Fossil evidence reveals how plants responded to cooling during the Cretaceous-Paleogene transition

Background Around the Cretaceous-Paleogene (K-Pg) boundary, an obvious global cooling occurred, which resulted in dramatic changes in terrestrial ecosystems and the evolutionary trends of numerous organisms. However, how plant lineages responded to the cooling has remained unknown until now. Between ca. 70–60 Ma Mesocyparis McIver & Basinger (Cupressaceae), an extinct conifer genus, was distributed from eastern Asia to western North America and provides an excellent opportunity to solve this riddle. Results Here we report a new species, Mesocyparis sinica from the early Paleocene of Jiayin, Heilongjiang, northeastern China. By integrating lines of evidence from phylogeny and comparative morphology of Mesocyparis, we found that during ca.70–60 Ma, the size of seed cone of Mesocyparis more than doubled, probably driven by the cooling during the K-Pg transition, which might be an effective adaptation for seed dispersal by animals. More importantly, we discovered that the northern limit of this genus, as well as those of two other arboreal taxa Metasequoia Miki ex Hu et Cheng (gymnosperm) and Nordenskioldia Heer (angiosperm), migrated ca.4–5° southward in paleolatitude during this time interval. Conclusions Our results suggest that the cooling during the K-Pg transition may have been responsible for the increase in size of the seed cone of Mesocyparis and have driven the migration of plants southwards. Electronic supplementary material The online version of this article (10.1186/s12870-019-1980-y) contains supplementary material, which is available to authorized users.


Background
The cooling from the Maastrichtian to the Paleocene that occurred during the Cretaceous-Paleogene (K-Pg) transition (ca. 70-60 Ma), has been recorded by land plant leaf data [1] and marine oxygen isotope records [2]. These dramatic environmental and climate changes [3][4][5] altered the composition of terrestrial ecosystems and the evolutionary trends of numerous organisms. Examples include the extinction of non-avian dinosaurs and the rise of mammals, the decline of gymnosperms, and the upsurge of angiosperms [6][7][8]. Some zoological cases indicated how animal taxa changed their morphological features during the K-Pg transition [8][9][10]. For instance, a previous study found that the maximum body size and dental complexity disparity of multituberculate mammals across the K-Pg boundary was associated with dietary expansion [8]. On the other hand, botanical evidence revealed regional vegetation change during this time interval [11,12] and provided estimates of paleotemperature in both hemispheres [2,[12][13][14]. Leaf physiognomic analysis showed that the 13°C mean annual temperature isotherm (mesothermal-microthemal boundary) of high latitudinal western North America moved southwards from ca. 65-75°N in the Maastrichtian to ca. 55°N in the Danian [15,16]. Besides, a study focused on the range changes of Metasequoia around the world since the Late Cretaceous has shown the knowledge of the distributional changes of this genus across the K-Pg transition [17]. Nevertheless, it has remained relatively unknown how specific plant taxa responded to the cooling during the K-Pg transition.
The extinct genus Mesocyparis McIver & Basinger (Cupressaceae) was distributed in eastern Asia and western North America from the Late Cretaceous to the Paleocene, ca. 70-60 Ma [18][19][20][21][22]. Here, we report new fossil material of this genus from the upper part of the Wuyun Formation, Jiayin County, Heilongjiang Province. The new Mesocyparis fossils from Northeast China, together with the other four species of this genus in the Northern Hemisphere, provide us with an opportunity to evaluate adaptions within the genus.  (Fig. 1b, c and Fig. 2b). Umbo near apex of scale, leaflike, erect, with apex tapered (Fig. 1b, c: arrow 2, 2a and 2b). Branches pinnate and opposite, forming flat frond-like sprays (Fig. 1d). Leaves on both vegetative and fertile shoots decussate, scaly and dimorphic with facial and lateral leaves (Fig. 1a, d, Fig. 2a and c).

Systematics
Holotype: compression fertile shoot with two mature seed cones and two immature seed cones, Palaeobotanical Museum of China, Institute of Botany, Chinese Academy of Sciences (IBCAS), specimen no. WY0801 ( Fig. 1)a, b and c.
Etymology: The specific name, sinica, refers to the fossil discovery in China.
Branching pinnate, flattened, branchlets plagiotropic, borne in opposite pairs, forming flat frond-like sprays, about 3.2 cm long. Basal pair of facial leaves arising from the axils of branch lateral leaves. Branchlets on same side parallel, 1.9-2.8 mm apart (Fig. 1d). Scale-like leaves dimorphic, facial leaves adpressed, lateral leaves folded along midrib.   There are some morphological differences between leaves of branches and branchlets ( Fig. 1e and f). Facial leaves of branchlets ovate to rhombic, 1.1-2.1 mm long, 0.8-1.6 mm wide, adpressed, thickened along midrib, apex acuminate to acute, rising above basal part of subsequent leaves. Lateral leaves of branchlets folded, falcate, 0.9-2.4 mm long, 0.4-0.8 mm wide, apex acute; most of them overlap basal part of subsequent lateral leaves (Fig. 1e). Facial leaves of branches 1.8-3.8 mm long, 0.4-0.6 mm wide, apex acuminate to acute, thickened slightly, rising slightly above basal part of subsequent leaves. Lateral leaves of branches folded, falcate, 1.7-2.7 mm long, 0.4-0.5 mm wide, with apex acute, and basal part covered by earlier facial leaves (Fig. 1f).

Comparison between M. sinica and other species of Mesocyparis
The newly-found fossils from Jiayin and other Mesocyparis records (Figs. 1 and 2) display significant differences ( Fig. 2 and Table 1): The seed cones in the Jiayin fossil were erect, with an acuminate apex, while those of M. borealis from Saskatchewan, Canada were reflexed, with an acute apex [18]. Seed cones from Jiayin were borne in opposite pairs, with cone scales ovate to obovate, and umbo near the apex of the cone scale, while the leaves of fertile shoots were scaly and dimorphic. In contrast, the seed cones of M. umbonata from Alberta, Canada were decussate, with peltate cone scales bearing a central umbo, and leaves of fertile shoots monotypic [22]. The cone scales of the Jiayin specimens are nearly equal in size, while the upper pair of cone scales of M. beringiana from Koryak, Russia was larger than the basal pair, while the apex of the umbo was acute. At the same time, the seed cones and foliage of M. beringiana were smaller than those of the other three species and the Jiayin fossils [19,20,22]. Most cone scales of M. rosanovii from the Amur Region, Russia were arranged in decussate pairs, only a few being borne in opposite pairs. Moreover, the cone scales were peltate, with an acute umbo apex, and the leaves of the fertile shoots monotypic.
Here we provide a key to the fossil species of Mesocyparis based on cone characters as below:

Phylogenetic position of Mesocyparis
Here we treat the Wuyun fossils as a new species based on a detailed morphological comparison, which shows two or more significant morphological differences between the Wuyun fossils and other Mesocyparis species (Fig. 2, Table 1  Our family-wide phylogenetic analysis indicates that Mesocyparis is a member of the Cupressoideae and clusters with Hesperocyparis, Callitropsis-Xanthocyparis, and Juniperus-Cupressus (BS = 51%) (Fig. 4). Mesocyparis shares whorled cone scales equal (Additional file 1: Char. 30:1 and Table S1), and ovule shape obpyriform (Additional file 1: Char. 34:2 and Table S1), with these five genera. The phylogenetic tree of Mesocyparis is shown in

Distributional changes during the K-Pg transition
Paleomaps [23] indicate that the landmasses of Eastern Asia and western North America were connected from the Late Cretaceous to the Paleocene. However, North America in the Late Cretaceous was divided by the Western Interior Seaway, while in the Paleocene western and eastern North America became contiguous due to the retreat of this seaway. Fossil records show Mesocyparis ranged from 59°N-76°N paleolatitude (Horseshoe Canyon, Alberta, Canada [18] and Kyoyak, Russia [18][19][20] during the Late Cretaceous (Fig. 6a) while in the Paleocene it ranged from 52 to 72°N paleolatitude (Amur, Russia [21] and Heilongjiang, China (this study) in Asia and Wyoming & Alaska, USA and Alberta, Canada [22] of western North America) (Fig. 6b). This Mesocyparis distribution (Fig. 6), combined with phylogenetic analysis (Fig. 5), reveals that the genus probably first occurred at high latitudes in the Northern Hemisphere and then divided into 2 clades -the East Asian (EA) clade and the Western North American (WNA) clade.

Morphological changes during the K-Pg transition
When plotting the scored 22 morphological characters against the species-level tree of Mesocyparis, we found some significant morphological changes in the genus from Maastrichtian to Paleocene: 1) seed cones became larger (average cone size increased about 130%, detail estimates in Methods), 2) the umbo shape on the scale changed from erect ( Fig. 2h and k) to reflexed (Fig. 2e), and 3) the umbo position on the cone scales moved from the middle (Fig. 2h and k) to the apex (Fig. 2e).
In general, the increasing size of the seed cone in Mesocyparis may be associated with the climate cooling   "*" indicating character differences between Mesocyparis sinica and other four Mesocyparis species from Maastrichtian to Paleocene across the K-Pg boundary. A negative correlation between the cone size and mean annual temperature (MAT) is apparent in modern Cupressus, one of the nearest living relatives of extinct Mesocyparis ( Fig. 7 and Additional file 4: Table S3). In addition, it has been observed that fruits and seeds of angiosperms in North America and Europe increased during the K-Pg transition [24]. Specific morphological features can be linked to a specific biological function, and may allow us to speculate about the reason behind Mesocyparis seed cones more than doubling in size across the K-Pg transition. The increasing size of seed cones might augment their visibility and attractiveness for herbivorous and/or omnivorous animals, thereby enhancing seed dispersal, which could expand the plant distribution and increase their ability to survive temporal and spatial environmental change. Today some bird species (such as azure-winged magpie and light-vented bulbul) feed on intact seed cones and we envisage similar feeding strategies existed in early birds during the K-Pg transition. The increasing size of the seed cone might be an adaptive mechanism for seed dispersal (i.e., conspicuously larger seed cones were more attractive for those cone-eating/removal animals). An animal's preference for larger seed cones might select and even amplify the increasing size of seed cones in Mesocyparis. Evidence has shown that the rapid radiation and diversification of birds [25,26] and mammals [27] that, occurred during this period produced numerous potential vectors for seed dispersal of both gymnosperms and angiosperms.

Cooling during the K-Pg transition drove Mesocyparis southwards
In general, the geographical distribution of plants at high latitudes is strongly constrained by the terrestrial thermal regime, particularly the mean coldest monthly temperature (MCMT) and/or the mean annual temperature (MAT) [28,29]. Thus, the northern limit of a specific terrestrial plant taxon can reflect temperature changes with relatively high sensitivity [30].
The cooling during the K-Pg transition (ca. 70-60 Ma, global average temperature fell by ca. 4°C) [31] could have driven the northern limit of Mesocyparis south by ca. 4°N from the Maastrichtian to the Paleocene (Fig. 8 and Additional file 5: Table S4), which means that the northern limit of survival for the plant retreated southwards by ca. 444 km. Similar responses are found in Metasequoia (Cupressaceae) and Nordenskioldia (Trochodendraceae). Metasequoia's northern limit moved south by ca. 4°( Fig. 8 and Additional file 6: Table S5), while Nordenskioldia's moved almost 5°( Fig. 8 and Additional file 7: Table S6). These findings indicate that the southward migration of the northern limit of Mesocyparis as driven by the cooling during the K-Pg transition is by no means unique.

Conclusions
We report a new fossil species, Mesocyparis sinica from the early Paleocene of Jiayin, Heilongjiang, northeastern China. By integrating lines of evidence from phylogeny and the comparative morphology of Mesocyparis, we found that during ca. 70-60 Ma, the size of the seed cone of Mesocyparis increased by ca. 130%, probably driven by the cooling during the K-Pg transition, which might be an effective adaptation for seed dispersal by animals. Meanwhile, our analyses indicate that the northern limits of this genus, as well as those of two other arboreal taxa Metasequoia (gymnosperm) and Fig. 4 The strict consensus tree obtained after addition of Mesocyparis (in bold) to the result of the two trees [48,49]. Numbers above the branches are bootstrap values (> 50%). Tax, Taxodioideae; Seq, Sequoioideae; Ath, Athrotaxidoideae; Tai, Taiwanioideae; Cun, Cunninghamioideae; Og, outgroups Nordenskioldia (angiosperm), have migrated ca. 4-5°s outhwards in paleolatitude during this interval, in association with the cooling. This is the first report to link plant evolutionary and ecological responses to the climate cooling during the K-Pg transition. This study not only enriches our understanding of how specific plant taxa responded to the climate changes during the K-Pg transition, but also provides a reversed case study for the scientific community to speculate about plant responses and distributional changes under global warming today.

Materials in this study
The fossil specimens of Mesocyparis sinica examined in this article are deposited in the National Museum of Plant History of China, Institute of Botany, Chinese Academy of Sciences: WY0801 (Figs. 1a, b and c), WY0802 (Figs. 1d, e and f), WY0803. These specimens were exposed and observed under a stereomicroscope, which identified by Dr. Yi-Ming Cui and Prof. Yu-Fei Wang. The characters were described following the terminology of [46]. Attempts were made to extract structure of seed cones under micro-CT and cuticles of leaf, but these efforts were unsuccessful.
Morphological and distributional information for the other four species of extinct Mesocyparis were collected from literature: M. borealis from [18], M. umbonata from [22], M. beringiana from [19,20,22], and M. rosanovii from [21].  The morphological and distributional information of extant Cupressus were measured and collected from specimens in the Herbarium of the Institute of Botany, Chinese Academy of Sciences, Beijing (PE) (Additional file 1: Table S1).

Phylogenetic analysis
We first used the "backbone constraint" tree approach [47] to determine the phylogenetic position of Mesocyparis in the Cupressaceae s.l. The constrained tree is modified from the two trees of Mao et al. [48] and Yang et al. [49], in which significant conflicting nodes (BS ≥ 85%) between the two trees were collapsed. The morphological matrix was modified from Farjon [46] and included 53 characters (Additional file 1: Table S1). Following the family-wide analysis, we selected Juniperus monticola, Xanthocyparis vietnamensis, and Hesperocyparis arizonica as outgroups and built the species-level relationships of Mesocyparis. All five species of Mesocyparis were included. The 22 morphological characters were coded based on the literature [18][19][20][21][22] and our observations (Additional file 3: Table S2). Phylogenetic analysis for each matrix was conducted using maximum parsimony method in PAUP* version 4.0b10 [50]. Heuristic searches were performed with 1000 random sequence addition replicates, tree-bisection-reconnection branch swapping, MulTrees in effect, and steepest descent off. Bootstrapping was carried out with 1000 replicates, using a heuristic search strategy.

Estimates of average cone size changes during the K-Pg transition
Here we use a volume formula of an ellipsoid to estimate the average cone size of each species in Mesocyparis, for the shapes of the seed cones in this genus are ovoid to ellipsoidal. Therefore, we set the seed cone length (scl) as the polar-radius of the ellipsoid and seed cone width (scw) as the two equatorial-radii. The estimated volume (V) of Mesocyparis is expressed by the following ellipsoid volume equation:  [31] so the increase in volume of Mesocyparis from the Maastrichtian to the Paleocene was about 136%.

Correlational analysis of seed cone sizes of Cupressus and temperature
To explore the influence of climate change on the morphology of seed cones in Mesocyparis, we calculated the potential relationship between the seed cone sizes of extant Cupressus and temperatures in their habitats. We examined 45 samples of five Cupressus species (C. chengiana S. Y. Hu, C. funebris Endl., C. duclouxiana Hickel, C. torulosa D. Don and C. gigantea Cheng et L. K. Fu) naturally occurring in China from PE Herbarium, which have female cones and entire information on locality and elevation. The diameters of the female cones were measured to represent their seed cone sizes. Mean annual temperature (MAT) used the climate data set of New et al. [51] and then corrected for the altitude of the plant locality using a temperature lapse rate of 5°C /km (Additional file 4: Table S3).

Paleogeographic analysis
The coordinates of paleolatitude and paleolongitude were compiled from previous reports of Mesocyparis [18][19][20][21][22], Metasequoia (Cupressaceae, gymnosperm) [17] and Nordenskioldia (Trochodendraceae, angiosperm) [52] in the literature, and converted from today's latitude and longitude to those inferred for the Cretaceous and Paleocene using "Point Tracker for windows" software, to plot the sites on paleogeographic maps representing two intervals of geologic time. The base maps with paleocoastlines were modified from paleogeographic maps of the late Cretaceous (ca. 94 Ma) and the Paleocene (ca. 60 Ma) from Scotese [23].