. The basic principle of CA is the assumption that the climatic requirement of a fossil taxon was similar to that of its NLR. The climatic parameters in the modern geographic distributions of all NLRs were compared [36], and then the coexistence intervals of the climatic tolerances of the fossil taxa were obtained. The modern climatic parameters of NLRs used in CA were extracted from Surface Meteorological Data of China [37]. Meanwhile, the MAT values of NLRs from the Palaeoflora Database were also adopted to obtain the climatic parameters [38]. It resulted in seven palaeoclimatic parameters for Wutu: MAT = the mean annual temperature, MWMT = the mean temperature of the warmest month, MCMT = the mean temperature of the coldest month, DT = the difference in temperature between the coldest and warmest months, MAP = the mean annual precipitation, MMaP = the mean maximum monthly precipitation and MMiP = the mean minimum monthly precipitation.
Based on the study of modern floras, Grimm and Denk [39] referred to the CA as a less reliable approach for climate reconstructions. However, the CA was earlier tested with modern vegetation [35,40], and was applied on Cenozoic floras of Central Europe to obtain the continuous continental climate curve which closely correlated with the evolution of marine temperature [41]. According to our works by using the CA in the last more than 10 years, we found that this method needs to be improved to some extent, but it is still very useful for reconstructing the Cenozoic climatic changes in East China.

Palynological assemblages
Forty-seven different palynomorphs were identified in the Wutu section, consisting of 28 angiosperms (48.36%), 7 gymnosperms (49.51%), 10 pteridophytes (1.99%) and 2 algae (0.14%) ( Table A in S1 File). The angiosperms were assigned to 20 families and one to ambiguous type. The gymnosperms belonged to 4 families and one to ambiguous type. The pteridophytes belonged to 8 families and 2 to ambiguous type.
Of the 25 collected samples 11 were rich enough to perform the TILIA analysis recognizing three zones from bottom to top, based on the RA (Fig 6).
The RA of megathermic and mega-mesothermic elements represents a total of 1.79%. The RA of microthermic and meso-microthermic elements is 56.27% (Table B in S1 File).
In comparison with Zone 1, the average RA of megathermic and mega-mesothermic elements increased to 17.28%, while those of microthermic and meso-microthermic elements decreased to 21.64% (Table B in S1 File).
In comparison with Zone 2, the RA of megathermic and mega-mesothermic elements decreased to 3.06%, while those of microthermic and meso-microthermic elements increased to 48.06% (Table B in S1 File).

Palaeovegetation succession
The whole palynological assemblage suggests that the Early Eocene vegetation of Wutu was composed of a mixed deciduous broad-leaved and coniferous forest growing under warm temperate condition. The co-occurence of megathermic and microthermic elements might suggest the presence of zonal vegetation in the hills nearby. The megatherm and mega-mesotherm grew sparsely in the valleys and/or basins. The mesotherm probably grew on the hillsides at low altitudes. The microtherm and meso-microtherm were likely to occupy the hills at higher altitudes.
Based on the palynological data from the three zones, the vegetation succession in Wutu during the Early Eocene is divided into three periods and described as follows (Fig 7): Period 1 (Zone 1): The conifers dominated the mixed needle-and broad-leaved forest during this period and the vegetation was characterized by low plant diversity.
Mesothermic and meso-microthermic elements such as Pinus, Betula and Corylus dominated the forest. Thermophilous elements (Taxodiaceae and Castanopsis) and psychrophilous elements (Pinus, Abies) were present. Hygrophilous elements such as Alnus and Taxodiaceae grew sparsely around the paleolake or surrounding wetland. Xerophilous element (Ephedra) may be occurred on the dry slopes.
Period 2 (Zone 2): In contrast to the low plant diversity during the Period 1, the diversity of plant increased. Instead of conifers, broad-leaved trees such as Corylus and Betula predominated in the mixed needle-and broad-leaved forest during this period.
In comparison with the Period 1, the diversity and RA of thermophilous elements (Taxodiaceae, Castanopsis, Euphorbiaceae, Magnoliaceae, Arecaceae and Bignoniaceae) increased, while the RA of psychrophilous elements (Pinus, Abies) decreased sharply. The hygrophilous elements (e.g. Alnus, Taxodiaceae) increased dramatically, with xerophilous elements (Ephedra and Artemisia) growing sparsely in the herbaceous layer of the forest. In comparison with the Period 2, thermophilous elements like Euphorbiaceae and Arecaceae decreased, while the psychrophilous elements such as Picea and Abies increased  dramatically. Hygrophilous elements (e.g. Alnus, Taxodiaceae) decreased, indicating a dryer condition.

Palaeoclimate and climatic fluctuations in Wutu
Based on the NLRs of 33 spermatophyte taxa from the whole pollen assemblage (Table C in S1 File), seven climate parameters of the Wutu section were estimated by applying the CA method ( Figure  The new parameters were compared with the current meteorological data in Wutu. The comparison revealed that the median values of MAT, MCMT, MAP, MMaP and MMiP were higher, and the median values of MWMT and DT were lower than today (Table D in S1 File). This indicates that the Early Eocene climate in Wutu was warmer and wetter, with weaker seasonality than today.
The palaeoclimatic fluctuations were inferred from the changes of RA of megathermic/ microthermic elements (Fig 8) and hygrophilous/xerophitous elements during the Periods 1-3. Period 1 was characterized by a warm temperate and humid climate. Period 2 was characterized by a relatively warmer and wetter climate, and Period 3 was cooler and drier again. Taken as a whole, the palynological data suggests that the Early Eocene climate in Wutu did not experience strong fluctuations during the three periods.

The climate in East China during the last 65 million years
The palaeoclimate analysis developed from qualitative to quantitative in recent years. On the long-term scale, Mosbrugger et al. [41] studied the climatic changes of Central Europe during the last 45 million years, but more quantitative reconstructions of continental palaeoclimates need to be carried out. For now, the data on long-term climatic changes of East China are still missing. Unfortunately, no basin with the whole Cenozoic stratigraphic sequence like in Central Europe was found yet in East China, so 25 localities (including Wutu) from different latitudes were used here to reconstruct the Cenozoic palaeoclimate in East China (Fig 1). By comparing the palaeoclimatic parameters, this research represents an attempt in exploring the climatic changes in East China during the last 65 million years.
The climatic changes in East China during the Cenozoic may be illustrated by the comparison of climatic parameters obtained from 40 fossil floras [12,13,14,16,29,32,42,43,44] (including this study) (Table E in S1 File). Table F Table G in S1 File shows the comparison of climatic parameters after considering the latitudinal temperature gradients. The following three temperature curves of East China were obtained (Fig 9, Table H in S1 File):  MAT curve showed a series of warming and cooling fluctuations. Taken as a whole, MAT curve indicated a general cooling trend in East China during the last 65 million years with MAT dropped by 9.3°C (from 16.9 to 7.6°C). This cooling trend coincided with the ocean temperature changes but with weaker amplitude (Fig 9) [1]. Based on the global marine oxygen isotope record, Zachos et al. [1] obtained the ocean water temperature curve. Although since the Early Oligocene the variability in oxygen isotope record mostly reflected the changes in Antarctica and Northern Hemisphere ice volume, it still reveals the global cooling trend [1].   (Fig 9).
The comparison between the three temperature curves of East China suggests that during the last 65 million years, the changing trend of MCMT and MWMT coincided with MAT changes.

Comparison with the Cenozoic climatic changes in Central Europe
The Cenozoic temperature curves of East China were compared with those from Central Europe [41]. It revealed that:  (Fig 9).
The Cenozoic cooling trend in East China was thus gentler than in Central Europe and mainly due to an obvious cooler climate in East China than the subtropical climate in Central Europe during the Early-Middle Eocene. This phenomenon may be related to the palaeogeography during the Eocene. Some authors have already pointed out the predominance of the large benthic foramifera Nummulites induced by the warm seawater all along the Tethys palaeomargins during the Eocene [45,46]. Following global palaeogeographic reconstructions [47], East China was influenced by the northern Pacific oceanic circulation during the Early-Middle Eocene, while Europe was influenced by the warm Tethys Ocean causing the subtropical climate.

Conclusions
The comprehensive palaeoecological and palaeoclimatic studies of the pollen samples from the Wutu Formation (Early Eocene) lead to the following conclusions:  (Song 1999). Table D, Comparison between the seven climatic parameters in the early Eocene of Wutu and the current meteorological data (▲-median value of the climatic parameters; &-mean value of the climatic parameters). Table E, List of fossil localities in East China (Site numbers as in Fig 1).