As a famous edible mushroom, Morchella owns important ecological functions and has high commercial value around the world (Dissanayake et al., 2021; Wu H. et al., 2021; Yu et al., 2022). It was popular research in taxonomy, species diversity, distribution, ecological diversity, phylogeny, biogeography, and artificial cultivation of Morchella species (Annette et al., 1978; Dahlstrom et al., 2000; Hao et al., 2019; Liu et al., 2019; Ali et al., 2021; Cao et al., 2022; Deng et al., 2022). The distribution of Morchella exhibits a high level of cryptic speciation and provincialism due to phenotypic plasticity and unreliable morphological species recognition (O’Donnell et al., 2011; Du et al., 2012a, 2015, 2018; Richard et al., 2015;). There are 72 phylogenetically distinct species in the world that have been recognized in this genus based on GCPSR (Loizides et al., 2015, 2016; Yatsiuk et al., 2016; Baroni et al., 2018; Du et al., 2019). In China, which was known as the center of Morchella species diversification and rich floristic diversity, a total of 16 Elata clades and 27 Esculenta clades phylospecies have been recorded (Du et al., 2012a,b, 2019). The phylogenetic species diversity of the Morchella in the Qinghai-Tibet Plateau subkingdom (QTPs) is not yet known, though.

Great changes in crustal movement on earth occurred during the Phanerozoic Paleozoic (4.6 billion years ago), after occurring of biological explosions and forming of thick sedimentary limestone (Mittermeier et al., 2011; Kate, 2022). The QTPs have been uplifted steadily and rhythmically since the end of the Early Tertiary period, which is famous as the “Third Pole” of the earth (Mao et al., 2021; Spicer et al., 2021; Xiong et al., 2022); and were known as the ecological barrier of China and even Asia based on complex topography, variable plateau climate, and rich ecosystem (Sun et al., 2012; Qin et al., 2015; Miehe et al., 2019; Liu et al., 2022). Several fossil records of QTPs illustrated the cradle of mammalian fauna and mountain flora in the Ice age (Deng et al., 2020; Mao et al., 2021; Wu Y. D. et al., 2021). For fungus in the QTPs, the opportunities for genetic variation and speciation were strongly increased via the isolation of high-altitude geographic and geological complexity. Furthermore, the QTPs are also called a refugium created by microclimatic variations that provided some protection, and situ speciation and relic persistence in the early originated lineages (Shrestha et al., 2010; Yuan, 2015; Yan et al., 2017; Phonepaserd et al., 2019; Mao et al., 2021). The rapidly radiational differentiation of Morchella species was reported in North America, Asia, and Europe. M. rufobrunnea (Rufobrunnea clade), as the oldest taxon of the genus Morchella and might diverge into the basal lineage, originated in western North America in the late Jurassic (O’Donnell et al., 2011; Du et al., 2012a, 2015; Loizides et al., 2016, 2021). During the emergence of the Mid-Continental Seaway and the subsequent uplift of the Rocky Mountains, the ancestors of the Esculenta and Elata clades spread to eastern North America from western North America in the early Cretaceous (Sanmartín et al., 2001; Donoghue, 2008; Du et al., 2015). After that, Morchella experienced widespread extinction due to the new folding of the Rocky Mountains and the uplift of the Sierra Madre Oriental Range in central North America. It is also speculated that the Morchella species spread to Europe and Asia from North America via the Thulean North Atlantic Land Bridge and the Beringian Land Bridge (Du et al., 2015). During the middle Miocene to the Pleistocene, the Morchella species in East Asia and Europe rapidly evolved under the gradually cooling climate and environmental heterogeneity caused by the rise of the Qinghai-Tibetan Plateau (O’Donnell et al., 2011; Du et al., 2012a, 2015). However, further information regarding the differentiation, speciation, origination, and evolution of Morchella in the QTPs is still unclear.

In this study, ITS rDNA sequences of 174 Morchella individuals collected from QTPs were generated for the aims of (i) investigating phylogenetic species diversity and geographic distribution of Morchella in the QTPs by using genealogical concordance phylogenetic species recognition (GCPSR); (ii) estimating divergence times of Morchella species lineages in the QTPs; (iii) defining the geographic distributions of ancestor lineages of the Morchella in the QTPs; and (iv) estimating divergence times and reconstructing ancestral regions for world-widely distributed species of Morchella.

This is the first time report the phylospecies diversity of Morchella in the QTPs used GCSPR, and the results indicate that there is 18 phylospecies of Morchella in QTPs, which far exceeds the number of published taxa for these areas (Du et al., 2012b, 2016, 2019). Generally, both Esculenta and Elata clades have preferred habitats, known as phylogenetic niche conservation (PNC; Donoghue, 2008). We investigated the occurrence and preferred habitats of Morchella in the QTPs and found that occurred in primary forests with high vegetation coverage, deciduous forests, or mixed coniferous and broad-leaved forests, such as the primary forests of Qilian Shan, Belong River, Mote, and Shangri-la, all of which belong to the eastern or northwestern margin of the QTPs. In addition, about 90% of the species are found in temperate deciduous forests, while 80% of the species were found in coniferous forests (Figure 1). It can be inferred that the occurrence of Morchella in the QTPs was accompanied by the establishment of the temperate deciduous biome and coniferous biome in the late Cretaceous (O’Donnell et al., 2011; Du et al., 2012a, 2015; Chen et al., 2015).

The maximum crown age of Morchella in the QTPs was estimated to be around the Late Cretaceous (98.62 Mya). The majority of intracontinental range expansions within Morchella in the QTPs appear to have taken place relatively recently between the middle Oligocene and Miocene. During that time, the uplift of the southwest block (Yunnan-Guizhou Plateau) and the eastern margin of the QTP resulted in the distribution of plant diversity in this region. Previous studies have shown that the Beringian Land Bridge (BLB) plays a key role in the spread of Morchella species from North America to Asia in the early Cretaceous, which was a natural route linking Eurasia and North America (O’Donnell et al., 2011; Du et al., 2012a, 2015; Loizides et al., 2016, 2021). Palaeobotanical data indicate that the BLB route played a crucial role in plant dispersal for Populus, Lonicera, Leibnitzia, and Linnaea (Wang et al., 2009; Qiu et al., 2011; Wen et al., 2014; Du et al., 2015; Wang et al., 2015; Wu et al., 2017). The divergence events exhibited by the plant taxa (Pinus, Abies, and Picea) confirm the geological events associated with species diversification, and those indigenous florae provide native secluded habitats for ancestral species of Morchella in QTPs (O’Donnell et al., 2011; Loizides et al., 2021). The Beringian Land Bridge as a species expansion channel is a critical driver of Morchella migrating to the QTPs between the Cretaceous and Paleocene when plant diversity was already established on QTPs, which means that the QTPs had formed a variety of suitable habitats for living organisms before in the Paleocene.

The differentiation of the Morchella is strongly linked to the geological movements of the QTPs (Figure 5). Palaeogeographical evidence suggests that plate activity and plateau uplift of QTP resulting from the collision are divided into four stages (Shi et al., 1998; Ding et al., 2022). (i) Late Cretaceous, the ancient Mediterranean crust subducted to the Eurasian continental crust, blowing the prelude to the plateau uplift. At this stage, Morchella of western North America is diverging from its closest relatives in the early Cretaceous, which are divided into Rufobrunnea, Esculenta, and Elata clades, and palaeogeological events played a driving role in the dispersal of Morchella (O’Donnell et al., 2011). (ii) Tertiary Period (Including the Paleocene, Eocene, and Oligocene), the Indian plate collided with the Eurasian plate, opening the prelude to the plateau uplift. The Indian plate rapidly drifted northward, the Indo-Pak subcontinent and the subcontinent were getting closer, and the ancient Mediterranean crust gradually disappeared during the Oligocene. In our data, M. norvegiensis = M. eohespera/Mel-19 was differentiated at 50.24 Mya with the new uplift belts of Tengchong-Bango formatted and the uplift area of Songpan-Ganzi shrank to the east during Eocene (Wang et al., 2008, 2022). Eocene (55.8 ± 0.2) was a key period for Asian paleoenvironmental changes and was characterized by a warmer climate than any other interval in the Cenozoic (Shi et al., 1998; Zachos et al., 2001; Hoorn et al., 2012). M. norvegiensis = M. eohespera/Mel-19 in the middle latitudes region were differentiated at 34.24 Mya with the further uplifted of Kunlun-Algin-Qilian during the Oligocene, which has been called the beginning of the present ‘icehouse’ epoch (Ling et al., 2021; Dieter et al., 2022). (iii) Neogene, the Indian and Tarim plates compressed and subducted to the QTPs with greater stress, and the plateau was greatly uplifted, forming the QTPs and the Himalayas. Our results indicate that 9 of the 18 QTPs Morchella species lineages (i.e., 50%) diversified between the middle Miocene and the present (Figure 4). (iv) Quaternary Period, the plateau was greatly uplifted, forming the present towering QTP. The Esculenta clade of Morchella in the QTPs was undergoing differentiation at this time. Tectonic activity and climate change in geological periods can form geographical isolation barriers to promote species differentiation and increase diversity, and can also reduce biological diffusion barriers to expand species distribution areas and increase biological exchanges between different regions (Che et al., 2010; Antonelli et al., 2018; Rahbek et al., 2019; Ding et al., 2020). Generally speaking, the formation and evolution of Morchella phylospecies in the QTPs were affected by the tectonic uplift of the QTPs and the geological movements of QTPs were an important force for the differentiation of the Morchella species (Merckx et al., 2015; Dai et al., 2019). The early marginal geological movement of the QTPs caused strong habitat fragmentation and rapid expansion of dry and cold habitats, which further strengthened the monsoon climate in East Asia and promoted the differentiation of microorganisms in the QTPs (An et al., 2001; Yang, 2005; Wan et al., 2014; Liu et al., 2016).

At least three geographic distribution patterns were discovered to correspond to the following phylogeographic structures. (i) the wide distributions around the QTPs: geographic locations of 4 endemic species, such as Mel-13, Mel-14, Mel-19, and M. esculenta, cross over three regions (Qinghai, Gansu, and Tibet in high, middle, and low latitudes respectively) of the QTPs. (ii) provincialism in the QTPs: the specific local distributions of two species in the Elata clade (M. pulchella/Mel-31, M. purpurascens/Mel-20) were unique (only in the Tibet region). (iii) the long-distance dispersal with latitude-based distributions: the divergence of Mel-13 and Mel-14 experienced two dispersal and vicariance events in three regions of the QTPs (Figure 6A). Mel-13 diverged in the mid-latitudes at 40.63 Mya and subsequently differentiated to the higher-latitudes at 34.76 Mya, which was based on latitude long-distance dispersal. Mes-19 also experienced dispersal and vicariance events in mid-latitudes and lower-latitudes, although this species originated from low latitudes (Yunnan) of the southwestern QTP (Figure 6B). To sum up, long-distance dispersals based on latitudes may have contributed to the current disjunct distribution ranges of Morchella, which is supported by biogeographic studies of plants and animals on the QTPs (Figure 7). The spread and differentiated way of Morchella in the global range, including intercontinental, intracontinental, and putative transoceanic long-distance dispersals, was slightly different from our result in QTPs (Du et al., 2012; Quan et al., 2014).

The complex topography and a special eco-climate of QTPs provide multiple periglacial microrefugia for Morchella. The Qilian Shan, located in the northeastern of the QTP, was defined as the ancestral region of the Elata clade as early as the Eocene. The Qilian Shan is an intraplate orogenic belt that experienced multiple episodes of fold and thrust deformation throughout the Mesozoic and Cenozoic periods, which controlled the evolution of regional climatic conditions in a broad region in inland Asia since the Miocene (Jia et al., 2022). The Shangri-La region in the southeast of the QTPs, which was the ancestors’ region for the Esculenta clade of Morchella, not only served as an important glacial refugium but also as a center of diversification for a variety of plants and animals (Suzuki et al., 2003; Yang et al., 2008, 2009; Harrison and Noss, 2017). The QTPs contain several important biodiversity hotspots, particularly along its southeastern margin (the Henduan Mountains and the eastern Himalayas), which were proposed to be glacial refugia for alpine hepialid species that effectively avoided extinction during the Quaternary glacial period (Hewitt, 2000; Dai et al., 2019).

Multiple pieces of evidence suggest that the Elata clade originated from QTPs, whereas the origin of the Esculenta clade was diverse. Our results show that the worldwide distribution species was an initial differentiation time of 73.78 Mya–35.67 Mya earlier than Central Asia, Europe, and South Africa (Figure 8). During the Early Tertiary (65.5–23.03 Mya), the Indian plate collided with the Eurasian plate opening the prelude to the plateau uplift, which is the emergence time of modern organisms (Liu and Hu, 2003). For example, in M. norvegiensis = M. eohespera/Mel-19, the divergence time was estimated at 52.25 Mya, which was earlier than that of Europe (Supplementary Figure S4). The Morchella was out of QTPs probably because of Eurasia’s collision and plate migration. On the other hand, the probability of QTPs as the most likely ancestral region for these widely distributed species is greater than that of Europe, North America, and South Africa (Table 2). All world-widely phylospecies have experienced historical extinction events that may be caused by the palaeogeology events and tectonic movement of the QTPs during the Eocene, followed by the differentiation and expansion of the two clades of Morchella (Figure 8). For the Elata clade, Morchella originated from Qilian Shan and spread along the Himalayas on the edge of QTPs to surrounding Europe and South Africa. However, after the Esculenta clade originated from Shangri-La and spread to other regions of Asia, it was subsequently blocked by the geographical isolation formed in the Quaternary, which led to the diversification of the origin of the Esculenta clade. This result further enriches the theory of species origin in QTPs, except for the reported species such as Floccularia luteovirens, Ophiocordyceps sinensis, Saccharomyces pastorianus, etc. (Quan et al., 2014; Dai et al., 2019; Wu Y. et al., 2021; Bai, 2022; Guo et al., 2022).

In this study, a total of 216 individuals were identified 18 Morchella phylospecies of two clades (Elata and Escuelnta clade), and the results indicate that the Morchella’s phylogenetic diversity within the QTPs far exceeds the number of published taxa for these areas. The divergence time of the 18 Morchella phylospecies occurred in the Eocene-Pliocene period (50.24–4.2 Mya), it was strongly related to the geological strike-slip and the uplift movements of the QTPs, suggesting that the geographical movements had a large influence on the differentiation of the Morchella. Furthermore, the reconstructed ancestral areas of the Elata and Esculenta clades indicate that the northwestern and southeastern regions of QTPs are the likely ancestral area, which also has been suggested to coincide with the glacial refugia in the Quaternary. Moreover, we confirmed that Elata and Esculenta of Morchella originated from QTPs and spread out with plateau geological uplift, transoceanic or transcontinental long-distance dispersal, orogeny, or geographical movements during the Cenozoic Era. The origin of the Esculenta clade is not unique. These results offer strong evidence for the theory of the origin of species in the QTPs.

The data presented in the study are deposited in the NCBI repository, accession number of those data was list in Supplementary Table S1.

QM: conceptualization, methodology, and writing-original draft. ZX: supervision, validation, and writing review and editing. HX, JG, and QP and TM: writing-review and editing. YT, BW, YM, SY, JY, YD, FZ, and TG: investigation. All authors contributed to the article and approved the submitted version.

This work was supported by the Natural Science Planning Project of Qinghai Province (Grant no. 2021-HZ-802), and the first batch of central forestry and grassland ecological protection and restoration funds in 2021 (2021-87).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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