ZSC: for review purposes only - please do not distribute Geographic differentiation and cryptic diversity in the monocled cobra, Naja kaouthia (Elapidae) from Thailand Journal: Zoologica Scripta Manuscript ID ZSC-03-2019-0036.R2 Manuscript Type: Original Manuscript Date Submitted by the n/a Author: Review Copy Complete List of Authors: Ratnarathorn, Napat; UCL, Cell and Developmental Biology; Chulalongkorn University, Biology Harnyuttanakorn, Pongchai; Chulalongkorn University, Biology Chanhome, Lawan; Thai Red Cross Society, Snake Farm, Queen Saovabha Memorial Institute Evans, Susan; UCL, Cell and Developmental Biology Day, Julia; UCL, Genetics, Evolution, and Environment Monocled cobra, snakes, molecular phylogenetics, mitochondrial DNA, Keywords: phylogeography, cryptic species ZSC submitted manuscript Page 1 of 43 ZSC: for review purposes only - please do not distribute 1 2 3 4 5 1 Geographic differentiation and cryptic diversity in the 6 7 2 monocled cobra, Naja kaouthia (Elapidae) from Thailand 8 9 10 11 3 Napat RATNARATHORNa,b,c, Pongchai HARNYUTTANAKORNb, Lawan CHANHOMEc, Susan E. 12 4 EVANSa, and Julia J. DAYd 13 14 a Department of Cell and Developmental Biology, University College London, London WC1E 6BT, 15 5 16 6 UK 17 7 b Department of Biology, Faculty of Science, Chulalongkorn University, Pathum Wan, Bangkok, 18 19 8 10330, Thailand 20 9 c Snake Farm, Queen Saovabha Memorial Institute, Pathum Wan, Bangkok, 10330, Thailand 21 d Review Copy 22 10 Department of Genetics, Evolution and Environment, University College London, London WC1E 23 11 6BT, UK 24 25 26 27 12 Running title: Geographic differentiation in the monocled cobra 28 Ratnarathorn et al. 29 13 30 31 32 14 Corresponding authors: 33 Dr Julia DAY, Tel: 0044-(0)20-7679-0159 Email: [email protected] 34 15 35 16 Dr Napat RATNARATHORN, Tel: 0066-(0)80-016-7789 Email: [email protected] 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ZSC submitted manuscript ZSC: for review purposes only - please do not distribute Page 2 of 43 2 1 Ratnarathorn et al. 2 3 4 17 Abstract: South-east Asia has an exceptionally high diversity of snakes, with more 5 6 18 than 250 snake species currently recorded from Thailand. This diversity likely 7 8 19 reflects the diverse range of geographical and climatic conditions under which they 9 10 20 live, but the evolutionary history and population genetics of many snake species in 11 21 South-east Asia has been little investigated in comparison with morphological 12 13 22 studies. Here, we investigated genetic variation in the monocled cobra, Naja 14 15 23 kaouthia, Lesson, 1831, across its distribution range in Thailand using 16 24 mitochondrial-DNA (Cytochrome b, control region) for ~100 individuals, and the 17 18 25 nuclear DNA gene (C-mos) for a small subset. Using population genetic and 19 20 26 phylogenetic methods, we show high levels of genetic variation between regional 21 Review Copy 22 27 populations of this non-spitting cobra, including the north-eastern, north-central, 23 28 and southern regions, in addition to a population on Pha-ngan Island, 150 km 24 25 29 offshore from the southern peninsula. Moreover, inclusion of the north-eastern 26 27 30 population renders N. kaouthia paraphyletic in relation to other regional Naja 28 29 31 species. The north-eastern population is therefore probably specifically distinct. 30 32 Given that these cobras are otherwise undifferentiated based on colour and 31 32 33 general appearance to the ‘typical’ cobra type of this region; they would represent 33 34 34 a cryptic species. As has been shown in other animal groups from Thailand, it is 35 35 likely that the geographic characteristics and/or tectonic alteration of these regions 36 37 36 have facilitated high levels of population divergence of N. kaouthia in this region. 38 39 37 Our study highlights the need for dense sampling of snake populations to reveal 40 41 38 their systematics, plan conservation, and facilitate anti-snake venom development. 42 43 Keywords: Monocled cobra, snakes, molecular phylogenetics, phylogeography, 44 39 45 40 cryptic species 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ZSC submitted manuscript Page 3 of 43 ZSC: for review purposes only - please do not distribute 3 1 Ratnarathorn et al. 2 3 4 41 Introduction 5 6 42 7 8 43 South-east Asia has an exceptionally high diversity of snakes (Jitakune, 2004), 9 10 44 with more than 250 snake species currently recorded from Thailand (Cox et al., 11 45 2013). This diversity likely reflects the diverse range of geographical and climatic 12 13 46 conditions under which they live (Mott, 2010), but the evolutionary history and 14 15 47 population genetics of many snake species in South-east Asia has been little 16 48 investigated in comparison with morphology-based studies. For example, 17 18 49 phenotypic polymorphisms at the population level have been found in snakes living 19 20 50 between different geographical regions of South-east Asia e.g. Asiatic cobra 21 Review Copy 22 51 species in the genus Naja (Wüster et al., 1995), the monocled cobra, Naja 23 52 kaouthia, the King cobra, Ophiophagus hannah (Jitakune, 2004), and Kruki 24 25 53 snakes, Oligodon (Cox et al., 2013), but their genetic variation is generally lacking. 26 27 54 Such morphology-based studies have formed the basis for understanding 28 29 55 biodiversity, but where species occur over a wide range, with distinct populations, 30 56 cryptic species may be missed. 31 32 57 The monocled cobra (Naja kaouthia) is reportedly the only species of non- 33 34 58 spitting cobra that occurs throughout South-east Asia and some parts of China and 35 59 India (Uetz and Hošek, 2014), in which the type locality is listed as “Bengale” 36 37 60 (Lesson 1932: 314), India. Due to the large distribution area of N. kaouthia, 38 39 61 variation in adult characteristics and morphological divergence in populations have 40 41 62 repeatedly been found (e.g. Wüster et al., 1995; Wallach et al., 2014). The adult 42 63 variation may reflect environmental (e.g. tropical savanna, rain forest) and 43 44 64 geophysical differences resulting from mountain, river, and land-ocean partitions. 45 46 65 In Thailand, Naja kaouthia shows high levels of variation among regional 47 48 66 populations in features such as colouration (Cox, 1991; Wüster et al., 1995; 49 67 Jitakune and Chanhome, 1996; Jitakune, 2004) and toxin components (e.g. Tan et 50 51 68 al., 2017). Field observations also supported that the colour and pattern of N. 52 53 69 kaouthia (north: black; central: black and brown; south: olive), and timing of their 54 70 reproductive period (north-central: Oct/Nov - Mar/Apr and south: Apr – Sep) vary 55 56 57 58 59 60 ZSC submitted manuscript ZSC: for review purposes only - please do not distribute Page 4 of 43 4 1 Ratnarathorn et al. 2 3 4 71 among regions in Thailand. However, these differences have not been investigated 5 6 72 from a genetic perspective to test if phenotypic variation is reflected by genetic 7 8 73 variation. 9 10 74 The relationship between environmental conditions and population genetic 11 75 diversification is well documented in Naja atra, the closest relative of N. kaouthia. 12 13 76 The genetic study of Lin et al. (2014) revealed three distinct populations of N. atra 14 15 77 across East Asia (n = 285) and suggested that lineage diversification was caused 16 78 by the development of the Nanling and Luoxiao mountain ranges (the ranges run 17 18 79 from west to east in Southern China, and continue north into Central China). 19 20 80 Wüster and colleagues (1989, 1992, 1995) examined the systematics of the genus 21 Review Copy 22 81 Naja in Asia, and variation of N. kaouthia across its range, and these studies have 23 82 formed the basis of subsequent discussions on the evolution of cobras in South- 24 25 83 east Asia. Analysis of N. kaouthia based on meristic and linear measurements 26 27 84 found no differentiation (Wüster et al., 1995), while ‘typical’ and Suphan (white) 28 29 85 phase cobras were found to be genetically identical, although only four samples of 30 86 N. kaouthia were included. Other investigations of genetic differentiation in snake 31 32 87 taxa have generally yielded inconclusive results such as Elapidae in Asia, Africa, 33 34 88 and America, (Slowinski and Keogh, 2000), sequences of Caenophidian snakes 35 89 from GenBank (Kelly et al., 2003; Vidal et al., 2007), sequences of Colubroidea 36 37 90 snakes from GenBank (Lawson et al., 2005; Yan et al., 2008), and many snake 38 39 91 species in Thailand (Laopichienpong et al., 2016). 40 41 92 As shown for N. atra (Lin et al., 2014), our prediction was that 42 93 geographically separated regions (e.g. Woodruff, 2003; Hughes et al., 2003) and 43 44 94 temporal geological changes had the potential to facilitate divergence of N. 45 46 95 kaouthia in Thailand (e.g. Buddhachat and Suwannapoom, 2018). The northern 47 48 96 region is a mountainous area covered by many high, parallel mountain ranges that 49 97 extend from the north, alternating with many deep valleys and steep rivers. It is 50 51 98 also drained by rivers that unite in the lowlands of the central region. Further south, 52 53 99 the Himalayan-Tanoasri mountain range stretches from the central region into the 54 100 southern peninsula region. Similarly, the north to south Phetchabun, Dong Phaya 55 56 57 58 59 60 ZSC submitted manuscript Page 5 of 43 ZSC: for review purposes only - please do not distribute 5 1 Ratnarathorn et al.