A 'little dragon' from Kunming City: a new green pit viper from Yunnan Province, China (Squamata, Viperidae, Trimeresurus)

A ‘little dragon’ from Kunming City: a new green pit viper from Yunnan Province, China (Squamata, Viperidae, Trimeresurus) Yuhao Xu1, Jundong Deng1, Tierui Zhang2, Tan Van Nguyen3,4, Shiyang Weng5, Nikolay A. Poyarkov6, Gernot Vogel7, Fanyue Sun1, Chencan Liao8,9, Lifang Peng1 1 State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining 810016, Qinghai, China 2 The Anhui Provincial Key Laboratory of Biodiversity Conservation and Ecological Security in the Yangtze River Basin, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, China 3 The School of Medicine & Pharmacy, Duy Tan University, Da Nang 550000, Vietnam 4 Center for Entomology & Parasitology Research, Duy Tan University, Da Nang 550000, Vietnam 5 Institute of Plateau Biology of Xizang Autonomous Region, Lhasa 850008, Xizang, China 6 Department of Vertebrate Zoology, Lomonosov Moscow State University, Leninskiye Gory, GSP–1, Moscow 119234, Russia 7 Society for Southeast Asian Herpetology, D-69115 Heidelberg, Baden-Württemberg, Germany 8 Ailaoshan Subtropical Forest Ecosystem Observation and Research Station of Yunnan Province, Puer 676200, Yunnan, China 9 Xi Shuang Ban Na Tropical Botanical Garden, Chinese Academy of Sciences, Dai Autonomous Prefecture of Xishuangbanna 666303, Yunnan, China https://zoobank.org/E3A021AB-E3A6-434C-89B6-5C5E163B34C4 Corresponding authors: Lifang Peng ([email protected]); Tan Van Nguyen ([email protected]) Academic editor: Justin Bernstein ♦ Received 23 October 2025 ♦ Accepted 7 November 2025 ♦ Published 18 November 2025 Abstract A new species of the genus Trimeresurus Lacépède is described from Kunming City, Yunnan Province, China, based on an integrative analysis of morphological and molecular data. The new species, Trimeresurus loong sp. nov., is assigned to the subgenus Viridovipera Malhotra & Thorpe and can be distinguished from its congeners by the following combination of characters: the first supralabial completely separated from the nasal scale; a short and spinose hemipenis; moderate adult body size; dorsal scales in 19 (21 or 23)-19-15 rows; 150–151 ventral scales in males, and 157–158 in females; 67–68 subcaudals in males, and 57–64 in females; and distinct body color patterns. Molecular analyses based on the mitochondrial 16S, cyt b, and ND4 gene fragments indicate that Trimeresurus loong sp. nov. is genetically divergent from all currently recognized congeners, showing uncorrected p-distances ranging from 6.7–11.2% in cyt b and 6.1–8.3% in ND4. The discovery of this species brings the total number of recognized Trimeresurus species to 57, including nine recorded from Yunnan Province, further emphasizing the complex topography and exceptional level of endemism. Key Words Hemipenis, molecular phylogeny, morphology, taxonomy, Trimeresurus loong sp. nov., Southwestern China, systematics Introduction The genus Trimeresurus Lacépède, commonly known as Asian green pit vipers, represents one of the most species-rich and taxonomically challenging groups within the subfamily Crotalinae Oppel. These venomous snakes are of considerable medical importance and are widely distributed across tropical and subtropical Asia, from the Himalayas and southern China to mainland and insular Southeast Asia, extending as far as Timor and the Lesser Sunda Islands (Gumprecht et al. 2004; Malhotra and Thorpe 2004a; David et al. 2011, 2023; Poyarkov et al. 2023; Mirza et al. 2023). Species of Trimeresurus exhibit remarkable ecological and morphological diversity, Zoosyst. Evol. 101 (4) 2025, 2267–2293|DOI 10.3897/zse.101.175879 Copyright Xu, Y. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. zse.pensoft.net Xu, Y. et al.: A new Trimeresurus species from Yunnan, China2268 which, together with sexual dimorphism and geographic variation, have long complicated their taxonomy. Extensive revisions during the past two decades have resulted in the recognition of multiple lineages and numerous new species, particularly from Indochina and southern China (Vogel et al. 2023; Idiiatullina et al. 2023, 2024a–c; Pawangkhanant et al. 2025; Xu et al. 2025). At present, 57 valid species are recognized in the genus (Mirza et al. 2023; Idiiatullina et al. 2023, 2024a–c; Pawangkhanant et al. 2025; Uetz et al. 2025). Despite numerous taxonomic revisions in recent years, species diversity within Trimeresurus remains incompletely resolved. High morphological similarity among species, coupled with pronounced sexual dimorphism and geographic variation, often obscures diagnostic differences and conceals cryptic lineages (e.g., Malhotra and Thorpe 2004a,b; Rathee et al. 2022; Nguyen et al. 2024; Idiiatullina et al. 2023, 2024a–c; Liang et al. 2025; Xu et al. 2025). According to the classification proposed by Mirza et al. (2023), Trimeresurus is currently divided into six subgenera: Trimeresurus sensu stricto, Parias Gray, Popeia Malhotra & Thorpe, Himalayophis Malhotra & Thorpe, Sinovipera Guo & Wang, and Viridovipera Malhotra & Thorpe (see also Idiiatullina et al. 2024b; Pawangkhanant et al. 2025; Xu et al. 2025). Members of Viridovipera are defined by two key morphological features: the first supralabial scale is completely separated from the nasal scale, and the hemipenis is short and spinose (Malhotra and Thorpe 2004a; Dawson et al. 2008; Mirza et al. 2023). Currently, eight species are assigned to this subgenus, including T. mayaae Rathee, Purkayastha, Lalremsanga, Dalal, Biakzuala, Muansanga & Mirza [type locality: Champhai District, Mizoram State, India], T. medoensis Zhao [type locality: Motuo (Medog) County, Xizang Autonomous Region, China], T. nujiang Liang, Ding, Vogel, Chen & Wu [type locality: Cikai Town, Gongshan County, Nujiang Lisu Autonomous Prefecture, Yunnan Province, China], T. pretiosus Xu, Nguyen, Wang, Zhang, Poyarkov, Wei, Vogel, Peng & Weng in Xu, Nguyen, Wang, Zhang, Poyarkov, Wei, Vogel, Li, Deng, Sun, Peng & Weng [type locality: Xiayadong Township, Yadong County, Xigaze City, Xizang Autonomous Region, China], T. stejnegeri Schmidt [type locality: Shaowu City, Nanping Prefecture, Fujian Province, China], T. truongsonensis Orlov, Ryabov, Bui & Ho [type locality: Phong Nha-Ke Bang National Park, Quang Binh Province (now Quang Tri Province), Vietnam], T. vogeli David, Vidal & Pauwels [type locality: Khao Yai National Park, Nakhon Ratchasima Province, Thailand], and T. yunnanensis Schmidt [type locality: Tengchong City, Baoshan City, Yunnan Province, China] (see Rathee et al. 2022; Liang et al. 2025; Xu et al. 2025). Yunnan Province, situated at the junction between the Palearctic and Oriental Realms, is recognized as one of China’s major biodiversity hotspots. It harbors a particularly rich pit viper fauna, including several narrowly distributed Trimeresurus species. Currently, eight species of Trimeresurus are recorded from Yunnan Province: three of the subgenus Trimeresurus (T. albolabris Gray, T. guoi Chen, Shi, Vogel & Ding in Chen, Shi, Gao, Vogel, Song, Ding & Dai, and T. caudornatus Chen, Ding, Vogel & Shi in Chen, Yu, Vogel, Shi, Song, Tang, Yang, Ding & Chen); two of Popeia (T. popeiorum Smith and T. lanna Idiiatullina, Nguyen, Pawangkhanant, Suwannapoom, Chanhome, Mirza, David, Vogel & Poyarkov); and three of Viridovipera (T. stejnegeri Schmidt, T. yunnanensis Schmidt, and T. nujiang Liang, Ding, Vogel, Chen & Wu) (e.g., Idiiatullina et al. 2024b; Nguyen et al. 2024; Liang et al. 2025). Although Yunnan has been the focus of numerous herpetological studies, recent surveys have revealed that many montane valleys and forested gorges in southwestern China remain insufficiently explored. During fieldwork conducted in 2025 in Qinglong Town, Anning City, Kunming City, Yunnan Province, China, we discovered a population of green pit vipers that could not be assigned to any known species. Morphological examination and mitochondrial DNA analyses (16S, cyt b, and ND4 genes) confirmed that this population represents an undescribed species of Trimeresurus. We therefore describe it herein as a new species. Material and methods Sampling and specimen preservation Fieldwork was conducted in forested areas of Anning City, Kuming City, Yunnan Province, China in May and September 2025 (Fig. 1). Geographic coordinates and elevation were recorded using the TwoStep Outdoor Assistant v7.9.13 (Shenzhen 2bulu Information Technology Co., Ltd., China). Specimens were located using snake hooks, photographed in life, and humanely euthanized with a buffered MS-222 (tricaine methanesulfonate) solution. Subsequently, specimens were fixed in 10% formalin and transferred to 75% ethanol for longterm preservation. Liver tissue samples were collected fresh, preserved in 95% ethanol, and stored at –20 °C for molecular analyses. All voucher specimens were deposited in the herpetological collection of Qinghai University, Qinghai Province, China (QHU). All procedures followed the regulations of the Wildlife Protection Law of China and were approved by the Institutional Ethics Committee of Qinghai University (Protocol No. PJ202501-89). DNA extraction, amplification, and sequencing Total genomic DNA was extracted from ethanol-preserved liver tissues using the QIAamp DNA Mini Kit (QIAGEN, Changsheng Biotechnology Co. Ltd., Changchun, China). Three mitochondrial DNA (mtDNA) fragments, including 16S ribosomal RNA (16S), cytochrome b (cyt b), and NADH dehydrogenase subunit 4 (ND4), were am- Zoosyst. Evol. 101 (4) 2025, 2267–2293 zse.pensoft.net 2269 plified using polymerase chain reaction (PCR). The amplification of 16S was conducted with primers 16S1LM (5′-CCGACTGTTGACCAAAAACAT-3′) and 16SH1 (5′-TCCGGTCTGAACTCAGATCACGTAGG-3′), following the protocol of Nguyen et al. (2020). For cyt b, we used primers L14910 (5′-GACCTGTGATMTGAAAACCAYCGTTGT-3′) and H16064 (5′-CTTTGGTTTACAAGAACAATGCTTTA-3′), as described by Burbrink et al. (2000). The ND4 fragment was amplified using Trim-ND4F (5′-CACCTATGACTACCAAAAGCTCATGTAGAGC-3′) and Trim-ND4LEUR (5′-CATTACTTTTACTTGGATTTGCACCA-3′), following Salvi et al. (2013). The PCR thermal cycling profile was as follows: initial denaturation at 95 °C for 3 min; followed by 36 cycles of denaturation at 95 °C for 30 s, annealing at 48 °C for 45 s, and extension at 72 °C for 1 min; with a final extension at 72 °C for 10 min; and a final hold at 4 °C. PCR products were sequenced by Shanghai Map Biotech Co., Ltd. (Shanghai, China). Raw sequences were assembled using SeqMan (DNASTAR; Burland 2000), and newly generated sequences were submitted to DDBJ (Table 1). Phylogenetic analysis For phylogenetic analysis, a total of 235 sequences were included (Table 1), of which 21 were newly generated in this study, while 214 were retrieved from two public databases: GenBank and GenBase (National Genomics Data Center (NGDC), Beijing Institute of Genomics, Chinese Academy of Sciences / China National Center for Bioinformation; Bu et al. 2024; CNCB-NGDC Members and Partners 2025). These included representatives from 34 species of Trimeresurus and two outgroup taxa: Craspedocephalus puniceus (Boie) and Peltopelor malabaricus (Jerdon). Sequences were aligned in MEGA X (Kumar et al. 2018). The Maximum Likelihood (ML) analysis was conducted in IQ-TREE v1.6.12 (Nguyen et al. 2015) using the best-fit model GTR + F + I + G4 for all three fragments (16S, cyt b, and ND4), as determined by ModelFinder for IQ-Tree in PhyloSuite 1.2.3 according to Akaike information criterion (AIC) (Akaike 1973; Akaike 1974; Kalyaanamoorthy et al. 2017; Zhang et al. 2020). Node support was assessed using both the Ultrafast Bootstrap Figure 1. Map showing the type localities of the new species and other Trimeresurus species of the subgenus Viridovipera. Red star: Trimeresurus loong sp. nov. from Anning, Kunming, Yunnan, China; brown circle: T. cf. loong from Huili, Sichuan, China; yellow circle: T. mayaae from Champhai, Mizoram, India; blue circle: T. medoensis from Motuo (Medog), Xizang, China; pink circle: T. nujiang from Gongshan, Yunnan, China; green circle: T. pretiosus from Xiayadong, Yadong, Xizang, China; purple circle: T. stejnegeri from Shaowu, Fujian, China; orange circle: Trimeresurus sp. from Chengdu, Sichuan, China; black circle: T. truongsonensis from Phong Nha-Ke Bang NP, Quang Tri, Vietnam; white circle: T. vogeli from Khao Yai NP, Nakhon Ratchasima, Thailand; and grey circle: T. yunnanensis from Tengchong, Yunnan, China. zse.pensoft.net Xu, Y. et al.: A new Trimeresurus species from Yunnan, China2270 Table 1. NCBI / DDBJ / NGDC accession numbers, localities, and voucher information for all specimens used in this study. * = DDBJ Accession Number, and # = NGDC Accession Number. NO. Species name Locality Voucher NO. 16S cyt b ND4 References Genus Trimeresurus Subgenus Viridovipera 1Trimeresurus loong sp. nov. Anning, Kunming, Yunnan, China QHU R2025027 LC899328*LC899334*LC899341* This study 2Trimeresurus loong sp. nov. Anning, Kunming, Yunnan, China QHU R2025028 LC899329*LC899335*LC899342* This study 3Trimeresurus loong sp. nov. Anning, Kunming, Yunnan, China QHU R2025029 LC899330*LC899336*LC899343* This study 4Trimeresurus loong sp. nov. Anning, Kunming, Yunnan, China QHU R2025030 LC899331*LC899337*LC899344* This study 5Trimeresurus loong sp. nov. Anning, Kunming, Yunnan, China QHU R2025031 LC899332*LC899338*LC899345* This study 6Trimeresurus loong sp. nov. Anning, Kunming, Yunnan, China QHU R2025032 LC899333*LC899339*LC899346* This study 7T. cf. loong Huili, Sichuan, China GP 37 EU443812 EF597522 EF597527 Dawson et al. (2008) 8T. cf. loong Huili, Sichuan, China GP 38 EU443814 EF597523 EF597528 Dawson et al. (2008) 9Trimeresurus sp. Mt. Emei, Sichuan, China CIB CB2120 C_AA122998.1#C_AA122989.1#C_AA123004.1#Unpublished 10 Trimeresurus sp. Mt. Yuping, Sichuan, China CIB CB2121 C_AA122999.1#C_AA122990.1#C_AA123005.1#Unpublished 11 Trimeresurus sp. Mt. Emei, Sichuan, China CIB CB2127 C_AA123000.1#C_AA122991.1#C_AA123006.1#Unpublished 12 T. mayaae Champhai, Mizoram, India NCBS NRC-AA-0012 – OM966859 – Rathee et al. (2022) 13 T. mayaae Ri-Bhoi, Meghalaya, India BNHS 3658 – OM966860 – Rathee et al. (2022) 14 T. mayaae Ri-Bhoi, Meghalaya, India VR/ERS/ZSI/833 – OM966862 – Rathee et al. (2022) 15 T. mayaae Manipur, India MZMU 2970 – OQ968476 – Elangbam et al. (2023) 16 T. mayaae Manipur, India MZMU 2971 PP566116 – – Elangbam et al. (2023) 17 T. medoensis Motuo, Xizang, China SYS r001831 / CHS 824 MK194252 MK201553 – Li et al. (2020) 18 T. medoensis Motuo, Xizang, China KIZ YPX46122 MW020095 MW111479 – Che et al. (2020) 19 T. medoensis Motuo, Xizang, China KIZ YPX46123 MW020331 MW133479 – Che et al. (2020) 20 T. medoensis Motuo, Xizang, China ANU ZR24025 – PX068371 PX094008 This study 21 T. medoensis Motuo, Xizang, China ANU ZR24026 – PX068372 PX094007 This study 22 T. medoensis Motuo, Xizang, China ANU ZR24072 – PX068373 – Xu et al. (2025) 23 T. cf. medoensis Putao, Kachin, Myanmar AM B416 / CAS 221528 AY352735 AY352765 AY352831 Malhotra and Thorpe (2004a) 24 T. cf. medoensis Northeast India V18 MG995794 MG995819 MG995834 Unpublished 25 T. nujiang Fugong, Yunnan, China ANU ZR24133 PX061893 PX068374 PX094009 Xu et al. (2025) 26 T. nujiang Gongshan, Yunnan, China CIB DLR353 PV994716 PX021391 PX021401 Liang et al. (2025) 27 T. nujiang Gongshan, Yunnan, China CIB DLR365 PX021397 PX021407 PV994722 Liang et al. (2025) 28 T. nujiang Gongshan, Yunnan, China CIB DLR375 PX021398 PX021408 PV994723 Liang et al. (2025) 29 T. nujiang Gongshan, Yunnan, China CIB DLR380 PX021399 PX021409 PV994724 Liang et al. (2025) 30 T. pretiosus Yadong, Xizang, China QHU R2025019 PX061894 PX068368 PX094011 Xu et al. (2025) 31 T. pretiosus Yadong, Xizang, China QHU R2025020 PX061895 PX068369 PX094010 Xu et al. (2025) 32 T. pretiosus Yadong, Xizang, China QHU R2025021 PX061896 PX068370 PX094012 Xu et al. (2025) 33 T. stejnegeri Mt. Wuyi, Fujian, China GP 816 – KX019140 KX019294 Guo et al. (2016) 34 T. stejnegeri Mt. Jinggang, Jiangxi, China GP 662 – KX019120 KX019274 Guo et al. (2016) 35 T. stejnegeri Qimen, Anhui, China GP 470 – KX019094 KX019248 Guo et al. (2016) 36 T. stejnegeri Jinhua, Zhejiang, China GP 633 – KX019111 KX019265 Guo et al. (2016) Zoosyst. Evol. 101 (4) 2025, 2267–2293 zse.pensoft.net 2271 NO. Species name Locality Voucher NO. 16S cyt b ND4 References 37 T. stejnegeri Youxi, Fujian, China GP 755 – KX019131 KX019285 Guo et al. (2016) 38 T. stejnegeri Dehua, Fujian, China GP 2435 – KX019054 KX019209 Guo et al. (2016) 39 T. stejnegeri Taichung, Taiwan, China AM TST23 – AF277689 EU443799 Creer et al. (2003); Dawson et al. (2008) 40 T. stejnegeri Mt. Diaoluo, Hainan, china GP 45 – KX019088 KX019242 Guo et al. (2016) 41 T. stejnegeri Qiongzhong, Hainan, China GP 46 – KX019089 KX019243 Guo et al. (2016) 42 T. stejnegeri Qiongzhong, Hainan, China GP 48 – KX019104 KX019258 Guo et al. (2016) 43 T. stejnegeri Cat Ba NP, Hai Phong, Vietnam GP 881 – KX019145 KX019299 Guo et al. (2016) 44 T. stejnegeri Phia Oac-Phia Den NP, Cao Bang, Vietnam ROM 35321 – KT216408 KT216457 Guo et al. (2016) 45 T. stejnegeri Nonggang, Guangxi, China GP 2461 – KX019056 KX019211 Guo et al. (2016) 46 T. stejnegeri Nanchuan, Chongqing, China GP 1227 – KX019007 KX019162 Guo et al. (2016) 47 T. stejnegeri Leishan, Guizhou, China GP 1887 – KX019034 KX019189 Guo et al. (2016) 48 T. stejnegeri Luoding, Guangdong, China KIZ 09939 – KX019158 KX019312 Guo et al. (2016) 49 T. stejnegeri Enshi, Hubei, China GP 2011 – KX019040 KX019195 Guo et al. (2016) 50 T. truongsonensis Phong Nha-Ke Bang NP, Quang Tri Vietnam AM B659 / VNUH 190606 EU443818 EU443815 EU443816 Dawson et al. (2008) 51 T. truongsonensis Khammouane, Laos NAP-09115 – PX068365 PX094015 Xu et al. (2025) 52 T. vogeli Nakhon Ratchasima, Thailand AM B97 AY059562 AY059574 AY059596 Malhotra and Thorpe (2004a) 53 T. vogeli Nakhon Ratchasima, Thailand NAP-08227 – PX068366 PX094013 Xu et al. (2025) 54 T. vogeli Nakhon Ratchasima, Thailand NAP-08228 – PX068367 PX094014 Xu et al. (2025) 55 T. yunnanensis Tengchong, Yunnan, China QHU R2025033 LC899641*LC899340*LC899347* This study 56 T. yunnanensis Loei, Thailand AM A164 AF517181 AY352766 AF517224 Malhotra and Thorpe (2004a); Creer et al. (2003) 57 T. yunnanensis Pu Mat NP, Nghe An, Vietnam AM B174 AY059563 AY059573 AY059595, Malhotra and Thorpe (2004b) 58 T. yunnanensis Jingdong, Yunnan, China GP 851 – KT216392 KT216441 Guo et al. (2015) 59 T. yunnanensis Lincang, Yunnan, China GP 3507 KT216339 KT216384 KT216433 Guo et al. (2015) 60 T. yunnanensis Lincang, Yunnan, China GP 3509 KT216341 KT216386 KT216435 Guo et al. (2015) 61 T. yunnanensis Longling, Yunnan, China KIZ 05089 KT216352 KT216399 KT216448 Guo et al. (2015) 62 T. yunnanensis Jingdong, Yunnan, China KIZ 047083 KT216351 KT216398 KT216447 Guo et al. (2015) 63 T. cf. yunnanensis Honghe, Yunnan, China GP 2532 KT216331 KT216376 KT216425 Guo et al. (2015) 64 T. cf. yunnanensis Pingbian, Yunnan, China GP 3273 KT216334 KT216379 KT216428 Guo et al. (2015) 65 T. cf. yunnanensis Pingbian, Yunnan, China GP 3275 KT216336 KT216381 KT216430 Guo et al. (2015) 66 T. cf. yunnanensis Mengzi, Yunnan, China GP 3564 KT216344 KT216389 KT216438 Guo et al. (2015) 67 T. cf. yunnanensis Mengzi, Yunnan, China GP 3589 KT216345 KT216390 KT216439 Guo et al. (2015) Subgenus Himalayophis 68 T. arunachalensis Northeast India APF/SFRI-1871 MK722155 MK720609 – Captain et al. (2019) 69 T. tibetanus Nepal AM B258 AY352715 AY352749 AY352810 Malhotra and Thorpe (2004a) Subgenus Parias 70 T. flavomaculatus Mindanao, Philippines AM B4 AY352734 AY352764 AY352830 Malhotra and Thorpe (2004a) zse.pensoft.net Xu, Y. et al.: A new Trimeresurus species from Yunnan, China2272 NO. Species name Locality Voucher NO. 16S cyt b ND4 References 71 T. hageni Songhkla, Thailand AM B33 AY059552 AY059567 AY059585 72 T. malcolmi Mt. Kinabalu, Sabah, Malaysia AM B349 AY371786 AY371832 AY371861 Malhotra and Thorpe (2004a) 73 T. mcgregori Batan Is., Philippines AM B289 AY371795 AY371831 AY371858 Malhotra and Thorpe (2004a) 74 T. schultzei Palawan, Philippines AM B210 AY352725 AY352756 AY352819 Malhotra and Thorpe (2004a) 75 T. sumatranus Bengkulu, Sumatra, Indonesia AM B367 AY371791 AY371824 AY371864 Malhotra and Thorpe (2004a) Subgenus Sinovipera 76 T. sichuanensis Sichuan, China GP7 / YBU030116 HQ850449 HQ850447 HQ850446 Guo and Wang (2011) Subgenus Popeia 77 T. lanna Doi Inthanon NP, Chiangmai, Thailand AUP-00061 OR471637 OR470571 OR470534 Idiiatullina et al. (2024b) 78 T. nebularis Cameron Highlands, Pahang, Malaysia AM B345 AY371775 AY371811 AY371849 Sanders et al. (2006) 79 T. phuketensis Phang Nga, Thailand AM B467 AY371781 AY371807 AY371851 Sanders et al. (2006) 80 T. popeiorum Yingjiang, Yunnan, China DL2017070101 MH779887 MH779875 MH779879 Chen et al. (2019) 81 T. sabahi sabahi Mt. Kinabalu, Sabah, Malaysia AM B344 AY371771 AY371815 AY371842 Malhotra and Thorpe (2004a) 82 T. tenasserimensis Suan Phueng, Ratchaburi, Thailand ZMMU Re-17669 PP032802 OR999089 PP032781 Idiiatullina et al. (2024b) Subgenus Trimeresurus 83 T. albolabris Shek Kwu Chan, Hong Kong, China AM A157 AY352744 AF171884 AY352839 Malhotra and Thorpe (2000, 2004a) 84 T. cantori Nicobar Is., India AM A85 AY352741 AF171889 AY352836 Malhotra and Thorpe (2000, 2004a) 85 T. caudornatus Yingjiang, Yunnan,China AR1238 MK575042 MK575036 MK575038 Chen et al. (2020) 86 T. ciliaris Thum Khao Ting, Trang, Thailand ZMMU Re-17661 OR471621 OR470557 OR470538 Idiiatullina et al. (2023) 87 T. erythrochloris Tham Si Va Cave, Klong Hat, Sa Kaeo, Thailand RIM-0079 PQ654052 PQ658816 PQ658818 Pawangkhanant et al. (2025) 88 T. ayeyarwadyensis Rangoon, Myanmar AM A209 AF517174 AF171900 AF517217 Creer et al. (2003) 89 T. kanburiensis Khao Yai NP, Kanchanaburi, Thailand ZMMU Re-17667 – OR470579 OR470553 Idiiatullina et al. (2023); Idiiatullina et al. (2024a) 90 T. macrops Bangkok, Thailand ZMMU Re-17856 – PP766219 PP779475 Idiiatullina et al. (2024c) Out group 91 Craspedocephalus puniceus Indonesia AM B213 AF517177 AF517192 AF517220 Creer et al. (2003) 92 Peltopelor malabaricus Tamil Nadu, India AM A218 AY059564 AY059569,AY059587 Malhotra and Thorpe (2004a) Approximation (UFB) and the SH-like approximate likelihood ratio test (SH). UFB values were calculated with 5000 bootstrap replicates, with values ≥ 95% considered strong support; and SH was conducted with 1000 replicates, and values ≥ 80% were regarded as well supported (Stephane et al. 2010; Hoang et al. 2018). The Bayesian Inference (BI) analysis was conducted via MrBayes v3.2.7a (Ronquist et al. 2012) under the best-fit model GTR + F + I + G4 for all three fragments (16S, cyt b, and ND4), which was calculated according to BIC as well by ModelFinder for Mr-Bayes in PhyloSuite 1.2.3 (Zhang et al. 2020). In the BI analysis, three independent runs were conducted with 1 × 107 generations and sampled every 1000 generations, with the first 25% of samples discarded as burn-in. Nodes with Bayesian posterior probabilities (PP) ≥ 0.95 were considered strongly supported (Huelsenbeck et al. 2001). The phylogenetic tree was visualized using FigTree v.1.4.4 (Rambaut 2018). Uncorrected pairwise genetic distances (p-distances) among species were also calculated using MEGA X (Kumar et al. 2018). Morphological and hemipenial examination A total of 31 morphological characters were recorded for each specimen (following Vogel et al. 2023; Xu et al. 2025). Measurements were taken with a Mitutoyo digital calipers (CD-15AX) to the nearest 0.1 mm, except for body and tail lengths, which were measured to the nearest millimetre with a measuring tape. The number of ventral scales was counted according to Dowling (1951). Half ventrals were counted as one. The first enlarged shield Zoosyst. Evol. 101 (4) 2025, 2267–2293 zse.pensoft.net 2273 anterior to the ventrals was regarded as a preventral and was present in all examined specimens. The first scale under the tail meeting its opposite was regarded as the first subcaudal, and the terminal scute was not included in the number of subcaudals. The dorsal scale rows were counted at one head length behind the head, at mid-body, and at one head length before the vent. In the number of supralabials touching the subocular, those only touching the presubocular were not included. Infralabials were considered to be those shields that were completely below a supralabial and bordering the mouth gap. The first sublabial was defined as the scale that starts between the posterior chin shield and the infralabials and that borders the infralabials. Values for paired head characters were recorded on both sides of the head and were reported in a left-right order. In addition, we paid special attention to diagnostic color pattern characters, such as eye color, postocular streak, ventrolateral stripe, tail coloration, and overall body coloration. The sex was determined by dissection of the ventral tail base. Comparative morphological data were compiled from the literature, including Schmidt (1925,1927), Maki (1931), Pope (1935), Zhao et al. (1998), David et al. (2001a, b, 2002), Ao et al. (2004), Orlov et al. (2004), David and Mathew (2005), Zhao (2006), Guo et al. (2009), Teynié and David (2014), Nguyen et al. (2018), Che et al. (2020), Rathee et al. (2022), Elangbam et al. (2023), Nguyen et al. (2025), Liang et al. (2025), and Xu et al. (2025). Hemipenial morphology The preparation, measurement, and description of hemipenes followed the methodologies of Zhang et al. (1984), Myers and Cadle (2003), Jiang (2010), and Ren et al. (2022, 2025). Hemipenial material was obtained from the left side of fresh or preserved adult male specimens. Everted hemipenes were reinflated using colored petroleum jelly, and images were captured using a digital camera. The measurement characters of the hemipenis are as follows: HTL = hemipenial total length; HTW = hemipenial total width; HCL = hemipenial truncus length; and SPBD = sulcus spermaticus bifurcation distance, measured from the base of the hemipenis to the bifurcation point of the sulcus spermaticus in the vertical direction. The following ratios were used for comparative purposes: HCL/HTL = ratio of truncus length to total length; and SPBD/HCL = ratio of sulcus bifurcation distance to truncus length, to evaluate the relative position of the sulcus bifurcation point along the hemipenial truncus. Specimen information of Viridovipera species used in hemipenial comparisons conducted in this study are provided below: Trimeresurus medoensis (N = 1): QHU R2025040 (adult male), collected on June 2018, by Jundong Deng, from Beibeng Township, Motuo (Medog) County, Nyingchi City, Xizang Autonomous Region, China (approx. 29.23°N, 95.18°E). Trimeresurus pretiosus (N = 2): QHU R2025019 (adult male), collected on 22 June 2025 by Zhenqi Wang, Yuhao Xu, Fanyue Sun, and Lifang Peng, from Xiayadong Township, Yadong County, Xigaze City, Xizang Autonomous Region, China (27.262°N, 89.016°E; elevation 1824 m asl.); QHU R2025020 (adult male), collected on 22 June 2025, shares the same locality and collector as QHU R2025019. Trimeresurus stejnegeri (N = 1): QHU R2025037 (adult male), collected on September 2025 by Yuhao Xu, from Liandu District, Lishui City, Zhejiang Province, China (28.499°N, 119.922°E; elevation 526 m asl.). Trimeresurus yunnanensis (N = 1): QHU R2025035 (adult male), collected on July 2025 by Jundong Deng, from Lincang City, Yunnan Province, China (approx. 23.91°N, 100.15°E). Abbreviations Morphological descriptions and morphometry follow standardized abbreviations revised according to Darko et al. (2022). The morphometric characters are as follows: SVL = snout-vent length; TAL = tail length; TL = total length; TAL/TL = ratio of tail length to total length; HL = head length; HW = head width; ESD = eye-snout distance, measured from the tip of the snout to the anterior edge of eye; ED = eye diameter; EN = eye to nostril distance, measured from the anterior margin of the eye to the posterior margin of the nostril; SOL = subocular length; and SOW = subocular width. The scalation characters are as follows: VS = number of ventral scales; SC = number of subcaudal scales; CP = cloacal plate; SL = supralabials; IL = infralabials; DSR = dorsal scale rows; ASR = anterior dorsal scale rows; MSR = dorsal scale rows at midbody; PSR = posterior dorsal scale rows; PRO = preoculars; PO = postoculars; SO = supraocu-lars; SBO = suboculars; IOS = interorbital scales, number of scales at the narrowest point between the orbits; SpOC = number of dorsal head scales surrounding the supraocular; NS = Nasals; INS = internasals; NINN = number of scales separating the internasals; KTEM = keeling of the temporal scales, and KOCC = keeling of the occipital scales. Other abbreviations: AR = Autonomous region; NP = National Park; NR = Nature Reserve; Mt = Mountains; asl. = above sea level; WS = Wildlife Sanctuary. Result Phylogenetic relationships The concatenated sequence alignment was 2,314 base pairs (bp) in length (16S = 508 bp; cyt b = 1,034 bp; and ND4 = 772 bp). Both ML and BI analyses yielded congruent topologies (Fig. 2). According to our mtDNA-based phylogeny, all putative Trimeresurus species cluster into a single clade. However, relationships among species within zse.pensoft.net Xu, Y. et al.: A new Trimeresurus species from Yunnan, China2274 GP 662 GP 755 GP 470 GP 633 GP 2435 AM TST23 GP 816 GP 45 GP 46 GP 48 GP 1227 GP 2011 GP 1887 KIZ 09939 GP 881 GP 2461 ROM 35321 GP 3509 GP 3507 KIZ 05089 QHU R2025033 KIZ 47083 GP 851 AM A164 AM B174 GP 2532 GP 3564 GP 3589 GP 3275 GP 3273 ANU ZR24133 CIB DLR353 CIB DLR380 CIB DLR365 CIB DLR365 AM B659 ZMMU NAP-09115 AM B97 ZMMU NAP-08227 ZMMU NAP-08228 ANU ZR24025 ANU ZR24026 ANU ZR24072 KIZ YPX46122 KIZ YPX46123 SYS r001831 NCBS NRC-AA-0012 MZMU 2970 MZMU 2971 BNHS 3658 VR/ERS/ZSI/833 AM B416 V18 QHU R2025020 QHU R2025021 QHU R2025019 QHU R2025028 QHU R2025032 QHU R2025027 QHU R2025029 QHU R2025030 QHU R2025031 GP 37 GP 38 CIB CB2127 CIB CB2120 CIB CB2121 AM A85 T. cantori AM A209 T. ayeyarwadyensis AM A157 T. albolabris AR 1238 T. caudornatus RIM 0079 T. erythrochloris ZMMU Re-17856 T. macrops ZMMU Re-17667 T. kanburiensis ZMMU Re-17661 T. ciliaris GP 7 T. sichuanensis AM B4 T. flavomaculatus AM B289 T. mcgregori AM B210 T. schultzei AM B367 T. sumatranus AM B349 T. malcolmi AM B33 T. hageni AUP-00061 T. lanna AM B467 T. phuketensis AM B345 T. nebularis DL2017070101 T. popeiorum AM B344 T. sabahi sabahi ZMMU Re-17669 T. tenasserimensis APF/SFRI-1871 T. arunachalensis AM B258 T. tibetanus AM B213 Craspedocephalus puniceus AM A218 Peltopelor malabaricus 0.03 86/92/0.72 99/100/1.0 79/90/0.99 99/99/1.0 100/100/1.0 75/95/0.79 100/100/1.0 97/99/1.0 80/94/0.81 100/100/1.0 100/100/1.0 87/95/0.93 100/100/0.97 –/62/0.65 100/100/1.0 89/98/0.95 96/99/1.0 95/98/0.96 99/95/0.95 100/100/1.0 100/100/1.0 100/100/1.0 82/74/0.75 83/92/0.97 100/100/1.0 85/84/0.50 100/100/1.0 51/70/0.74 95/99/0.95 68/97/0.76 100/100/1.0 76/97/0.68 93/100/0.78 100/100/1.0 –/64/0.79 84/77/1.0 100/100/1.0 69/83/0.74 83/86/0.96 99/100/1.0 99/100/1.0 –/53/– 75/89/– 72/86/0.52 73/73/0.93 80/78/0.97 100/100/1.0 82/67/0.75 87/72/0.78 99/100/1.0 Genus Trimeresurus Viridovipera Trimeresurus Sinovipera Parias Popeia Himalayophis T. stejnegeri T. yunnanensis T. cf. yunnanensis T. T. nujiang truongsonensis T. vogeli T. medoensis T. mayaae T. cf. medoensis T. pretiosus T. loong sp. nov. T. cf. loong Trimeresurus sp. Figure 2. Phylogram of the genus Trimeresurus inferred from three mitochondrial (16S/cyt b/ND4) fragments. The branch support values are presented with the SH-like approximate likelihood ratio test (SH)/Ultrafast Bootstrap Approximation (UFB)/Bayesian posterior probabilities (PP); the ones lower than 50 or 0.5 are displayed as “–”. the genus remain poorly resolved. The monophyly of the subgenera Parias and Popeia was strongly supported (SH 99/ UFB 100 / PP 1.0; SH 100 / UFB 100 / PP 1.0 respectively), while subgenus Himalayophis and Viridovipera received moderate support (SH 87 / UFB 72 / PP 0.78; SH 82 / UFB 74 / PP 0.75, respectively). The subgenus Trimeresurus is not recovered as monophyletic in analysis and is clearly divided into two deeply divergent clades. Within the subgenus Viridovipera, phylogenetic relationships among species are generally well resolved (Fig. 2). Two specimens AM B416 (= CAS 221528) from Kachin State, Myanmar and V18 from Northeast India, previously identified as T. medoensis, form a distinct clade that is sister to the clade comprising T. medoensis and T. mayaae. Therefore, we tentatively refer to these specimens as T. cf. medoensis. In addition, five specimens Zoosyst. Evol. 101 (4) 2025, 2267–2293 zse.pensoft.net 2275 from southeastern Yunnan Province, China (GP 2532, GP 3273, GP 3275, GP 3564, and GP 3589), previously identified as T. gumprechti, form a sister clade to T. yunnanensis with moderate support (SH = 75 / UFB = 97 / PP = 0.79). The uncorrected p-distances between the two clades range from 4.0–6.2% for cyt b and 2.9–5.1% for ND4. As morphological data for this population are not yet available, we tentatively refer to these specimens as T. cf. yunnanensis. The six unidentified Trimeresurus specimens from Anning, Kunming City, Yunnan Province form a well-supported monophyletic clade (SH = 100 / UFB = 100 / PP = 1.0). This clade is sister to two specimens from Huili City, Sichuan Province (GP 37 and GP 38), which had long been misidentified as T. yunnanensis. Together, they form a distinct subclade with three additional specimens from western Sichuan Province (CIB CB2120, CIB CB2121, and CIB CB2127), with moderate support in the ML analysis (SH = 85 / UFB = 84), but low support in the BI analysis (PP = 0.5). This entire assemblage clusters at the outer margin of the clade formed by all other currently recognized species of Viridovipera. Uncorrected p-distances for the cyt b and ND4 gene fragments are summarized in Table 2 and Table 3, respectively. Among currently recognized Viridovipera species, interspecific genetic distances ranged from 4.9% (T. stejnegeri and T. yunnanensis) to 12.8% (T. vogeli and T. yunnanensis) for the cyt b gene (Table 2), and from 3.1% (T. stejnegeri and T. yunnanensis) to 11.0% (T. pretiosus and T. vogeli) for the ND4 gene (Table 3). The Anning population exhibited substantial genetic divergence from all other recognized congeners, with uncorrected p-distances ranging from 6.7% (vs. T. mayaae) to 11.2% (vs. T. stejnegeri) for the cyt b gene, and from 6.1% (vs. T. yunnanensis) to 8.3% (vs. T. medoensis) for the ND4 gene. In addition, specimens from Huili City, Sichuan Province showed cyt b divergence of 4.8–4.9% and ND4 divergence of 4.1–5.2% from the specimens from Anning, Yunnan Province. These values approach the minimum interspecific divergence observed within Viridovipera for cyt b, and exceed the minimum observed for ND4. Meanwhile, specimens from western Sichuan Province differed from the Anning population by 7.0–7.1% in cyt b and 5.9–6.1% in ND4, clearly exceeding the minimum interspecific distances within the subgenus. Liang et al. (2025) assessed the taxonomic status of Trimeresurus gumprechti David, Vogel, Pauwels & Vidal [type locality: Phu Luang WS, Loei Province, Thailand], based on four mitochondrial gene fragments (12S, 16S, cyt b, and ND4) together with morphological data. Their analyses revealed that the topotypes of T. gumprechti clustered with the topotypes of T. yunnanensis, and that the genetic divergence between the two (0.0–1.6% in cyt b) was markedly lower than typical interspecific divergence within the subgenus Viridovipera. Consequently, T. gumprechti was treated as a junior synonym of T. yunnanensis. Our phylogenetic analyses yielded similar results in this regard. However, the phylogenetic topology presented by Liang et al. (2025) placed the subgenus Trimeresurus nested within what was previously considered the subgenus Viridovipera, forming a sister group to T. medoensis, T. mayaae, and Trimeresurus sp. (Huili City, Sichuan Province, China). Based on this result, they annotated Viridovipera on their phylogenetic tree as comprising only the clade including T. stejnegeri, T. yunnanensis, T. vogeli, T. truongsonensis, and T. nujiang, thereby excluding T. medoensis, T. mayaae, and Trimeresurus sp. (Huili City, Sichuan Province, China) from the subgenus. This arrangement is incongruent with both previous studies and our own morphological and molecular evidence (Creer et al. 2003; Malhotra and Thorpe 2004a, b; Dawson et al. 2008; Guo et al. 2015, 2016; Rathee et al. 2022; Xu et al. 2025; this study). A likely explanation for this inconsistency is that Liang et al. (2025) included only three species of the subgenus Trimeresurus, namely T. septentrionalis Kramer, 1977, T. guoi, and T. purpureomaculatus (Gray, 1832), and the phylogenetic resolution in this part of the tree is rather weak. Therefore, the overall phylogenetic framework proposed by Liang et al. (2025) warrants additional verification with expanded sampling. Furthermore, detailed morphological examinations confirmed that the specimens from Anning, Kunming, Yunnan are diagnosably distinct from all known Viridovipera species. However, due to the lack of available morphological data for the Huili and Chengdu in Sichuan Province specimens, we are currently unable to determine their exact taxonomic status. Based on the combined molecular and morphological evidence, we describe the population from Anning City, Kunming City, Yunnan Province, China as a new species, and the Huili population is tentatively reassigned to this new species, while the western Sichuan population is herein treated as Trimeresurus sp. Taxonomy Trimeresurus loong Xu, Deng, Zhang, Nguyen, Poyarkov, Vogel & Peng, sp. nov. https://zoobank.org/B557FA3A-3B3A-47C2-A9F8-239824C7DE63 Tables 4, 5; Figs 3, 4A, B, 5–8, 9B, C, 10A, B, 11A1, A2 Type material. Holotype • QHU R2025027, adult male, collected from Qinglongxia, Anning City, Kunming City, Yunnan Province, China (25.0590°N, 102.3561°E; elevation 1,822 m asl.), on 15 September 2025 by J.D. Deng and Y.H. Xu. Paratypes (n = 5) • QHU R2025031 (adult female), with same data as the holotype. • QHU R2025028 (adult male), collected on 22 September 2025 by J.D. Deng and Y.H. Xu; • QHU R2025029 (subadult male) and • QHU R2025032 (adult female), collected on 27 May 2025 by J.D. Deng; • QHU R2025030 (subadult male), collected on 21 September 2025 by J.D. Deng and Y.H. Xu; all from the same locality as the holotype. Diagnosis. The new species Trimeresurus loong sp. nov. is distinguished from all of its congeners by a combination of the following morphological characters: (1) first zse.pensoft.net Xu, Y. et al.: A new Trimeresurus species from Yunnan, China2282 Table 4. Main measurements and meristic characters of the type series of Trimeresurus loong sp. nov. Specimen voucher QHU R2025027 QHU R2025028 QHU R2025029 QHU R2025030 QHU R2025031 QHU R2025032 Type Holotype Paratype Sex Male Male Subadult male Subadult male Female Female SVL (mm) 517 601 402 335 657 632 TAL (mm) 129 147 96 83 135 146 TL (mm) 646 748 498 418 792 778 TAL/TL 0.20 0.20 0.19 0.20 0.17 0.19 HL (mm) 25.8 29.0 20.7 19.3 36.6 34.6 HW (mm) 17.7 20.9 14.4 13.9 27.6 20.9 HW/HL 0.69 0.72 0.70 0.72 0.75 0.60 ESD (mm) 8.6 9.5 6.6 5.7 11.4 8.9 EN (mm) 5.4 6.6 4.5 4.0 7.7 6.7 ED (mm) 3.3 3.6 2.4 2.7 4.2 4.3 ED/ESD 0.38 0.38 0.36 0.47 0.37 0.48 ED/HL 0.13 0.12 0.12 0.14 0.11 0.12 NS 1, undivided 1, undivided 1, undivided 1, undivided 1, undivided 1, undivided INS Slightly enarged Slightly enarged Slightly enarged Slightly enarged Slightly enarged Slightly enarged NINN 2 small + 1 large 2 small + 1 large 2 2 2 2 PRO 3 3 3 3 3 3 PO 2/2 4/4 2/2 3/3 2/2 3/3 SO 1/1 3/2 1/1 1/1 1/1 1/1 SOL 4.3 – 3.6 3.4 5.4 4.3 SOW 1.9 – 1.3 1.2 2.1 2.1 SBO 1/1 1/1 1/1 1/1 1/1 1/1 IOS 9 10 7 9 10 11 SpOC 8/8 8/7 7/8 9/8 6/5 6/7 KTEM Smooth Smooth Smooth Smooth Smooth Smooth KOCC Slightly keeled Smooth Slightly keeled Slightly keeled Slightly keeled Smooth SL 10/10 11/10 10/10 10/10 11/11 10/10 IL 12/11 12/11 12/12 11/12 13/14 12/12 DSR 19-19-15 21-19-15 23-19-15 19-19-15 21-19-15 19-19-15 VS 150+2 151+2 150+3 150+2 158+3 157+2 SC 67, paired 68, paired 67, paired 67, paired 57, paired 64, paired VS+SC 217 219 217 217 215 221 CP 1 1 1 1 1 1 Eye color Amber with a faint reddish hue Amber with a faint reddish hue Amber Amber Yellowish Yellowish Postocular streak Thin and white, with a few scale margins tinged with yellow White Thin and white, with the upper margins of a few scales tinged with reddishbrown Thin and white, with a few scale margins tinged with yellow Absent Absent Ventrolateral stripe Yellow+white+red+brown Thin, white and tinged with yellowish margins Yellow+white+red+brown Yellow+white+red+brown White Absent while only limited information is available for females due to the small sample size. This species exhibits sexual dichromatism. In males, the eyes are amber in color. Among the two adult specimens (QHU R2025027 and QHU R2025028), the irises exhibit a slight reddish hue, whereas in the two subadult specimens (QHU R2025029 and QHU R2025030), the eyes are purely amber. The postocular streak is narrow and white, with a few scale margins slightly tinged with yellow or reddish-brown. The ventrolateral stripe runs along the first dorsal scale row and is variable in coloration among males, occurring in two forms. In most male specimens, it consists of a narrow white band bordered dorsally by a faint yellowish margin and ventrally by a thin, red line, with a diffuse brownish tinge below the red line. However, in QHU R2025028, the ventrolateral stripe is only a narrow white band bordered on both sides by faint yellowish margins. In some individuals, the upper and lower margins of the first dorsal scale row remain green. In females, the eyes are yellowish. The postocular streak is absent in both specimens. The ventrolateral stripe is absent in QHU R2025032, while in QHU R2025031 it is narrow, white, and restricted to the middle portion of the first dorsal scale row. In all individuals, the dorsal surfaces of the head and body are grass green. In males, the lateral body coloration Zoosyst. Evol. 101 (4) 2025, 2267–2293 zse.pensoft.net 2283 Figure 7. Paratypes of Trimeresurus loong sp. nov. A. Dorsal view of the head; B. Lateral view of the head; C. Ventral view of the head; D. Lateral view of the body; E. Ventral view of the body. A1–E1. QHU R2025028, adult male; A2–E2. QHU R2025029, subadult male; A3–E3. QHU R2025030, subadult male; A4–E4. QHU R2025031, adult female; A5–E5. QHU R2025032, adult female. Photographs by Y.H. Xu. is slightly paler than the mid-dorsal region, and the lateral dorsal scales are variably speckled with fine creamish-blue or white spots. In contrast, females exhibit a uniformly grass-green coloration across the entire dorsum. The dorsal surface of the tail is distinctly bicolored: green anteriorly, gradually transitioning to nearly uniform orange or light orange-red posteriorly, with the tail tip being blackish brown (Fig. 6B–E). The ventral surface of the head and the central portion of the anterior ventral body are noticeably paler, appearing pale whitish-green or creamish-yellow. Posterior to midbody, the ventral coloration becomes uniformly yellowish-green or light green. The anterior portion of ventral surface of the tail is pale green, while the posterior portion is light orange-red. Main characters of the pattern in preservative. The description is based on a subadult male specimen (QHU R2025029) and a female specimen (QHU R2025032), both collected in May 2025. After approximately five months in alcohol, the dorsal surface of the head had turned olive-yellow and that of the body yellow in both specimens. The eyes had become pale gray, and the supralabials as well as the ventral surface of the head had faded to milky white. The lateral portions of the ventral body were pale yellow, while the entire central ventral surface had turned creamish-yellow. In the male specimen, the posterior portion of the ventral body was slightly yellowish-green. The orange-red area of the tail remained relatively well defined. In the male specimen, the ventrolateral stripe had changed to white dorsally and brownish with a slight reddish hue ventrally. Distribution and natural history. Currently, Trimeresurus loong sp. nov. is certainly known only from the type locality, Qinglongxia, Anning City, Kunming City, Yunnan Province, China, at an elevation of approximately 1,822 m asl. All specimens were encountered during the first half of the night, coiled within shrubs on hillsides near streams in well-preserved broad-leaved forest (Fig. 10A–C), when nighttime temperatures ranged 15 °C ~ 18 °C. The female specimen QHU R2025031 defecated immediately when disturbed during capture. Its feces contained numerous bird feathers, suggesting that small birds may constitute part of the adult diet of this species (Fig. 10D). Comparisons. Trimeresurus loong sp. nov. is assigned to the subgenus Viridovipera based on a combination of diagnostic characters, including its phylogenetic position, the complete separation of the first supralabial from the nasal scale, and a short spinose hemipenis, all of which are characteristic features of Viridovipera (e.g., Nguyen et al. 2025; Xu et al. 2025). Accordingly, morphological comparisons are restricted to congeners within Viridovipera, which currently comprises nine recognized species and represents the most relevant group for differential diagnosis. The principal characters distinguishing Trimeresurus loong sp. nov. from these congeners are summarized in Table 5 and illustrated in Fig. 10. zse.pensoft.net Xu, Y. et al.: A new Trimeresurus species from Yunnan, China2284 Figure 8. Coloration of the paratypes of Trimeresurus loong sp. nov. in life. A. Lateral view of the head; B. Dorsolateral view; C. Ventral view. A1–C1. QHU R2025028, adult male; A2–C2. QHU R2025029, subadult male; A3–C3. QHU R2025030, subadult male; A4–C4. QHU R2025031, adult female. Photographs by Y.H. Xu. Morphologically, Trimeresurus loong sp. nov. is most similar to T. yunnanensis (distributed in central to southwestern Yunnan Province of China, the Mandalay Region and Shan State of Myanmar, northern Laos, northern and central Thailand, and northern to central Vietnam), but it can be clearly distinguished from the latter by the following combination of characters: (1) smaller maximum SVL in females (657 mm vs. 804 mm); (2) lower total number of VS+SC in males (217–219 [mean 217.5 ± 1.0] vs. 221–231 [mean 225.9 ± 3.9]); (3) irises amber in males vs. bright or deep red irises observed in males of T. yunnanensis (Fig. 10A, B, K); (4) the ventrolateral stripe in the males of the new species is slender and limited to the first dorsal scale row occurring in two forms, either a narrow white band bordered dorsally by a faint yellowish margin and ventrally by a red line with a diffuse brownish tinge below the red line, or a narrow white band bordered by faint yellowish margins on both sides, while in the males of T. yunnanensis the stripe is broader and more vivid, occupying the entire first dorsal scale row and the lower margin of the second row, even extending onto the lateral edges of the ventrals, consisting of a thick red lower border and a white upper zone with sharply separated colors visible from a distance; (5) the new species has a anteriorly green coloration of tail, posteriorly gradually transitioning to a uniformly orange or light orange-red. In contrast, T. yunnanensis shows dark red blotches forming large, irregular patches with uneven margins on the anterior part of the tail (Fig. 11). In hemipenial morphology, Trimeresurus loong sp. nov. can be distinguished from T. yunnanensis by shorter hemipenial truncus and a higher degree of bifurcation (HCL/HTL ratio 0.62–0.66 vs. 0.69), a more distal sulcus spermaticus bifurcation point (SPBD/HCL ratio 0.43–0.49 vs. 0.30), and a greater number of enlarged spines (14–19 per side vs. 9–10, excluding the smaller spines lining the margin of the sulcus spermaticus) (Fig. 4A, B, F). Zoosyst. Evol. 101 (4) 2025, 2267–2293 zse.pensoft.net 2285 Figure 9. Habitat and field observations of Trimeresurus loong sp. nov. A. The microhabitat of the new species in Anning, Kunming, China; B. Holotype QHU R2025027 in life, in situ; C. Paratype QHU R2025028 in life, in situ; D. Feces of the paratype QHU R2025031. Photographs by Y.H. Xu (A–C) and J.D. Deng (D). Trimeresurus loong sp. nov. differs from T. mayaae (distributed across the Indian States of Manipur, Meghalaya, Mizoram, Assam, Nagaland, West Bengal, Sikkim, Bhutan, and Chin State of Myanmar) by the following combination of characters: larger maximum SVL in females (657 mm vs. 590 mm); lower number of VS in males (150–151 [mean 150.3 ± 0.5] vs. 153–163 [mean 158.1 ± 3.0]); higher number of SC in females (57–64 [mean 60.5 ± 4.9] vs. 53–55 [mean 54.0 ± 1.0]); and higher number of VS+SC in females (215–221 [mean 218.0 ± 4.2] vs. 205–208 [mean 206.7 ± 1.5]). In addition, females of Trimeresurus loong sp. nov. have yellowish irises, distinctly differing from the greenish eyes of T. mayaae. Trimeresurus loong sp. nov. differs from T. medoensis (distributed in the Xizang AR of southwestern China, and possibly in northern Myanmar and northeastern India) by the following combination of characters: slight larger maximum SVL in both sexes (601 mm in males, 657 mm in females vs. 553 mm in males, 624 mm in females); higher number of VS in both sexes (150–151 [mean 150.3 ± 0.5] in males, 157–158 [mean 157.5 ± 0.7] in females vs. 146–151 [mean 148.6 ± 1.8] in males, 145–147 [mean 146.0 ± 1.0] in females); greater number of SC in males (67–68 [mean 67.3 ± 0.5] vs. 55–59 [mean 57.0 ± 1.4]); and higher number of VS+SC in both sexes (217–219 [mean 217.5 ± 1.0] in males, 215–221 [mean 218.0 ± 4.2] in females vs. 201–208 [mean 205.6 ± 2.9] in males, 204–206 [mean 205.0 ± 1.0] in females). The new species also possesses a greater number of SL (10–11 vs. 8–9) and IL (11–14 vs. 8–10). It further differs in having more dorsal scale rows at midbody (19 vs. 17) and posteriorly (15 vs. 13, rarely 11). Eye coloration also differs: males of new species have amber irises with a slight reddish hue, and females yellowish, whereas both sexes of T. medoensis possess green or yellowish-green eyes (Fig. 10A, B, E). In addition, the ventrolateral stripe in females of new species is absent or uniformly white, contrasting with the red-and-white stripe of T. medoensis. In hemipenial morphology, Trimeresurus loong sp. nov. can be distinguished from T. medoensis by longer hemipenial truncus and a lower degree of bifurcation (HCL/HTL ratio 0.62–0.66 vs. 0.53), and a more distal sulcus spermaticus bifurcation point (SPBD/HCL ratio 0.43–0.49 vs. 0.21) (Fig. 4A–C). Trimeresurus loong sp. nov. differs from T. nujiang (distributed across several localities within the Nujiang River Basin of northwestern Yunnan Province, China, including Gongshan, Fugong, and Lushui Conties) by the following combination of characters: slightly smaller maximum SVL in both sexes (601 mm in males, 657 mm in females vs. 694 mm in males, 682 mm in females); lower number of zse.pensoft.net Xu, Y. et al.: A new Trimeresurus species from Yunnan, China2286 Table 5. Summary of morphological characters in members of the subgenus Viridovipera. Notes: Diagnostic characters distinguishing Trimeresurus loong sp. nov. are indicated in bold. Trimeresurus Sources Max SVL (mm) VS SC VS+SC ASR MSR PSR SL IL ♂♀♂ ♀ ♂ ♀ ♂ ♀ T. loong sp. nov. (19) 601 657 150–151 [150.3 ± 0.5] 157–158 [157.5 ± 0.7] 67–68 [67.3 ± 0.5] 57–64 [60.5 ± 4.9] 217–219 [217.5 ± 1.0] 215–221 [218.0 ± 4.2] 19–23 19 15 10 or 11 11–14 T. mayaae (7), (8), (9), (14), (15), (16) 610 590 153–163 [158.1 ± 3.0] 152–153 [152.7 ± 0.6] 54–69 [61.1 ± 4.5] 53–55 [54.0 ± 1.0] 211–231 [218.9 ± 6.1] 205–208 [206.7 ± 1.5] 19–28 19–21 15–17 8–10 10–13 T. medoensis (4), (13), (16) 553 624 146–151 [148.6 ± 1.8] 145–147 [146.0 ± 1.0] 55–59 [57.0 ± 1.4] 58–60 [59.0 ± 1.0] 201–208 [205.6 ± 2.9] 204–206 [205.0 ± 1.0] 17 (rarely 19) 17 13 (rarely 11) 8 or 9 8–10 T. nujiang (17) 694 682 164–173 [168.7 ± 3.6] 165–168 [166.6 ± 1.3] 59–68 [62.8 ± 3.9] 57–60 [58.2 ± 1.1] 226–241 [231.3 ± 6.8] 222–226 [224.8 ± 1.8] 19–21 (rarely 22) 19 15 9 or 10 11–13 T. pretiosus (18) 516 512 140–143 [141.5 ± 2.1] 142 56–58 [57.0 ± 1.4] 54 198–199 [198.5 ± 0.7] 196 19 19 15 8 or 9 10 or 11 T. stejnegeri (1), (3), (16) 635 670 154–178 [164.5 ± 4.8] 155–173 [163.9 ± 3.5] 60–80 [70.3 ± 4.2] 58–70 [63.4 ± 2.9] 218–256 [234.9 ± 7.7] 218–237 [227.5 ± 4.5] 21–25 21–23 15 9–12 10–14 T. truongsonensis (8), (11), (16) 521 488 170–190 [179.8 ± 8.7] 165–167 [166.0 ± 1.0] 65–71 [67.4 ± 2.5] 61–70 [66.7 ± 4.9] 235–259 [247.2 ± 10.7] 228–235 [232.7 ± 4.0] 21 21 15 9–11 11–13 T. vogeli (5), (16) 661 947 157–169 [162.8 ± 3.7] 157–173 [164.2 ± 5.8] 63–71 [67.0 ± 2.3] 59–70 [63.0 ± 3.8] 221–238 [229.8 ± 4.9] 218–238 [227.7 ± 6.3] 21–23 21 (rarely 20) 15 9–11 11–16 T. yunnanensis (2), (3), (6), (10), (12), (17) 602 804 151–164 [158.2 ± 3.9] 150–164 [157.9 ± 3.6] 61–71 [66.1 ± 2.9] 52–65 [57.8 ± 3.6] 221–231 [225.9 ± 3.9] 211–223 [216.4 ± 3.7] 19–21 19–21 15–17 9–11 10–13 Trimeresurus Eye color in life Postocular streak in life Ventrolateral stripe in life Tail red Tail red pattern Body colouration in life ♂ ♀ ♂ ♀ ♂ ♀ T. loong sp. nov. amber, sometimes with a slight reddish hue yellowish thin and white, with a few scale margins slightly tinged with yellow or reddish-brown absent yellow + white + red + brown or thin, white with yellowish margins absent or white, thin yes orange or light orange-red, uniform, blackish brown at the tip uniformly grass-green T. mayaae rusty or greenish greenish none or red + white absent red + white, wide pale yellow + white, thin yes rusty red, forming blotches anteriorly uniformly grass-green T. medoensis green or yellowish green green or yellowish green none or faint white absent red + white, wide red + white yes red to dark rusty red, sometimes forming blotches anteriorly uniformly grass-green T. nujiang golden yellow golden yellow none absent dark red + white faint white yes reddish-brown with dark brown at the tip uniformly grass-green T. pretiosus reddish-brown orange-yellow none or very faint, thin white absent red + white, wide white, thin yes brick-red, sometimes forming blotches anteriorly uniformly grass-green T. stejnegeri bright red or orange-red (rarely yellow) yellow or orange red + white or white absent or white, thin red + white, wide red + white or white yes light orange-red to red, uniform or forming blotches anteriorly uniformly grass-green T. truongsonensis greenishyellow greenishyellow none absent red + brown, wide red + brown no – greenish blue with brown broad bands T. vogeli light orange light orange none or faint white absent red + white, wide pale yellow + white, thin no – uniformly grass-green T. yunnanensis bright or deep red golden yellow red + white or white or absent absent or white, thin red + white, wide absent or white or pale green, thin yes dark red, with large anterior blotches and irregularly shaped margins uniformly grass-green Sources: (1) = Maki 1931; (2) = Pope 1935; (3) = Zhao et al. 1998; (4) = David et al. 2001a; (5) = David et al. 2001b; (6) = David et al. 2002; (7) = Ao et al. 2004; (8) = Orlov et al. 2004; (9) = David and Mathew 2005; (10) = Guo et al. 2009; (11) = Teynié and David 2004; (12) = Nguyen et al. 2018; (13) = Che et al. 2020; (14) = Rathee et al. 2022; (15) = Elangbam et al. 2023; (16) = Nguyen et al. 2025; (17) = Liang et al. 2025; (18) = Xu et al. 2025; (19) = this study. Zoosyst. Evol. 101 (4) 2025, 2267–2293 zse.pensoft.net 2287 Figure 10. Comparison of the lateral head view of males of species in the subgenus Viridovipera in life. A. Trimeresurus loong sp. nov., QHU R2025027, holotype; B. Trimeresurus loong sp. nov., QHU R2025028, paratype; C. Trimeresurus sp. from Chengdu, Sichuan, China; D. T. mayaae from Kangpokpi, Manipur, India; E. T. medoensis, QHU R2025040, from Motuo (Medog), Xizang, China; F. T. nujiang from Gongshan, Yunnan, China; G. T. pretiosus, QHU R2025020, from Yadong, Xizang AR, China; H. T. stejnegeri, QHU R2025037, from Lishui, Zhejiang, China; I. T. truongsonensis from Phong Nha-Ke Bang NP, Quang Tri, Vietnam; J. T. vogeli from Phong Nha-Ke Bang NP, Quang Tri, Vietnam; K. T. yunnanensis from Jingdong, Yunnan, China; L. T. cf. yunnanensis from Mt. Dawei, Honghe, Yunnan, China. Photographs by Y.H. Xu (A–C, E–L) and P. Shinde (D). VS in both sexes (150–151 [mean 150.3 ± 0.5] in males, 157–158 [mean 157.5 ± 0.7] in females vs. 164–173 [mean 168.7 ± 3.6] in males, 165–168 [mean 166.6 ± 1.3] in females); and lower number of VS+SC in both sexes (217– 219 [mean 217.5 ± 1.0] in males, 215–221 [mean 218.0 ± 4.2] in females vs. 226–241 [mean 231.3 ± 6.8] in males, 222–226 [mean 224.8 ± 1.8] in females). Eye coloration also differs, with males of the new species having amber irises with a slight reddish hue vs. yellow or mottled gray and flesh-colored in T. nujiang (Fig. 10A, B, F). In addition, a postocular streak is present in males of new species but absent or very faint in T. nujiang. Trimeresurus loong sp. nov. differs from T. pretiosus (distributed in Yadong County, Xigaze City, Xizang AR, China) by the following combination of characters: having a larger maximum SVL in both sexes (601 mm in males, 657 mm in females vs. 516 mm in males, 512 mm in females); a higher number of VS (150–151 [mean 150.3 ± 0.5] in males, 157–158 [mean 157.5 ± 0.7] in females vs. 140–143 [mean 141.5 ± 2.1] in males, 142 in female); a greater number of SC (67–68 [mean 67.3 ± 0.5] in males, 57–64 [mean 60.5 ± 4.9] in females vs. 56–58 [mean 57.0 ± 1.4] in males, 54 in female); a higher number of VS+SC (217–219 [mean 217.5 ± 1.0] in males, 215–221 [mean 218.0 ± 4.2] in females vs. 198–199 [mean 198.5 ± 0.7] in males, 196 in female); a greater number of supralabials (10 or 11 vs. 8 or 9); in possessing all subcaudal scales paired (vs. partly single); and eye coloration in males is zse.pensoft.net Xu, Y. et al.: A new Trimeresurus species from Yunnan, China2288 Figure 11. Comparison of male coloration between Trimeresurus loong sp. nov. and T. yunnanensis. A1. Trimeresurus loong sp. nov., QHU R2025028; A2. Trimeresurus loong sp. nov., QHU R2025029; B1. T. yunnanensis, QHU R2025035, from Lincang, Yunnan, China; B2. T. yunnanensis, unvouchered individual from Jingdong, Yunnan, China. The white arrows indicate the dorsal surface of the tail, and blue arrows indicate the ventrolateral stripe. Photographs by Y.H. Xu. amber with a slight reddish hue (vs. brick red) (Fig. 10A, B, G). In hemipenial morphology, Trimeresurus loong sp. nov. can be distinguished from T. pretiosus by longer hemipenial truncus and a lower degree of bifurcation (HCL/HTL ratio 0.62–0.66 vs. 0.47–0.53), and a more distal sulcus spermaticus bifurcation point (SPBD/HCL ratio 0.43–0.49 vs. 0.28–0.36) (Fig. 4A, B, D). Trimeresurus loong sp. nov. differs from T. stejnegeri (restricted to eastern and southern China, including Taiwan Province, northern Vietnam, and northeastern Laos) by the following combination of characters: lower VS in males (150–151 [mean 150.3 ± 0.5] vs. 154–178 [mean 164.5 ± 4.8]); lower number of VS+SC in males (217– 219 [mean 217.5 ± 1.0] vs. 218–256 [mean 234.9 ± 7.7]); and fewer dorsal scale rows at midbody (19 vs. 21–23). In males, the irises are amber with a slight reddish hue, and the ventrolateral stripe is slender and confined to the first dorsal scale row, occurring in two forms: either a narrow white band bordered dorsally by a faint yellowish margin and ventrally by a red line with a diffuse brownish tinge below the red line, or a narrow white band bordered by faint yellowish margins on both sides in Trimeresurus loong sp. nov. In contrast, in T. stejnegeri, the irises are orange-red or red (Fig. 10A, B, H), and the ventrolateral stripe consists of a red lower border and a white upper zone, with the two colors sharply separated and clearly visible from a distance. In females, the ventrolateral stripe is uniformly white, whereas in T. stejnegeri it is white or red-and-white. In hemipenial morphology, Trimeresurus loong sp. nov. can be distinguished from T. stejnegeri by shorter hemipenial truncus and a higher degree of bifurcation (HCL/HTL ratio 0.62–0.66 vs. 0.69), and a more distal sulcus spermaticus bifurcation point (SPBD/HCL ratio 0.43–0.49 vs. 0.28) (Fig. 4A, B, E). Trimeresurus loong sp. nov. differs from T. truongsonensis (restricted to central Vietnam and central Laos) by the following combination of characters: a larger maximum SVL in both sexes (601 mm in males, 657 mm in females vs. 521 mm in males, 488 mm in females); a lower number VS (150–151 [mean 150.3 ± 0.5] in males, 157– 158 [mean 157.5 ± 0.7] in females vs. 170–190 [mean 179.8 ± 8.7] in males, 165–167 [mean 166.0 ± 1.0] in females); a greater number of SC (67–68 [mean 67.3 ± 0.5] in males, 57–64 [mean 60.5 ± 4.9] in females vs. 56–58 [mean 57.0 ± 1.4] in males, 54 in the female); and consequently a lower number of VS+SC (217–219 [mean 217.5 ± 1.0] in males, 215–221 [mean 218.0 ± 4.2] in females vs. 235–259 [mean 247.2 ± 10.7] in males, 228–235 [mean 232.7 ± 4.0] in females); and fewer dorsal scale rows at midbody (MSR 19 vs. 21). Eye coloration is amber with a slight reddish hue in males and yellowish in females, contrasting with the greenish-yellow eyes of both sexes in Zoosyst. Evol. 101 (4) 2025, 2267–2293 zse.pensoft.net 2289 the latter species (Fig. 10A, B, I). The body is uniformly bright grass-green, whereas T. truongsonensis exhibits a greenish-blue dorsum with broad brown crossbands. Finally, Trimeresurus loong sp. nov. differs from T. vogeli (distributed in the southeastern part of central Thailand, central and southern Laos, Cambodia, and central and southern Vietnam) by the following combination of characters: smaller maximum SVL in both sexes (601 mm in males, 657 mm in females vs. 661 mm in males, 947 mm in females); lower number of VS in males (150–151 [mean 150.3 ± 0.5] vs. 157–169 [mean 162.8 ± 3.7]); lower number of VS+SC in males (217–219 [mean 217.5 ± 1.0] vs. 221–238 [mean 229.8 ± 4.9]); fewer dorsal scale rows at midbody (MSR 19 vs. 21, rarely 20); and eye coloration in males is amber with a slight reddish hue (vs. light orange or flesh-colored) (Fig. 10A, B, J). Discussion Pholidosis (i.e., the pattern and arrangement of scales) has long been considered one of the most important diagnostic features for species identification and classification in snakes. However, in the genus Trimeresurus, scale counts are often highly conserved, making taxonomic delimitation particularly challenging (Guo et al. 2009; David et al. 2011; Sumontha et al. 2021; Idiiatullina et al. 2024b; Nguyen et al. 2025; Pawangkhanant et al. 2025). Many closely related species are nearly indistinguishable based on scalation characters alone, especially within the subgenus Viridovipera, where several taxonomic units had long been misidentified as T. stejnegeri or T. yunnanensis (Schmidt 1927; Pope 1935; Smith 1943; Zhao et al. 1998; Creer et al. 2003; Malhotra and Thorpe 2004a, b; Zhao 2006; Guo et al. 2009; Rathee et al. 2022; Wu et al. 2023; Liang et al. 2025). For example, T. mayaae, T. nujiang, and the newly described Trimeresurus loong sp. nov. were previously regarded as geographically distinct populations of T. yunnanensis (Zhao et al. 1998; Malhotra and Thorpe 2004a, b; Zhao 2006; Rathee et al. 2022; Wu et al. 2023; Liang et al. 2025). In recent years, the incorporation of molecular phylogenetic data has gradually uncovered the true diversity within Viridovipera, revealing that this subgenus harbors a greater number of cryptic species than previously recognized. Nonetheless, due to significant overlap in pholidosis among many taxa, the inclusion of more diverse morphological characters is essential for accurate species identification and classification. One such promising character set is coloration pattern, which has shown potential in resolving taxonomic ambiguities in this group. Sexual dichromatism is common in Viridovipera, with males typically exhibiting more vivid and distinctive coloration patterns that are often stable within species. As such, comparisons of male coloration traits can provide important diagnostic clues for species delimitation. Three recently described species, T. mayaae, T. pretiosus, and T. nujiang in Viridovipera have also employed coloration features as key diagnostic characters (Rathee et al. 2022; Liang et al. 2025; Xu et al. 2025). Trimeresurus loong sp. nov. is likewise distinguished by its unique coloration traits. Although it was once misidentified as T. yunnanensis, the two species can be readily distinguished in males by differences in eye coloration, as well as in the color pattern of the ventrolateral stripe and dorsal tail. In addition, hemipenial morphology serves as another effective diagnostic feature. Although the hemipenes of Viridovipera species have been described in previous studies (Pope 1935; Malhotra and Thorpe 2004a; Orlov et al. 2004; Dawson et al. 2008; Guo et al. 2009; Rathee et al. 2022; Liang et al. 2025; Xu et al. 2025), most accounts were based on incompletely everted or poorly preserved specimens, thereby limiting their diagnostic value. A comprehensive interspecific comparison has remained lacking. In this study, we compared the hemipenes of five species (Fig. 4) within the subgenus Viridovipera and found that the primary differences involve the degree of bifurcation, the relative position of the sulcus spermaticus bifurcation point, and the number of enlarged spines. While Guo et al. (2009) described variation in the zonation of the spine and calyx regions among several species, our results indicate that these features are, in fact, remarkably similar across taxa. The previously reported discrepancies may result from methodological differences in hemipenis preparation, including the use of inflation materials and preservation state, which can distort the apparent shape (Myers and Cadle 2003). Moving forward, hemipenial morphology should be documented across a broader range of species within Viridovipera, and the terminology and preparation techniques should be standardized to ensure consistent taxonomic interpretations. Taken together, both morphological and molecular evidence consistently support the distinctiveness of Trimeresurus loong sp. nov. within Viridovipera. The new species Trimeresurus loong sp. nov. represents a distinct evolutionary lineage within the subgenus Viridovipera. Phylogenetic analyses based on mitochondrial DNA (16S, cyt b, and ND4 genes) recovered T. loong sp. nov. as sister to T. cf. loong from Huili City, Sichuan Province, China, with moderate statistical support (SH = 83; UFB = 92; PP = 0.97). Together, they form a clade with an unidentified Trimeresurus lineage from western Sichuan Province, China. This entire assemblage occupies a peripheral position relative to the remaining recognized species of Viridovipera, and Trimeresurus loong sp. nov. exhibits substantial genetic divergence from all currently recognized congeners, with uncorrected p-distances of at least 6.7% (cyt b) and 6.1% (ND4). This level of divergence, combined with consistent morphological differentiation, supports its recognition as a valid species. Moreover, the specimens previously recorded as T. yunnanensis by Schmidt (1927) and Pope (1935) from “Yunnan-fu” (now Kunming City, Yunnan Province; specimen MCZ 14671, a paratype of T. yunnanensis), those listed by Zhao et al. (1998) from Kunming (specimens KIZ 72001, KIZ 73008, and KIZ 85702), as well as specimen NHMUK 1904.11.29.29 from “Yunnan-fu” housed at the Natural History Museum, London, may likewise represent Trimeresurus loong sp. nov. (Table 6). However, due to the zse.pensoft.net Xu, Y. et al.: A new Trimeresurus species from Yunnan, China2290 lack of direct examination, the coloration of these specimens from Kunming City and its surrounding areas remains uncertain. Further reassessment is therefore necessary to determine whether they indeed represent Trimeresurus loong sp. nov., or whether the two species exhibit partially overlapping distributions. Liang et al. (2025) recently synonymized T. gumprechti with T. yunnanensis on the basis of minimal genetic divergence and overlapping morphological variation. Nevertheless, some specimens included in their comparative dataset appear to have been assigned to different taxa. For instance, ZFMK 92790 and ZFMK 92791 from Phuoc Binh National Park, Ninh Thuan Province (currently Khanh Hoa Province), southern Vietnam, were labeled as T. gumprechti but seem morphologically consistent with T. vogeli (T.V. Nguyen, pers. obs.). Likewise, CAS 241182 from Moenyin Township, Myitkyina District, Kachin State, northern Myanmar, appears referable to T. popeiorum (see Idiiatullina et al. 2024b), and ZFMK 92793 from Chu Yang Sin National Park, Dak Lak Province, southern Vietnam, initially identified as T. stejnegeri, also corresponds to T. vogeli. Such errors may have affected their taxonomic interpretation and highlight the need for a thorough re-examination of comparative material used in their study. Considering the recent descriptions of T. nujiang and T. pretiosus, it is evident that southwestern China harbors multiple cryptic lineages of the subgenus Viridovipera with restricted distributions. The discovery of Trimeresurus loong sp. nov. from Kunming City, Yunnan Province, further supports the view that T. yunnanensis sensu lato represents a complex of closely related species that warrants comprehensive revision using both genomic and morphological data. The discovery of Trimeresurus loong sp. nov. underscores the taxonomic complexity of the T. yunnanensis group and the importance of integrative approaches in resolving cryptic diversity among Asian green pit vipers. Biogeographically, the occurrence of Trimeresurus loong sp. nov. in the Anning City, Kunming City adds an important element to our understanding of Viridovipera diversification in Yunnan Province. This area represents a transitional zone between the Red River drainage and the Yunnan-Guizhou Plateau, characterized by dissected montane terrain and broad-leaved forests or coniferous forests. The restricted range of Trimeresurus loong sp. nov. contrasts with the more westernly species T. nujiang and the Himalayan occurrence of T. pretiosus. Together, these species reveal a clear geographic and elevational gradient of diversification within the Viridovipera, likely influenced by complex orogeny, climatic oscillations, and ecological isolation. The narrow ventrolateral stripe, light orange red tail, and amber iris of Trimeresurus loong sp. nov. may represent local adaptive features associated with its mid-montane forest environment. With the discovery of Trimeresurus loong sp. nov., the total number of Trimeresurus species recorded from Yunnan Province now rises to nine, including two species endemic to Yunnan, namely Trimeresurus loong sp. nov. and T. nujiang. These endemics further emphasize Yunnan’s exceptional biogeographic heterogeneity and its role as a core diversification center for Asian green pit vipers. From a conservation perspective, Trimeresurus loong sp. nov. is currently known only from its type locality in Qinglongxia, Anning City, Kunming City, Yunnan Province, China, at an elevation of approximately 1,822 m asl. Given its highly restricted known distribution and the absence of population data, the species should be provisionally classified as Data Deficient (DD) under IUCN Red List criteria. Targeted field surveys are urgently needed to evaluate its actual distribution, population size, and ecological requirements. Because green pit vipers are medically important, accurate taxonomic recognition of the T. yunnanensis complex is also critical for clinical identification and venom management. In conclusion, Trimeresurus loong sp. nov. represents another lineage within the rapidly diversifying subgenus Viridovipera radiation of southwestern China. The discovery of this species, together with T. nujiang and T. pretiosus, highlights Yunnan and adjacent Himalayan regions as key centers of speciation for Asian green pit vipers. Integrative taxonomic approaches that combine morphological, molecular, and ecological data will be essential to fully resolve the complex evolutionary history of this medically significant group. This finding further reinforces the importance of southwestern China as a hotspot of pit viper diversification and highlights the need for continued integrative surveys in montane ecosystems. Acknowledgements We are deeply grateful to Dr. Bo Cai (CIB, China) and Parag Shinde (India) for providing valuable information and photographs of Trimeresurus spp. We sincerely thank Dr. Jinlong Ren (CIB, China) for his invaluable assistance in the preparation and morphological description of the hemipenes. T.V. Nguyen thanks Dr. Patrick David (MNHN, France) for sharing taxonomic Table 6. Specimens from Kunming, Yunnan, China previously assigned to Trimeresurus yunnanensis. Remark: N/a = not available. Collection number Sex SVL (mm) TAL (mm) ASR MSR PSR VS SC SL IL Source KIZ 72001 ♂417 100 20 19 15 157 71 9/10 11/11 Zhao et al. (1998) KIZ 73008 ♀457 99 19 19 15 150 57 10/11 12/11 Zhao et al. (1998) KIZ 85702 ♀617 141 21 19 15 158 65 10/10 11/11 Zhao et al. (1998) MCZ 14671 ♂N/a N/a 21 19 15 160 68 N/a N/a Schmidt (1927); Pope (1935) NHMUK 1904.11.29.29 ♀652 137 21 20 15 156 55 10/10 12/12 This study Zoosyst. Evol. 101 (4) 2025, 2267–2293 zse.pensoft.net 2291 information and helpful discussions regarding Trimeresurus species. N.A. Poyarkov thanks S.S. Idiiatullina (MSU, Russia) for help and assistance. We also thank Justin M. Bernstein (AMNH, USA) and the anonymous reviewer for constructive comments on an earlier version of the manuscript. This research was funded by the National Natural Science Foundation of China [32301325], the Open Project of State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University [2025-KF-02], and the Russian Science Foundation (RSF grant Nº 22-14-00037-P, for supporting the work of Dr. Nikolay A. Poyarkov). References Akaike H (1973) Information theory and an extension of the maximum likelihood principle. 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