Posted on Authorea 2 Mar 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.161470351.19785260/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. otwsenCia oa f18smlsfo 9rgoswr nlzdfrbt iohnra and mitochondrial highly both three for revealed analyzed data nuDNA were and regions mtDNA 29 subspecies concatenated represented from the the samples of within lineages 128 analyses divergent of Phylogenetic of history total genes. demographic nuclear A and time, China. divergence southwestern genus speciation, the patterns, to phylogeographic belongs Shrew Himalayan The Abstract * China 610081, # Chengdu Forestry, of China. Academy 610065, Sichuan Chengdu 3 University, Sichuan China. Sciences, 610066, Life Chengdu of University, 2College Normal Sichuan Sciences, Life of 1College shaoying Liu the haijun in status. Jiang resulted species have to of may phylogeography elevated QTP and be the phylogenetic should of Molecular minor uplift the n. and S. periods and Miocene nigrescens. species, the propose S. putative We since of two change nigrescens. speciation of climate n. and species composed that S. diversification The be suggested and for it B. analyses to LGM also addtion, plotting and the In study appears skyline during A Our nigrescens nigrescens. Bayesian expansions Clades n. Clades. range n. two two S. and into S. of of demographic divided that Clades up supported were two also made Motuo the in modeling is of altitudes niche which status different ecological nigrescens, subspecies species at represented individuals the n. concatenated that group supported found the One S. methods, was two of subspecies nigrescens. on analyses represented S. phylogeographic based Phylogenetic the group 128 analyses, the within another of delimitation genes. investigated lineages total minors, nuclear divergent We A and highly n. mitochondrial three China. S. genus. revealed southwestern both data monotypic in for nuDNA nigrescens a analyzed and Soriculus were is mtDNA of regions which history 29 Soriculus, demographic from and genus samples time, the divergence to speciation, patterns, belongs Shrew Himalayan The Abstract 2021 2, March 3 2 1 Chen Shunde Jiang Haijun China southwest in Soriculus nigrescens of phylogeography and phylogenetic Molecular orsodn uhr hnsud:[email protected];Lusayn:[email protected] shaoying: Liu [email protected]; shunde: Chen Author: Corresponding iha cdm fForestry of Academy Sichuan University Sichuan University Normal Sichuan uhr otiue qal oti work. this to equally contributed Authors 1# 3* uchangkun Fu , 1 1 hnknFu Changkun , n hoigLiu Shaoying and , .n nigrescens n. S. .nigrescens S. 1# agkeyi Tang , 1 eiTang Keyi , 3 hc smd po w ldsAadB h pce delimitation species The B. and A Clades two of up made is which , n ru ersne subspecies represented group One . 1 ifengjun Li , Soriculus oiuu nigrescens Soriculus 1 egu Li Fengjun , 1 hc samntpcgns eivsiae the investigated We genus. monotypic a is which , 2 i fei Xie , 2 1 e Xie Fei , i boxin Qin , nsuhetChina southwest in 1 oi Qin Boxin , 1 hndan Chen , .n minors n. S. oiuu nigrescens Soriculus 1 1 hnshunde Chen , a Chen Dan , nte group another , 1 1* in , , Posted on Authorea 2 Mar 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.161470351.19785260/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. rm20 o21,attlo 2 ape fthe of samples 124 of total a 2019, to 2009 From differentiation alignment for and sequencing data Sampling, provide and the China methods and in and diversity Himalayas. genetic Himalayas Materials the the the in explore mammals in to small order formation of of in and diversity mechanism model evolution, differentiation species system of niche time and of the the lineages mechanism reshaped calculated its population, between population, the the relationship of the of structure dynamics genetic historical the the analyzed we study, on this data In research phylogenetic molecular of lack a , 2010). of Xie, synonym and (Smith Bhutan and divided and of 1842) (2003) classification India Motokawa The Nepal, dis- data, genus. China, the monotypic southwest phological affecting a in ( is factors distributed Shrew it key is Himalayan And impact the which The huge are Soricinae, shrews. et a humidity the Ye of had and to 2015; species it temperature al., and covered, that et number completely indicated Qu tribution, not (1987) 2014; was Reumer al., sheet by et ice Research Lei the 2014; although Jiang, age, and of Asian ice species. He the uplift the on 2001; of During The al., formation et genetic 2016a,b). and 2013). the (An distribution al., orogeny, organisms the al., affected existing period, profoundly et have extensive of glacial structure (Wu oscillations structure and 2000). climate Quaternary spatial vertebrates Quaternary endemism al., morphology, the violent thermostatic the of et the and and for level (Myers monsoon affect (QTP) high especially biodiversity events Plateau the high animals, climatic Qinghai-Tibet to have and of the due which Geological structure Asia of genetic East There 2014). all in and Jiang, world, 2015). biodiversity and the al., of (He species in et hotspot biodiversity of (Chen hotspots a diversity process ecological is species the 25 China biodiversity, of Southwest of research, of management biogeography total laws and of evolutionary a contents protection the are core the understanding the formulating of for and one significance formation, are climatic great and causes of of its Willows-Munro consequences is and 2011; which biodiversity genetic of the al., pattern 2015). exploring distribution et al., The for affected et Wierzbicki (Churchfield, models (Chen strongly 2010; vagility isolation were excellent low shrews topographic al., provide shrews and terrestrial and could et The changes of Shrews (Vega metabolism, evolution 2014). situations the high 2011). al., Topographic consequence, body, Matthee, a et and small As (Kent Climatic their mammals 2012). by topography, to al., small as et such due of Churchfield factors, Feng abilities evolution environmental 1990; 2014; historical affected dispersal and Jiang, strongly limited current and were Both have He are 2016b). habitat, 2009; 2016a, species (Avise, and many al., events climate of et paleogeological Ye structure and 2016; population al., and fluctuations variation et paleoclimate genetic by patterns, affected driving all speciation the that seems and It diversification the expansion Population in delimitation; resulted Species have Introduction demography; may Population QTP Shrew; Himalayan the The of Keywords: uplift the and periods of speciation Miocene expansions the range since and change demographic Bayesian of supported and Clades. also Clades species, two putative into for two modeling divided LGM niche the were the Motuo ecological of in during and status altitudes analyses different species at plotting the individuals skyline that supported found was methods, it two addtion, on based analyses, .sikimensis S. .n minor n. S. .radulus S. .nigrescens S. , .n pahari n. S. .n minor n. S. .n nigrescens n. S. hms 92 and 1922, Thomas, osn 80(ooaa 2003). (Motokawa, 1890 Dobson, . , .n caurinus n. S. hudb lvtdt pce status species to elevated be should epooethat propose We . .n.nigrescens n S. oiuu nigrescens Soriculus and , oiuu nigrescens Soriculus .n nigrescens n. S. oiuu nigrescens Soriculus .n centralis n. S. 2 oiuu irsesminor nigrescens Soriculus .n nigrescens n. S. a eadda eirsnnm of synonyms senior as regarded was notosubspecies, two into oiuu nigrescens Soriculus Mtkw,20) u urnl hr is there currently But 2003). (Motokawa, eecletdfo 8rgosi Tibet in regions 28 from collected were . u td losgetdta climate that suggested also study Our a ofsn eoe ae nmor- on Based before. confusing was . oiuu nigrescens Soriculus per ob opsdo two of composed be to appears a osdrda senior as considered was .n nigrescens n. S. .n nigrescens n. S. ry 82belongs 1842 Gray, ) n studied and , .aterrimus S. (Gray, In . Posted on Authorea 2 Mar 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161470351.19785260/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. n 2 00 for Two ( produce BPP size 2000) 1 in population analysis. (2, and ancestral B) 0 the the fine G algorithms Clade specify with in and that to 0 and included showed (G) algorithm ( priors A were analysis used Gamma-distributed original age nuDNA) We (Clade used the BPP. We + species as in results. 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No reuse without permission. — https://doi.org/10.22541/au.161470351.19785260/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. h N eut hwta h ouainepnintm a eearlier. Since be may period. time interglacial expansion last population the the the of Miocene. and that branch Late show maximum the each results glacial ENM to of last of back the range the size traced distribution of population the be LIG, fluctuations the can of the severe history B that the the Clade show that during and show ENM changed A time of divergence Clade of results of results branches The The two 1B). the (Fig between results differentiation prediction of good achieved ENM have the LIG) study, this In mismatch gene 19.0 nuclear modeling - of niche all (26.5 and Ecological and 4B). 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Data may be preliminary. hs h ieeteouinrtso tN n uN akr a edt h nossec fnuclear of inconsistency the to and lead conservative may markers be study. nuDNA the to this and of mask in mtDNA appeared structure revealed even of genetic nuDNA mitochondria rates may the The evolution evolution underestimated different which of 2007). the mtDNA, rate Thus, Brito, than existing high rate 1999; the excessively evolutionary (Hedrick, and lower the polymorphisms, have differentiation and enough high recognized, produce of well not signals be may nuDNA evolution cannot The of structure rate genetic sorting. genetic slow nuclear lineage The genetic less the incomplete differentiation. effective underestimated found which detect with We large mtDNA, consistent than relatively 2016b). rate is a evolutionary al., of lower structure as this et have (Ye and shallow, and conservative time be be mtDNA, it to sorting will appeared the DNA, long DNA mitochondrial relatively to nuclear a of compared of in pattern divergence observed pattern result mitochondrial the divergence will between in size the inconsistency results population that the sorting explain expected lineages to Ye be incomplete used 2014; can If often al., cause is et results. may Lin division nuclear markers 2012; lineages different and Brelsford, Incomplete of and incomplete (Toews 2016b). rate data The al., evolution gene et different factors. nuclear many the and by mitochondrial and between caused polymorphism inconsistencies be ancestral may of data ordering nuclear lineages and mitochondrial between inconsistency The distribution the with consistent clades. is two which pattern as a revealed data nuDNA the and for Cyt-B the on based analyses The climatic changed Pleistocene structure has of genetic mtDNA role range the and the predicted of Nuclear suggesting expansion the MYA, demographic revealed 0.02 the approximately analyses driving from ENMs in began changes Our events for expansion present past. the the the that and in LGM expansion clades the population two between indicated the analyses 2004; demographic 1996, for BSP (Hewitt, bottleneck America 2011). 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No reuse without permission. — https://doi.org/10.22541/au.161470351.19785260/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. pedxFgr n alslegends tables and Figure Appendix between distance Cyt-B 1:Mean of Table distribution mismatch of observed and plot line) skyline (solid Bayesian for Extended times 5A: model. divergence Fig BEAST and the analyses phylogenetic using Bayesian trees data species 4: gene delimitation Fig and species mtDNA+nuDNA of and nuDNA). 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S. .nigrescens S. ae nmrhlgcljdmn.I this In judgment. morphological on based . .nigrescens S. nTbtadYna rvne We province. Yunnan and Tibet in per ob opsdo two of composed be to appears ae ncmie genes. combined on based h ape olce in collected samples The . .n nigrescens n. S. nlddi hsstudy; this in included .n nigrescens n. S. .nigrescens S. The . .ni- S. based and , Posted on Authorea 2 Mar 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161470351.19785260/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. vs C 09 hlgorpy ersetadpopc.J igor 6 -5 di 10.1111/j.1365- (doi: 3-15. 36, Biogeogr. the J. of uplift prospect. phased and and retrospect monsoons Phylogeography: Asian (doi.org/10.1038/35075035) 2699.2008.02032.x) of 62-66. 2009. Evolution 411, JC. Nature 2001. Avise times. SC. Miocene Porter Late WL, since Prell plateau Himalaya-Tibetan JE, - Kutzbach ZS, MW670208 An MW670207; - MW670102 MW670101; References - MW670437. MW669978 31970399; - accessions MW670323 (31670388; Genbank MW670322; China sequences: of DNA Foundation Science Natural Accessibility National Data the by funded 32070424). was research (equal); This Supervision (lead); Resources Investiga-Funding (lead); (equal); administration (equal). acquisition draft Project Funding Writing-original (equal); (equal); (equal); Validation (equal); Methodology analysis acquisition Formal (equal); (equal). Funding (equal); editing (equal); tion (equal); & Conceptualization Investigation Writing-review Conceptualization curation (lead); (equal); shunde: administration curation Data shaoying: Chen Project Data (equal); Liu Investi- Methodology fei: dan: (equal); (equal); Chen Xie (equal). Investigation curation editing Data (equal). & boxin: (equal). editing Writing-review Qin fengjun: & editing Li Writing-review (equal). & (equal); editing (equal). Writing-review & gation cura- editing Writing-review (equal); Data (equal); & Investigation Investigation Writing-review keyi: (equal); (equal); Tang (equal); Investigation curation (lead). (equal); (equal); Data editing analysis analysis Formal & Formal Writing-review (equal); (equal); Visu- curation tion (supporting); (equal); Data Validation Methodology changkun: (equal); Fu (equal); Investigation Investigation (lead). (lead); draft analysis Writing-original Formal (lead); (equal); alization very curation study. Data are this We haijun: for Jiang China. sample of providing Foundation for Science Forestry of Natural contributions Academy National Author Sichuan the from from Rui funding Liao the to for grateful grateful very are We interests. Acknowledgements competing no have we all declare from We permission received We research. interests this Competing research. for conduct required to were managers approvals and committee landowners ethics parameters. or model for permits priors No different and using species HP putative H, 2 Ethics N, the for studty. probabilities Posterior present S6: in Table respectively. used diversity, nucleotide samples diversity, of haplotype reconstructions. haplotypes, statistics phylogenetic sequences, Sequence in used S5: model Table evolution molecular best The sequencing. S4: and Table PCR of for amplification used gene Primers and S3: specimen Table of numbers site, Sample S2: analysis. Table molecular for downloaded genes. Sequence nuDNA three S1: Table the of networks Median-joining S1: Fig 8 .nigrescens S. π . en h ubrof number the means Posted on Authorea 2 Mar 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161470351.19785260/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eK in,X.21.Syilnso otws hn.I noeve fpyoegahcpten.Chinese patterns. phylogeographic of overview an 10.1007/s11434-013-0089-1) I: (doi: China. and southwest 585–597. of shrews 59, islands Nectogalini Bull. Sky of Sci. 2014. phylogeny XL. 56(2):734- multi-locus Jiang, Evolution, K, A & He Phylogenetics 2010. Molecular the XL. evolution. 10.1016/j.ympev.2010.03.039). of and Jiang diversity (doi: speciation LK, genetic on 10.1038/hdy.2015.62)746. Lin high paleoclimate (doi: MC, preserved the Brandley refugia 116(1):23. of Heredity influences Interglacial YJ, China. Li 2016. southwest K, XL. of He Jiang mountains JT, the in Li shrew X, mole Chen Chinese NQ, L, Hu histories. Choleva K, independent G, He their the Gollmann 10.1111/j.1365-294X.2011.05175.x) supports of variegata M, phylogeography (doi: Bombina mitochondrial Stuglik 20(16). and and Ecology, W, Nuclear bombina Molecular Bombina 2011. Babik toads JM. SN, Szymura, fire-bellied Litvinchuk G, European Dzukic S, population T, Vukov Hofman change-driven D, climate K, Cogalniceanu Nadachowska and isolation A, Drainage Fijarczyk 2016. 10.1038/srep21811) ZL. (doi: Yang of with 21811. J, structures phylogenetics 6, genetic Qin 10.1093/molbev/mss075) Bayesian the JP, (doi: shape Xu 2012. 29(8). expansion Q, evolution, Zhao Andrew. and B, Rambaut biology Feng Molecular Dong, 1.7. Xie BEAST A, the Marc and Evolu- Suchard BEAUti Bmc J, trees. Alexei sampling long by Drummond analysis 10.1186/1471-2148-7-214) a evolutionary (doi: Bayesian Ma: (doi: 7(1):214. BEAST: 12 Biology, 267-277. 2007. tionary 214, at A. Letters, change Rambaut Science AJ, climate Planetary and Drummond Uplift-driven Earth plateau. 2003. Tibetan J. the of Li 10.1016/S0012-821X(03)00383-2) margin CN, NE the parallel Garzione from and X, record heuristics Fang new 10.1038/nmeth.2109) models, DL, more (doi: Dettman minimum 2: 772–772. new JModelTest 9, a 2012. Methods from D. Nat. Posada ago R, Myr computing. 10.1038/374049a0) Doallo 14 (doi: GL, (doi: Taboada before 49-52. D, uplift Darriba 1593–1602. 374, plateau Nature, Tibetan 121, for extension. Oikos east-west Evidence shrew, for common 1995. age phenomenon? K. the Hodges Dehnel’s of M, habits explain Coleman foraging shortage and food resources winter Food ) York. does 10.1111/j.1600-0706.2011.20462.x New (2012). Ithaca, JRE. Associates, araneus: Taylor Publishing Sorex L, Comstock Rychlik Shrews. Molecular S, of Churchfield History 2015. Natural The white-toothed S. of (1990). diversity Liu S. Xuming, cryptic Churchfield Wang Z, and phylogeny Yingting, Ban Multilocus Pu, Robert, B, 2019. Eulipotyphla, Murphy (Mammalia, Tu Shaoying. Mingkun, shrews Liu, Tang X, Yang, Xuelong, Liu Jiang, Zhang Zhu, Guo, NP, Liu Keji Jiao, Koju Qing 10.1016/j.ympev.2014.12.016) Shunde, L, (doi: Chen 10.1111/j.1365- Yang 84:245-253. X, Evolution (doi: Phylogenetics Jiang Molecular of 3423-3437. H, sequences. structure 16, Kai phylogeographic and Z, Ecol. phylogenetics West- Sun Mol among S, structure aluco). Chen genetic (Strix ex- microsatellite owls the and tawny at mitochondrial of delimitation 3556-3572. 294X.2007.03401.x) of populations species 67, patterns European of Evolution Contrasting ern Challenge 2007. skinks. The PH. sun Brito philippine 2013. in RM. change Brown 10.1111/evo.12219) morphological AC, (doi: without Diesmos diversification J, White tremes: J, 10.1093/oxfordjournals.molbev.a026036) Molec- A (doi: phylogenies. Barley intraspecific 37-48. inferring 16, for networks Evolution. Median-joining & Biology 1999. A. ular Rohl P, Forster HJ, Bandelt Crocidura ue indicum Tuber nCia di 10.21203/rs.2.14115/v1) (doi: China. in ) oe bedfordiae Sorex 9 ope nteHnda onan ein Rep. region. Mountains Hengduan the in complex ae nmtcodiladncerDNA nuclear and mitochondrial on based δ 8O 18 Posted on Authorea 2 Mar 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161470351.19785260/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. oiia) amla ilg,6() 7-8.(o:1.0811-07000) Indian 10.1078/1616-5047-00080 the (doi: and 178-180. 68(3), Plateau, Biology, Tibetan Mammalian the Soricidae). of 10.1029/93RG02030) uplift 2003. (doi: M. dynamics, Motokawa, 357-396. Mantle 31, 1993. Geophysics, of J. Palaeon- Reviews climate. Martinod monsoon. Asian P, East England of P, evolution Molnar Geomorphologic and ) Plateau (doi:10.1016/j.palaeo.2009.11.002 2004. 1-23. Tibetan XY. the 8, Wang of Electronica, HB, growth tologia Tan China Mio-Pliocene Z, Science 2005. ago. Li P. years Molnar HZ, million 10.1360/03yd0315) 14 Ma (doi: since 822-833. RX, Plateau 47, Qinghai-Tibet Zhu, northeastern Sciences, XD, the Earth of of Miao uplift ZS, Journal phased An of structure. evidence XY, genetic Wang Phylogeography multiple HY, population 2014. of Lu J. contemporary 10.1111/jbi.12163) Feng contribution KP, (doi: to the Sun 317-327. factors P, Region: 41, Soisook andintrinsic 2014, Oriental data. Biogeography, VD, refugia the Thong polymorphism Hipposideridae)in GJ, glacial DNA Lu (Chiroptera: Pleistocene TL, of armiger Jiang analysis Hipposideros LF, Li comprehensive of G, for Csorba software AQ, Lin a 10.1093/bioinformatics/btp187) v5: (doi: 44, DnaSP 1451-1452. Bulletin, 25, 2009. Science Bioinformatics, Chinese J. changes. environmental Rozas and Librado, Plateau Tibetan the (doi: of 10.1007/BF03182692) 149-161. Uplift (doi: 60, 1999. 2117-2124. response Zoology. X. in Current Fang birds J, glaciations. of Li Quaternary patterns and phylogeographical Plateau and Qinghai-Tibet diversification the Species 10.1093/czoolo/60.2.149) of 2014. uplift G. the Song geographic to YH, 1963– distinct 4, Qu in Evol. FM, Ecol. composition Lei scales. species spatial mammal multiple ) across and 10.1002/ece3.1072 factors (doi: Bird environmental volume1971. with (2014). relationships Journal, Y. their Carmel and Herpetological regions A, distribution. Bar-Massada fragmented variation R, with Kent morphological species of a patterns carbonelli, Geographic Podarcis 2010. 20(20):41-50. Neftal´ı lizard Sillero. A, the M in Asiatic Carretero of A, genus Zoological new Kaliontzopoulou comparisons. A 10.24272/j.issn.2095-8137.2018.058) morphological 2018. (doi: and molecular Xue-Long. 321-334. on Jiang (5), Feng, based Research Cheng Eulipotyphla) Shui-Wang, (Soricidae, He 53(6):904-913. shrew Peng, Biology, Chen short-tailed of Xing, Systematic Probabilities Chen Posterior Kai, He Models. Bayesian Substitution of Complex Properties interpolated Frequentist and 10.1080/10635150490522629) resolution Simple (doi: High 10.1002/joc.1276) 2004. Under Worldclim: (doi: Trees Bruce. 2005 25:1965-1978. Phylogenetic Climatology. Rannala, A. of Jarvis John, Journal PG, Huelsenbeck, International (doi: Jones areas. land JL, global 359(1442):183-195. Parra for SE, surfaces Sciences, Cameron Ages Biological RJ, speciation. Ice Transactions Hijmans and the Philosophical divergence of in Legacy Quaternary. role Evolutionary 10.1098/rstb.2003.1388) the their The and in Bmc ages, cillations 2004. ice loci. 10.1111/j.1095-8312.1996.tb01434.x) GM. of multiple (doi: Hewitt Society. consequences from Linnean genetic history the of Some size Journal population Biological 1996. of GM. inference Hewitt Bayesian 10.1186/1471-2148-8-289) (doi: 2008. 8:289-289. Biology conservation. AJ. Evolutionary and Drummond evolution J, in interpretation Heled their 10.2307/2640768) and (doi: loci variable 313-318. highly 53, Evolution Perspective: 1999. PW. Hedrick oiuu minor Soriculus osn 80 eirsnnmof synonym senior 1890, Dobson, 10 || eei osqecso lmtcOs- Climatic of Consequences Genetic .radulus S. hms 92(Insectivora, 1922 Thomas, Posted on Authorea 2 Mar 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161470351.19785260/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. hlgorpyo h oehre ie Vpr moye,Lnau 15):eiec o ihgenetic high for evidence Molecular uplift (1758)): Tibetan 2008. Linnaeus of W. ammodytes, Timing Mayer (Vipera L, viper 1993. Crnobrnja-Isailovi´c, Fumagalli nose-horned P. Tomovi´c, J the 10.1038/364050a0) Calsteren L of (doi: van phylogeography S, 50-54. S, Schweiger 364, Kelley S, Nature, Ursenbacher N. animals. rocks. Rogers volcanic in of discordance J, analysis nuclear Liu by and constrained C, mitochondrial Hawkesworth of S, biogeography Turner The 10.1111/j.1365-294X.2012.05664.x) 2012. (doi: . 21(16). 13, Ecology, A shrew-like biology, Molecular Brelsford Asian evolutionary , in BMC DPL diversity Toews cryptic conservation. and and phylogeny taxonomy 10.1186/1471-2148-13-232) Multilocus for evolution- (doi: implications molecular 232. 2013. Talpidae): MAGA5: Xue-Long. (Uropsilus, Jiang moles Kai, 2011. methods. ) He S. parsimony 10.1093/molbev/msr121 Tao, maximum Kumar Wan (doi: and M, 28:2731-2739. (doi: distance, Nei evolution evolutionary and G, likelihood, 641-653. biology maximum Stecher 235, Molecular using N, Letters, from analysis Peterson inferred genetics Science ago D, ary Planetary Ma Peterson 13.7 and since K, Earth Plateau Tamura Tibetan Fault. northern Tagh the from Altyn in uplift Reconstruction the 10.1016/j.epsl.2005.04.034) Tectonic along Haplotype 2005. 10.1086/379378) for deposits Z. (doi: An molasse Methods 73:1162-1169. R, Bayesian Zhu climatic Genetics of J, Human of Earth Sun of Comparison record Asian Journal A magnetic of American Journal rock Data. 2003. Neogene Genotype Plateau. P. Population Late Tibetan Donnelly the 2007. M, of Z. Stephens . 10.1016/j.jseaes.2006.10.004) uplift An Press to (doi: J, University related 324-332. Princeton Li sediments 30, N, China. Sciences, eolian Ishikawa of Chinese Mammals M, the Torii from New to variation X, Press, Guide Fang Laboratory A Y, Harbor 2010. Song Spring . Yan Cold Xie and ed. AT 4th Smith Cloning, 6(2):189-192. Molecular Paleobiologie, with Revuede 2006. Mammalia), DW. Soricidae. York. (Insectivora, of Russell Heterosoricidae subfamily J, the new the Sambrook and a on Soricidae Crocidosoricinae, events the the (doi: geological of of of description 179-192. Redefinition influence the 42, The 1987. Biogeogr. JWF. 2015. phylogeography. Reumer FM. comparative Lei, through PGP, a studied Ericson and birds Q, 10.1111/jbi.12407) Asian extensions Quan East new B, of Gao Maxent: endemism G, with Song distributions YH, species 10.1111/j.0906-7590.2008.5203.x) Qu of (doi: Modeling 31(2):161-175. Ecography, distribu- 2008. evaluation. geographic comprehensive Dudik. species 10.1111/j.1469- Miroslav of SJ, modeling (doi: entropy Phillips 10.1016/j.ecolmodel.2005.03.026) Maximum (doi: 177-191. 2006. MODELLING. 270(1), RE. ECOLOGICAL to Schapire so- London, tions. RP, reference of of Anderson special phylogenetics SJ, Society with Molecular Phillips Zoological sequences: 2010. the gene asi- LK. of b ) Lin northeastern Proceedings cytochrome 7998.2006.00125.x T, mitochondrial of (doi: Oshida soricinae. on P, history based the Vogel 157-162. (mammalia) Biogeographical MA, 39(2), shrews Iwasa Ecology, ricid M, Hasegawa Population 1997. SD, on NE. Ohdachi Researches Dokuchaev mammalia). H, Ma. 4 ) (insectivora, Abe at 10.1007/BF02765261 shrews records R, paleoclimate soricine ) in Masuda atic B26349.1 signal 10.1130/ ky S, (doi: 400 1338-1344. the Ohdachi 120, intensified Bulletin, uplift con- America Tibetan of for 2008. Society X. hotspots Fang Geological Biodiversity JW, 2000. King J. J, Kent Nie 10.1038/35002501) Da, (doi: GA, 403:853-858. Bonseca Nature, CG, priorities. Mittermeier servation RA, Mittermeier N, Myers 11 Posted on Authorea 2 Mar 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161470351.19785260/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eZ h ,Dmar ,Ce i,Ce P uW.21b hlgorpyo eiautcbug, semi-aquatic a of Phylogeography factors. 2016b. ecological WJ. and geographical Bu PP, historical, 10.1038/srep21932) (doi: of Chen 6(1):2193. evaluation Xin, Reports, an Chen entific Veliidae): J, (Hemiptera: (doi: motors Damgaard horvathi 6:33625. as Microvelia G, Reports, fluctuations Zhu entific climate Veliidae). Z, (Hemiptera: Pleistocene Ye Pseudovelia and genus islands Loci. the mountain on multiple Terrestrial study from 10.1038/srep33625) 2016a. case data A W. speciation: sequence Bu for P, DNA Chen using Z, delimitation Ye equal. 10.1093/molbev/msu279) 2013. species (doi: not YP. Unguided 31:3125-3135. are Zhang, Evolution 2014. things BS, Biology Yue all B. Molecular SF, building: Rannala Wu mountain Z, Christopher, to Yang Blair 10.1371/journal.pone.0055315) response (doi: (doi: W, in e55315. Robert 1976–1991. macaque 8(2): Murphy, rhesus 38, One, YQ, PLoS Chinese W Biogeogr. of QQ, J. genetics Li Ecological varius. J, heterogenei- Luo Myosorex habitat SJ, shrew, and Wu climate African to southern diversification the lineage 10.1111/j.1365-2699.2011.02543.x) 10.1111/j.1601- of Linking (doi: 2011. phylogeography 114–117. CA. 148, ty: Hereditas Matthee araneus)? S, (Sorex Willows-Munro shrew common the species of and zone 5223.2010.02224.x) hybrid analyses a phylogeographic in Macierzyn by flow T, Strza revealed la M, Moska Europe H, 10.1111/j.1600-0587.2010.06287.x) in Wierzbicki glacial (doi: minutus Northern 260–271. 2010. 33, Sorex J.B., Ecography shrew Searle, modelling. J.C., pygmy distribution Svenning, the Y., 46(3):1116- Nakazawa, for A., Evolution, refugia Lira-Noriega, & C., Phylogenetics Fløjgaard, Molecular R., Vega, peninsula. Balkan 10.1016/j.ympev.2007.11.002) the (doi: in 1128. refugia multiple and diversity ´ k .21.D qai aresrdc aemdae gene male-mediated reduce barriers aquatic Do 2011. A. ska 12 Posted on Authorea 2 Mar 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161470351.19785260/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 13 Posted on Authorea 2 Mar 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161470351.19785260/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 14