Oldest (ca. 518 Ma) Mariana type oceanic subduction initiation ophiolite: constraining initiation of modern oceanic plate tectonic regime

Initiation of stable Mariana type one-sided oceanic subduction zones requires rheologically strong oceanic lithosphere, which developed through secular cooling of Earth mantle. This enabled the development of focused high stress zones resulting in narrow weak zones of convergence with resultant oceanic subduction leading to mantle hydration and arc magmatism. Based on detailed study and identication of the oldest (518 Ma) Mariana type oceanic subduction initiation ophiolite (Munabulake ophiolite) on Earth from northern Tibet, along with compilation of oceanic subduction initiation ophiolites through Earth history, we argue for the initiation of modern plate tectonic regime by at least the early Cambrian. The mantle and crust members of the Munabulake ophiolite preserve a complete ophiolite stratigraphy. Blocks of layered marble and siliceous rocks interlayered with meta-basalt indicate a marine environment. Zircons from an olivine gabbro sample yield a concordant age of 518 Ma, along with mantle derived low δ 18 O (2.69 ‰ – 5.7 ‰ ) and high εHf(t) (11.1–13.6) values. The zircons also have varied H 2 O contents ranging from 109–1339 ppm with peaks at 260 and 520 ppm, indicative of hydration of mantle derived magma. The highly depleted peridotites display U–shaped REE patterns and varied Zr/Hf ratios, whereas spinel and olivine compositions within the peridotites indicate that they are residues of various degrees of melt extraction and evolved from abyssal to fore-arc peridotites. The crustal members of the ophiolite are mostly tholeiitic, display at REE patterns and lower HFSEs, comparable to transitional lavas associated with Mariana subduction initiation ophiolite. Some rocks from the crustal section of the ophiolite display NMORB-like compositions but are also characterized by depletion in HSFEs. Therefore, the Munabulake ophiolite displays a chemical duality and progressively evolved from MORB (mid-ocean ridge basalt) to SSZ (supra-subduction zone) compositions, consistent with observations from zircon Hf-O isotopes and H 2 O contents. Furthermore, the ophiolite was formed during subduction initiation of the Proto-Tethys Ocean at the northern Gondwana margin, and coincided with an inferred slab roll back event in the southern Gondwana margin at ca. 530 − 520 Ma, indicative of a time of global tectonic re-organization. The early Cambrian Munabulake ophiolite indicates comparable slab strength and conditions to those that characterize modern plate tectonics. Such a tectonic regime coincided with nal Gondwana assembly, and was associated with ca. 530 − 520 Ma global tectonic reorganization.


Introduction
The commencement and evolution of the plate tectonic regime on Earth is linked to secular mantle cooling and associated increasing lithospheric strength (1)(2)(3)(4). Some form of plate tectonics is generally inferred to have initiated in the late Archean, as mantle potential temperature dropped below ΔT = 250 °C (relative to present-day values) and lithosphere strength increased (4)(5). In the Neoproterozoic-Cambrian, during Gondwana assembly, mantle temperature dropped below ΔT = 80-100 °C, lithosphere strength further increased, enabling deep plate subduction and corresponding with the widespread record of high P/T UHP metamorphic assemblages, and is accepted as the start of a plate tectonic regime that continues to the present day (1,(6)(7). This period also marks signi cant changes in sur cial climate and biosphere, marking the beginning of the contemporary Earth (8 and references therein).
Subduction initiation is a key component of the plate tectonic paradigm and results in the closing of ocean basins, culminating in arc-continent and continent collisions, and exerts a major control on the modern Earth system through exchange between sur cial and solid Earth reservoirs (9). Key factors in the initiation of a stable modern, Mariana type, intra-oceanic subduction zone includes high slab strength, weak zones of focused stress, and mantle wedge hydration (2). It is characterized by the formation of a proto-arc ophiolite (also referred to as a subduction initiation ophiolite), subsequently preserved in the fore-arc of the resultant convergent plate margin, and contains boninites, basalts, gabbroic rocks, and mantle peridotites (10)(11)(12). The igneous sequences are derived from mantle sources that evolved from the combined effects of melt depletion and subduction-related metasomatism, and form within 7-10 Ma of subduction initiation (11)(12)(13). Ophiolites that form at the initiation of subduction are, however, rarely preserved (12). Examples include the Izu-Bonin-Mariana ophiolite (11,14), the late Cretaceous Tethyan ophiolites (13,15), the early Carboniferous Paleo-Asian Ocean ophiolite (16), and the early Ordovician Appalachian-Caledonian ophiolites (17)(18).
In this paper, we document the ca. 518 Ma Munabulake ophiolite from the Southern Altyn Terrane (also referred to as the Southern Altyn HP-UHP belt) in northern Tibet. Ophiolite stratigraphy, eld relations, ages, zircon Hf-O isotopes and H 2 O compositions, along with whole rock and mineral compositions argue for formation during intra-oceanic subduction initiation. This ophiolite is the oldest ophiolite formed during initiation of Mariana type oceanic subduction in Earth history, which we link to other subduction initiation ophiolites in Earth history and argue for high oceanic slab strength and initiation of modern plate tectonics at least since early Cambrian. The Munabulake ophiolite is also the rst record of the subduction initiation of the Proto-Tethys Ocean at the northern Gondwana margin (19)(20)(21), and coincides with a period of global tectonic re-organization.

Geological background
The Tarim Block lies between the Central Asian Orogenic Belt (CAOB) and the Tibetan Plateau (Fig. 1a).
The block is largely covered by desert but Precambrian rocks are exposed around its margins (22)(23)(24), and its southern margin is marked by the early Paleozoic Kunlun and Altyn orogenic belts (Fig. 1a, [25][26][27][28][29]. The Altyn Belt records multiple orogenic cycles, including Archean-Paleoproterozoic and latest Mesoproterozoic-Neoproterozoic events that were extensively overprinted in the early Paleozoic (24,(28)(29)(30)(31). Its northern boundary is the northern Altyn fault zone, but its southern margin is less well constrained and is offset by the Cenozoic reactivation of the Altyn fault zone associated with India-Asia collision (Fig.  1a, 26,31). The Altyn Belt is divisible into three Precambrian-early Paleozoic continental terranes (the north, central and south Altyn terranes) and two early Paleozoic ophiolite belts (the Hongliugou-Lapeiquan and southern Altyn ophiolitic belts) (Fig. 1a). The Hongliugou-Lapeiquan belt delineates the boundary between the north and central Altyn terranes, whereas the southern Altyn ophiolitic belt is located at the southeastern margin of the South Altyn Terrane (Fig. 1a). The boundary between central and south Altyn terranes is inferred to be an unnamed strike slip fault (Fig. 1a).
The North Altyn Terrane contains an assemblage of Archean to Paleoproterozoic igneous and metamorphic rocks which are considered to constitute the basement to the Tarim Block, and are unconformably overlain by Mesoproterozoic strata (24,32). The central Altyn Terrane, also referred to as the Milanhe-Jinyanshan terrane, is dominated by marble and meta-clastic sedimentary rocks that are covered by a thick layer of limestone-dolomite, both assigned to the Mesoproterozoic Jixian system, but latest investigations suggest Neoproterozoic to early Paleozoic ages (author unpublished data, 33). The units are unconformably overlain by limestone and clastic sedimentary rocks of the early Paleozoic Suoerkuli Group. Voluminous early Paleozoic granitoids dated at 522-430 Ma, mostly S-type, intrude into these sequences (34)(35). Meta-ma c arc sequences dated at ca. 520-510 Ma occur within the northern margin of the terrane (author unpublished data). The Southern Altyn Terrane consists primarily of the Altyn Complex, which consists of a suite of granitic gneisses and meta-sedimentary rocks, including ortho-and paragneisses, marbles, amphibolites, and minor meta-ma c-ultrama c rocks (30,32). The complex was considered to be Archean-Paleoproterozoic in age (32), but recent works indicate the complex contains latest Mesoproterozoic to early Paleozoic units, which experienced multiple stages of metamorphism and deformation at 500-430 Ma, as well as events at, or after, ca. 235 Ma (30, 36, author unpublished data). An inferred ophiolitic mélange of early Paleozoic age is also present in the complex. HP-UHP metamorphic rocks occur as lenses within the Altyn Complex, including UHP eclogite and kyanite-garnet bearing pelitic gneiss, HP-UHP garnet lherzolite (27)(28). These HP/UHP rocks yield metamorphic ages of ca. 509-475 Ma, with their protolith constrained to ca. 840-750 Ma (27). Granitic plutons with variable compositions (I-, S-and A-types) intrude into the Altyn Complex. Limited dating on these intrusions yield ages of 462-440 Ma (e.g. [34][35][36][37]. The southern Altyn ophiolite belt and the Hongliugou-Lapeiquan ophiolite belt are inferred to be fragments of the Proto-Tethys Ocean (20,28). Some recent work suggests these ophiolites were part of a single subduction zone repeated by oroclinal bending (20). The Hongliugou-Lapeiquan ophiolitic belt consists mainly of sheared and deformed ophiolitic rock units of both supra-subduction zone (SSZ) and mid-ocean ridge basalt (MORB) a nities, and are dated at 518 ± 4 Ma, 513 ± 3 Ma, and 479 ± 8 Ma (SHRIMP zircon U-Pb and LA-ICP-MS zircon U-Pb; [38][39]. Ophiolite components include serpentinized harzburgite and lherzolite, ma c-ultrama c cumulates, sheeted dykes, and pillow basalt (38). The South Altyn ophiolite belt, extending more than 700 km along strike from Mangya to Tula lies within the Altyn fault zone and contains ophiolite mélange and ysch. The sheared ophiolite sequences are not well preserved and occur as disrupted blocks of serpentinized dunite, harzburgite and some gabbroic rock, dated at 510 ± 1 and 501 ± 2 Ma (37 and references therein). Ca. 517 Ma and 503 Ma adakite also occurs within this ophiolite belt (e.g. 37 and references therein).

Stratigraphy of the Munabulake ophiolite in the South Altyn Terrane and sampling
The Munabulake ophiolite, occurs as a tectonic block within the Altyn Complex in the South Altyn Terrane (32). It was extensively sheared and deformed into an overall diamond shape. The ophiolite is thrust upon other units of the complex at its northern margin, whereas on its southwestern margin, a NW-SE directed sinistral strike slip ductile shear zone delineates the boundary with the remainder of the Altyn Complex (Fig. 1b, 1c). Original contacts between units within the complex remain unclear as they experienced long-term high-grade metamorphism and extensive deformation at ca. 500-420 Ma and in the early Mesozoic (27,31).
From southwest to northeast, and corresponding with the progression from base to top, the Munabulake ophiolite is composed of sheared serpentinite along the basal thrust, serpentinized dunite-harzburgite, pyroxene peridotite, olivine pyroxenite, gabbro, and meta-basaltic and meta-intermediate igneous suites, along with blocks of marble ( Fig. 1b, 2a, 2b). Fine grained siliceous rocks ranging in size from centimeters to meters thick are interlayered with the basaltic and intermediate igneous suites (Figs. 2c). They are interpreted to represent recrystallized chert and are indicative of a deep-sea marine environment. This is consistent with layered marbles that are also present in some localities (Fig. 2d). Ultrama c blocks of serpentinized dunite and pyroxene peridotite, up to 7 km thick, are exposed in the northwest segment of the ophiolite (Fig. 1b, 1c, 2b). An olivine pyroxenite block, approximately 2 km thick, also occurs between blocks of serpentinized dunite and pyroxene peridotite, whereas a harzburgite block is sandwiched between blocks of serpentinized dunite (Fig. 1b). The ultrama c rocks, generally serpentinized, are interpreted as the lower mantle components of the ophiolite. A structural block that consists mainly of meta-gabbro and meta-basaltic-intermediate suites occurs within the ductile shear zone along the southwest margin of the ophiolite (Fig. 1b, 2e

Results
Zircons from the meta-gabbro are euhedral grains and show banded zones in CL images, comparable to zircons crystallized from ma c magmas (Fig. 3). Thin metamorphic rims are visible but are too narrow for U-Pb age analyses (Fig. 3). The analyzed grains display Th/U ratios of 0.5-3.0 (Table S1), which are comparable to those of magmatic zircons. Forty-two analyses from the meta-gabbro sample 17SAT13-2 yielded a concordant 206 Pb/ 238 U age range of 543-506 Ma and a weighted mean age of 518 ± 2 Ma (MSWD = 0.94, n = 40) (Fig. 3).
Zircon grains from meta-gabbro sample 17SAT13-2 display present day 176 Hf/ 177 Hf ratios of 0.282792-0.282863, equivalent to εHf(t) values of 11.1-13.6 and Hf model ages of 566-670 Ma (Fig. 4a). Zircon grains from this sample also have δ 18 O compositions of +2.69 ‰ -+5.7 ‰ (Fig. 4b) that are comparable to, and lower than, mantle zircons (40). Thus, zircon Hf-O isotopes argue for crystallization of zircon grains from mantle derived ma c magmas. In addition, H 2 O-in-zircon contents of these grains range between 109 ppmw and 1339 ppmw, with two peaks at 260 and 520 ppmw. No obvious correlation between zircon δ18O values and H2O contents has been observed (Fig. 4b).
This study focuses on the immobile elements and element ratios, considering the potential for mobilization of large ionic radius elements during metamorphism and alteration. In addition, oxides in this study were recalculated on an anhydrous (volatile-free) basis, with the oxide sum normalized to 100 %.  (Fig. 5a, 5b). Two basaltic samples also display both LREE and HFSE depletions, whereas the gabbro sample shows minor at light rare earth element (LREE) enrichment (Fig. 5c, 5d).  (Fig. 5c, 5d).
Less serpentinized harzburgite samples 17SAT22-1 and 18SAT41-4 and olivine pyroxenite sample 18SAT41-5 were selected for olivine and spinel compositional analyses (Fig. 2f). The olivines display a uniform composition (Fig. 6) and in the pyroxenite has Fo values of 90.9-91.7 and NiO contents of 0.256-0.524 wt.%, whereas in the harzburgite shows Fo values between 89.9 and 91.9, and NiO contents of 0.25 and 0.46 wt.% (Fig. 6b). Peridotites spinels are resistant to secondary alteration processes and preserve environmental records of peridotites formation (reference). Oxidized rims can be observed in some spinels, so analyses were only undertaken on fresh cores. The analyzed spinels display a wide compositional range. The Cr# values of spines in the olivine pyroxenite (26-77) are lower than depleted harzburgite (71-84) and a linear trend of Cr# vs Mg# values for spinels can also be observed (Fig. 6c). TiO 2 contents are lower in the olivine pyroxenite (0-0.14 wt.%) than those in the harzburgites (0.11-0.20 wt.%) (Fig. 6d), whereas the NiO contents display a decrease from olivine pyroxenite to harzburgite.
Chemical duality of the Munabulake ophiolite: progressive evolution from MORB to SSZ compost ions during intra-oceanic subduction initiation The mantle members of the Munabulake ophiolite consists of residual dunite, harzburgite, and olivine pyroxenite (Fig. 1b). The negatively correlated Cr# vs Mg# values of spinels within harzburgite and olivine pyroxenite samples suggest that the investigated rocks represent residual mantle, with the olivine pyroxenite related to low degrees of partial melting and melt extraction and the harzburgite to higher degrees (Fig. 6b). The correlation between Mg# values of olivines and the Cr# values of spinels is conformable with the olivine-spinel mantle array (Fig. 6c), also indicating the peridotite samples are residues of various degrees of melt extraction. Spinel TiO 2 vs Cr# trend suggests a similar magma process (Fig. 6d). The studied residue ultrama c samples have higher Mg# (> 90) values than primitive mantle, and also display U-shaped REE patterns and that are indicative of melt introduction (Fig. 5c), consistent with high ux and volatile conditions in a supra-subduction zone setting. Melt or uid metasomatism can also be observed from tremolite and enclosure of olivine grains in orthopyroxene in peridotite, as are their varied Zr/Hf ratios. The magma evolution thus re ects progressive source depletion coupled with increasing melt or uid metasomatism. Therefore, compositions of ultrama c rocks and spinel-olivine minerals are consistent with progressive chemical evolution from abyssal to arcrelated peridotite (Fig. 6).  (Fig. S1a), where most of the samples lie above the mantle array, indicating Th enrichment in their mantle source, which is consistent with uid ux melting and was likely derived from subducted slab sediment.
Moreover, U/Th-Th/Nb and Ba/Th-La/Sm diagrams (not shown) also favor involvement of hydrous uids from altered oceanic crust. This chemical evolution trend indicates increasing source metasomatism by slab-derived material (elevating Th, U, and some LREE).
The analyzed samples plot in the SSZ type ophiolite eld in various tectonic discrimination diagrams, including Ta/Yb vs. Th/Yb and Ti vs. V (Fig. S1c). Their lower TiO2 (< 1.25 wt. %), Zr/Y (< 3), Nb/La (< 0.5), Hf/Th (mostly < 2), and Ta/Yb (< 0.1), along with higher Th/Nb (> 0.2) and Th/Yb (> 0.1) (Table S2), are comparable to those of oceanic arc basalts (41). Although these compositional features indicate metasomatism by a subducted slab, their at REE patterns argue against an island arc setting (Fig. 5a), as do their mostly tholeiitic compositions. In addition, even compared to lavas of nascent oceanic island arcs such as Saipan-Rota-Guam (42), the investigated ma c-intermediate samples lack marked Eu and Ti anomalies (Fig. 5b). This indicates no plagioclase fractionation and very minor rutile fractionation. These signatures suggest that no normal arc or felsic crust had formed at the time of ophiolite formation. In addition, the higher Y/Zr values of these igneous rocks argue for derivation from depleted mantle source (11), whereas Hf/Nb versus Zr/Nb suggests magma enrichment by subducted uids (43). The overall chemical signatures and evolution trends are comparable to crustal members of the IBM and Tethyan ophiolites (Fig. 5, S1; [13][14], consistent with the observations from Munabulake mantle end-members. More importantly, H 2 O contents in mantle zircons within the ophiolitic gabbro indicate similar processes, with two populations at 160-320 ppmw and 480-640 ppmw (Fig. 4b). The zircons with low H 2 O content are comparable to MORB zircons (44), whereas the zircons with higher water content indicate possible progressive magma hydration due to formation of a subduction zone. In addition to the mantle zircons in the ophiolite, zircons with lower δ 18 O isotopes (+2.69 ‰ -+5.0 ‰, peaked at 4.7‰) are also observed, which are comparable to altered oceanic crust (45). All the observations suggest linkages between progressive source depletion and metasomatism due to slab-derived uids, which is commonly expected for a subduction initiation ophiolite and is consistent with observations from mantle end-members. However, it is noteworthy that the ma c-intermediate units occur within a ductile shearing zone and were subjected to multi-stage higher grade metamorphism and shearing. Thus, detailed reconstruction of overall chemo-stratigraphy is di cult.
The overall ages and eld relations indicate that the 518 Ma Munabulake ophiolite is the oldest oceanic succession in the Southern Altyn, followed by establishment of an intra-oceanic arc system lasting some 20 Ma at least until ca. 500 Ma, as is inferred from the 510-500 Ma MORB type ma c-ultrama c suites and ca. 503-497 Ma arc type granitoids and adakite-diorite. Thus, the crustal and mantle members of the Munabulake ophiolite, along with available data across the south Altyn, agree with the subduction initiation signature of the 518 Ma ophiolite, which we conclude to be have been formed during subduction initiation of the Proto-Tethys Ocean, in a scenario similar to the IBM ophiolites (Fig. S1). The reported ca. 517 Ma oceanic type adakite was generated by partial melting of oceanic crust in a newly formed subduction zone. Moreover, given the large-scale sinistral shearing of the south Altyn fault zone, we infer that the Munabulake ophiolite should be a member of the south Altyn ophiolite belt and was offset to current location by strike-slip motion of the fault system.
Global plate re-organization at ca. 530-520 Ma during Gondwana assembly The Munabulake ophiolite dates the subduction initiation of the Proto-Tethys Ocean in the Altyn segment, but the overall subduction initiation of this ocean is not well constrained and earlier inferred individual subduction zones have largely been treated in isolation (e.g., 19,21,25,28,[46][47]. In addition, the ocean has been given a number of localized names adjacent to the variety of continental and arc related blocks in East Asia that are inferred to lie within the ocean. The early Paleozoic oceanic successions in these blocks are related to the evolution of the ocean and accretion of these blocks to the northern Gondwana margin (e.g., [19][20][21]47). Nevertheless, here in this study, a time-space plot of early Paleozoic ophiolites and trench-arc assemblages across East Asia blocks, along with related magmatic and metamorphic events, enables determination of overall timing of initial oceanic subduction of the Proto-Tethys Ocean (Fig. 7). In particular, elsewhere across East Asia, ages of initiation of subduction are inferred from the oldest arc magmatism, which are in accordance with stratigraphy and ages of ophiolite and metamorphic event (Fig. 7). We conclude that the main branch of the Proto-Tethys Ocean principally subducted northward and commenced at ca. 533 Ma in the West Kunlun segment (25)(26), at ca. 525-520 Ma in the Altyn-Qaidam-Qilian segments (28, 46; this study), and at ca. 515 Ma in the North Qinling segment (Fig. 7,  8; 47). Locally, possible isolated subduction zones in the Qiangtang and Indochina segments commenced at sometime around 535 Ma and 490 Ma (19,21,49), but these dates are not well constrained. Therefore, overall timing of oceanic subduction initiation of the Proto-Tethys Ocean displays an eastward younging direction from ca. 533 Ma in the west segment to ca. 515 Ma in the east most segment.
It is noteworthy that the timing of initial subduction of the Proto-Tethys Ocean coincides well with the timing of slab roll-back of the Paci c Ocean in the southern Gondwana margin (50) (Fig. 8). The extension regime in the southern Gondwana and expression regime in the northern Gondwana was thus strictly correlated, indicative of global plate re-organization at this time, which is linked to nal collisional assembly of Gondwana.

Subduction initiation ophiolites in Earth history: initiation of modern plate tectonic regime
Ophiolites formed during simultaneous subduction initiation of a modern Mariana type oceanic subduction zone include the 52 Ma Izu-Bonin-Mariana ophiolites and Tonga ophiolite in west Paci c (9,(11)(12)14), 100-90 Ma Tethyan ophiolites (13,15), the ca. 335 Ma Paleo-Asian Ocean ophiolite (16), 490-485 Ma Appalachian-Caledonian ophiolites (17)(18), and the 518 Ma Munabulake ophiolite (Proto-Tethys ophiolite) in northern Gondwana margin in this study (Fig. 8). Given the conditions required for simultaneous initiation of stable one-sided modern Mariana type oceanic subduction (2), the key factors of oceanic plate tectonic regime, slab strength in particular, must have been comparable at least since the early Cambrian time. This was controlled by progressive cooling of mantle which increased slab strength (3,51). High slab strength, along with comparable ophiolite characteristics and scenarios of simultaneous oceanic subduction initiation since the early Cambrian, coincides well with the inferred transition from early plate tectonic regime to modern plate tectonic regime in the Neoproterozoic-Cambrian (7,52). Earlier conclusions on the transition of tectonic regime in the Neoproterozoic-Cambrian are mostly based on records of the Phanerozoic low T/P UHP metamorphic rocks and geodynamic modelling results that indicate drops of mantle temperatures below ΔT= 80-100°C at this time (Fig. 9), and thus plate strength was strong enough for deep plate subduction (1,6). On the other hand, the subduction initiation ophiolites discussed in this study also argue for modern plate tectonics at least since the earliest Phanerozoic, consistent with signi cant drops of mean thermobaric ratios at 525 Ma (Fig. 9). In addition, recent works also favor formation of Earth inner core in the Ediacaran (53), which might have feedback effects on plate tectonic regime. We suggest that following the global plate reorganization at ca. 530-520 Ma during Gondwana assembly, a plate tectonic regime with products and slab strength similar to modern day Earth was achieved.

Conclusion
The disrupted and sheared stratigraphy in the Munabulake area of the south Altyn Terrane comprises a disrupted ophiolite with mantle related components of residual peridotites and crustal components of meta-ma c and intermediate igneous suites. The ophiolite is interpreted as formed during the initiation of subduction of the Proto-Tethys Ocean in the northern Gondwana margin and is dated at ca. 518 Ma.
Varied siliceous layers within ma c lavas and blocks of layered marble establish a deep marine environment. Geochemical, isotopic, and mineral compositional data constrains a chemical duality, with progressive evolution from MORB to SSZ and is analogous to the Mariana subduction initiation ophiolite. The Munabulake ophiolite is the oldest simultaneous subduction initiation ophiolite on Earth and corresponded to a period of global tectonic reorganization in Gondwana margins. Overall distributions and formation conditions of subduction initiation ophiolites on Earth argue for a modern type oceanic plate tectonic regime at least since the early Cambrian, and corresponding with nal Gondwana assembly.

Declarations Data availability
All data generated during this study are included in the supporting information.