Molecular Phylogenetics and Evolution 161 (2021) 107152

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Molecular Phylogenetics and Evolution

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Molecular phylogenetics of sub-Saharan African natricine snakes, and the biogeographic origins of the Seychelles endemic Lycognathophis seychellensis

V. Deepak a,*, Simon T. Maddock a,b,c, Rhiannon Williams a,d, Zoltan´ T. Nagy e, Werner Conradie f,g, Sara Rocha h, D. James Harris i, Ana Perera i, Vaclav´ Gvoˇzdík j,k, Thomas M. Doherty-Bone a,l, Rachunliu G. Kamei a, Michele Menegon m,n, Jim Labisko c,o,p, Charles Morel q, Natalie Cooper a, Julia J. Day p, David J. Gower a,c a Department of Life Sciences, Natural History Museum, London SW7 5BD, UK b School of Biology, Chemistry and Forensic Science, Wolverhampton University, WV1 1LY, UK c Island Biodiversity and Conservation Centre, University of Seychelles, Mah´e, Seychelles d NRA Environmental Consultants, Cairns, Queensland 4870, Australia e Independent Researcher, Berlin, Germany f Port Elizabeth Museum (Bayworld), Humewood, Port Elizabeth 6013, South Africa g Department of Nature Conservation Management, Natural Resource Science and Management Cluster, Faculty of Science, George Campus, Nelson Mandela University, George, South Africa h Biomedical Research Center (CINBIO), University of Vigo & Galicia Sur Health Institute, Vigo, Spain i CIBIO-InBIO, Centro de Investigaçao˜ em Biodiversidade e Recursos Gen´eticos, University of Porto, 4485-661 Vairao,˜ Portugal j Institute of Vertebrate Biology of the Czech Academy of Sciences, Brno, Czech Republic k National Museum, Department of Zoology, Prague, Czech Republic l Conservation Programs, Royal Zoological Society of Scotland, Edinburgh EH12 6TL, UK m Division of Biology & Conservation Ecology, Manchester Metropolitan University, UK n PAMS Foundation, P.O. Box 16556, Arusha, Tanzania o Durrell Institute of Conservation and Ecology, School of Anthropology and Conservation, University of Kent, Canterbury CT2 7NR, UK p Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, UK q Natural History Museum, Victoria, Mah´e, Seychelles

ARTICLE INFO ABSTRACT

Keywords: Phylogenetic relationships of sub-Saharan African natricine snakes are understudied and poorly understood, Biogeography which in turn has precluded analyses of the historical biogeography of the Seychelles endemic Lycognathophis Gondwana seychellensis. We inferred the phylogenetic relationships of Seychelles and mainland sub-Saharan natricines by Natricinae analysing a multilocus DNA sequence dataset for three mitochondrial (mt) and four nuclear (nu) genes. The Natricidae mainland sub-Saharan natricines and L. seychellensis comprise a well-supported clade. Two maximally supported Overseas dispersal Systematics sets of relationships within this clade are (Limnophis,Natriciteres) and (Afronatrix,(Hydraethiops,Helophis)). The relationships of L. seychellensis with respect to these two lineages are not clearly resolved by analysing concat­ enated mt and nu data. Analysed separately, nu data best support a sister relationship of L. seychellensis with (Afronatrix,(Hydraethiops,Helophis)) and mt data best support a sister relationship with all mainland sub-Saharan natricines. Methods designed to cope with incomplete lineage sorting strongly favour the former hypothesis. Genetic variation among up to 33 L. seychellensis from fiveSeychelles islands is low. Fossil calibrated divergence time estimates support an overseas dispersal of the L. seychellensis lineage to the Seychelles from mainland Africa ca. 43–25 million years before present (Ma), rather than this taxon being a Gondwanan relic.

1. Introduction both a remote, oceanic setting and continental origins as a fragment of Gondwana. Thus, its extant biota is a mosaic of ancient, relictual line­ The Seychelles archipelago is an unusual island system in that it has ages and more recent overwater arrivals (Ali, 2017). This provides an

* Corresponding author. E-mail address: [email protected] (V. Deepak). https://doi.org/10.1016/j.ympev.2021.107152 Received 14 April 2020; Received in revised form 8 March 2021; Accepted 8 March 2021 Available online 17 March 2021 1055-7903/© 2021 Elsevier Inc. All rights reserved. V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152 attractive system for studying evolutionary processes, though exploiting vertebral and hemipenial anatomy, and indicated a close relationship this system for evolutionary research depends on improved knowledge with (and possible membership of) a mainland sub-Saharan African of whether each resident lineage is ancient or relatively recent, as well as natricine radiation. However, those studies sampled only three of the six the status of populations on multiple Seychelles islands. Recently, some extant sub-Saharan African natricine genera (see Fig. 1 for distribution of this information has been generated for several lineages of Seychelles map) and only four of the 13 currently recognised species. Additionally, amphibians and reptiles (e.g., Brandley et al., 2005; Rocha et al., 2010, those previous studies disagreed as to whether L. seychellensis is more 2011, 2016a, b; Tolley et al., 2013; Maddock et al., 2014), but not yet for closely related to Afronatrix (Dubey et al., 2012; Pyron et al., 2013; any of the native snakes (see Williams et al., 2020 for the latest assess­ Zaher et al., 2019) or to Natriciteres (Guo et al., 2012). As far as we are ment of the non-native status of Seychelles Indotyphlops braminus). aware, there are no phylogenetic hypotheses that include all mainland The snake Lycognathophis seychellensis (Schlegel, 1837) is endemic to sub-Saharan African natricine genera such that, beyond the close the granitic islands of the Seychelles (Nussbaum, 1984). Several aspects inferred relationship of the only two previously sampled genera in of the systematics and historical biogeography of L. seychellensis remain molecular phylogenetic studies (Afronatrix and Natriciteres), there ap­ unclear. DNA sequence data (for two partial nuclear genes, cmos and pears to have been no previous consideration as to whether the main­ rag2) have been published for only one or two individuals of land sub-Saharan African natricines constitute a single radiation and L. seychellensis (Vidal et al., 2008). This species has been recorded from what their biogeographic origins might be. Finally, there are no pub­ six of the inner, granitic Seychelles islands (Fig. 1; Gerlach and Ineich, lished estimates of the age of the split between L. seychellensis and its 2006) and exhibits notable colour polymorphism (Nussbaum, 1984). closest extant relative. Having this temporal framework would allow a Other Seychelles reptile species have been found to display high levels of test of the hypothesis that the L. seychellensis lineage arrived in the variation within and among islands, with potential implications for Seychelles via overseas dispersal rather than being a Gondwanan relic. taxonomy and conservation management (Rocha et al., 2011, 2013, Here we present analyses of mitochondrial and nuclear DNA 2016; Valente et al., 2014; Harris et al., 2015). Thus, an inspection of sequence data in order to test the following hypotheses: (1) mainland possibly substantial intraspecific genetic variation in L. seychellensis is sub-Saharan African natricines are monophyletic, (2) L. seychellensis lies warranted. within the sub-Saharan African radiation, (3) intraspecific genetic Currently there are 252 recognised species in the colubrid subfamily variation within L. seychellensis is low across multiple Seychelles islands, Natricinae, with the majority of this diversity found in Asia (183 species and (4) the L. seychellensis lineage arrived in the Seychelles via oceanic ca. 72%), only 13 species (ca. 5%) in mainland sub-Saharan Africa, and dispersal rather than being an ancient, Gondwanan relic. none in Madagascar (Uetz et al., 2020). Previous molecular phylogenetic analyses (Vidal et al., 2008; Dubey et al., 2012; Guo et al., 2012; Pyron et al., 2013; Zaher et al., 2019) supported the natricine colubrid (natricid according to some classifications: e.g., Zaher et al., 2019) identity of L. seychellensis proposed by Dowling (1990) based on

Fig. 1. Global distribution of sub-Saharan African (including Seychelles) natricine genera based on data from GBIF, Nagy et al. (2014), Chippaux & Jackson (2019), Conradie et al. (2020) and specimen vouchers from Port Elizabeth Museum (PEM). These genera comprise 14 currently recognised extant species as follows: Afronatrix (1 species), Helophis (1), Hydraethiops (2), Limnophis (3), Lycognathophis (1), and Natriciteres (6). Occurrence data for the one Seychelles and six of the mainland sub-Saharan African natricine genera (all except Helophis) were downloaded from the GBIF database (GBIF, 2019). Distribution data for Helophis were taken from Nagy et al. (2014). Spatial data were mapped using ArcGIS v. 10.5. (ESRI, The Redlands, CA). Sampling localities in mainland sub-Saharan Africa are labelled with numbers corresponding to those in Table 1. Precise locality information for L. seychellensis is presented in Table 1.

2 V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152

2. Materials and methods analysis using MrBayes v. 3.2.6 on XSEDE (Ronquist et al., 2012). Par­ titionFinder2 (Lanfear et al., 2016) was used to determine best-fitgene 2.1. DNA sequence data generation and alignment and codon partitioning strategies using the greedy algorithm and BIC model selection, departing from gene and codon position blocks. RAxML To infer phylogenetic relationships and to assess intraspecificgenetic analyses applied the GTRGAMMA model (following Stamatakis, 2006) variation within the Seychelles endemic Lycognathophis seychellensis, we to each partition. For the BI analysis we executed two runs with four generated mitochondrial (mt) and nuclear (nu) gene sequence data for a Markov chains initiated from random trees and ran these for 10,000,000 total of 33 specimens of this species from across fiveof the six islands of generations each, sampling every 1000 generations. When the BI anal­ its known distribution (Table 1). This included 14 individuals from ysis was terminated, the standard deviation of split frequencies was less Mah´e, six from Praslin, six from Silhouette, two from La Digue, and five than 0.005, and the first 25% trees were discarded as “burn-in”. from Fr´egate. The eight partial genes sequenced were mt 16s (n = 30), Convergence was assessed for all parameters using Tracer v1.6 (Ram­ cytb (n = 29) and nd4 (n = 2), and nu cmos (n = 8), nt3 (n = 8), bdnf (n = baut et al., 2014) and the RWTY package (Warren et al., 2017). Bayesian 6), rag1 (n = 2), and prlr (n = 21). In addition to the new L. seychellensis analysis used best-fitmodels as determined by PartitionFinder. Support data, we generated 119 sequences for the three mt genes (16s, cytb, nd4) for internal branches was quantified using bootstrap proportions and four of the nuDNA loci (cmos, nt3, bdnf, rag1) for 27 other natricine (calculated from 500 replicates) and Bayesian posterior probabilities, snakes representing ten mainland sub-Saharan African species and five respectively. Asian species (Table 1). We compared the fitof the data to optimal and best suboptimal trees Genomic DNA was extracted from muscle, liver, tail tip or ventral for both ML and BI analyses for the 21-leaf dataset. For ML, we assessed ◦ scale clip tissue samples stored in absolute ethanol at 20 C, using the differences in likelihood between optimal and suboptimal trees using the DNeasy (QiagenTM) blood and tissue kit. Primers and annealing tem­ Approximately Unbiased (AU; Shimodaira, 2002) and Shimodaira- peratures for PCR amplification and for sequencing are reported in Hasegawa (SH; Shimodaira and Hasegawa, 1999) tests as imple­ Table S1. Bidirectional sequence chromatograms were edited and mented in the package CONSEL (Shimodaira and Hasegawa, 2001). For assembled using Chromas Lite v. 2.1.1 (Technelysium, 2012). Possibly BI, we used Bayes factors (Kass and Raftery, 1995), with Marginal heterozygous positions in nuDNA sequences were given ambiguity Likelihood estimate (MLE) scores estimated using path sampling and codes. MEGA v. 7.0 (Kumar et al., 2016) was used to assemble se­ steppingstone sampling methods as described by Baele et al. (2012, quences. Protein-coding sequences were checked for unexpected stop 2013). Using BEAST v 1.10.4, analyses applying each of the alternative codons by translating nucleotide alignments to amino acids. Newly tree topologies as constraints were each run for 150,000,000 genera­ generated data were combined with data from GenBank for other tions and sampled every 5,000. MLE scores were generated from 100 natricines and outgroups (Table 1), and aligned using the ClustalW al­ path steps for each run for 500,000 generations. gorithm (Thompson et al., 1994) with default settings as implemented in A species tree analysis of the 21-leaf dataset was carried out using a MEGA. Bayesian multispecies coalescent approach as implemented in Star­ BEAST2 (BEAST v2.5, Bouckaert et al., 2019). Input filesfor StarBEAST2 2.2. Molecular phylogenetics were prepared using BEAUti 2 (Drummond et al., 2012). Each terminal was assigned to a described species. Priors were set as follows: linked To infer the phylogenetic relationships of L. seychellensis and main­ tree model, strict clock, constant root population size model on species land sub-Saharan African natricines, we assembled a dataset of se­ tree, GTR model for each locus, analyses initiated with a random starting quences from GenBank (Benson et al., 2017) and our newly generated tree for each locus, population mean set to a uniform prior and a sequences for three mitochondrial (16s, cytb, nd4) and four nuclear lognormal prior for population size. The analysis was run for (cmos, nt3, bdnf, rag1) genes. Initially we ran analyses with a 157-leaf 100,000,000 generations sampling every 10,000 generations following dataset, comprising 155 natricines and two colubroid outgroups; the a 10% burn-in. Convergence was checked in Tracer V1.6 with ESS ≥ grayiine (grayiid) Grayia ornata and sibynophiine (sibynophiid) Sib­ 200. Resulting trees were summarised in LogCombiner 2.4.4 and ynophis subpunctatus (Table S2). Outgroup selection was based on the TreeAnnotator 2.5. (Bouckaert et al., 2019). Results were visualised in results of more broadly sampled molecular phylogenetic studies (Fig­ FigTree 1.4.3 (Rambaut, 2016) and DensiTree (Bouckaert and Heled, ueroa et al., 2016; Zaher et al., 2019; Lalronunga et al., 2020). The 155 2014). natricines comprised 26 sub-Saharan African (including Seychelles) We compared support for alternative resolutions of the relationships leaves representing all six genera and eleven of the 14 currently recog­ of L. seychellensis under ML and BI analyses of the 21-leaf, concatenated nised species. The other 129 natricine leaves were selected from across mt and nu dataset. Support values were read from majority rule partition the global geographic and phylogenetic diversity of the group based on tables in PAUP* (Swofford, 2000) for 500 RAxML bootstrap trees, 2,000 each leaf being represented by at least three of the seven genes included sampled (post burn-in) MrBayes BI trees, and 1,500 sampled (post burn- in the dataset. This sampling covered 30 of the 36 genera and 115 of the in) BEAST2 BI trees. For RAxML, analyses were carried out with data 245 species of extant natricines currently recognised (Uetz et al., 2020). partitioned by gene, and by gene and codon position. Based on results of maximum likelihood (ML) (Felsenstein, 1981) ana­ lyses of concatenated data for this 157-leaf dataset, in which sub- 2.3. Divergence time estimation & historical biogeography Saharan and Seychelles natricines were together maximally supported as monophyletic, we subsequently ran phylogenetic analyses for a more Our main aim was to test the hypothesis that the Seychelles natricine restricted 21-leaf dataset (Table 1, Table S2) in an attempt to run lineage reached the Seychelles via overseas dispersal, rather than being phylogenetic analyses using (hopefully, more realistic) models esti­ a Gondwanan relic. The geological timeframe for this test is as follows. mated for a more focused taxon set with more complete data. This Following the initial break up of eastern and western Gondwana from ca. comprised 17 Seychelles and mainland sub-Saharan African natricines 165 million years before present (Ma), Indo-Madagascar split from plus four outgroup natricines selected from among the likely closest Antarctica + Australia ca. 130–112 Ma, and Madagascar split from India relatives of the Seychelles + mainland sub-Saharan African clade. This + Seychelles from ca. 88 Ma (see Samonds et al., 2012). Finally, India dataset is also a combination of GenBank and newly generated se­ and Seychelles separated ca. 71–63 Ma (Collier et al., 2008; Minshull quences (Table S2). et al., 2008; Armitage et al., 2011; Ganerød et al., 2011: see Gower et al., Phylogenetic analyses were carried out using the CIPRES Science 2016). Gateway v3.3 (Miller et al., 2010). We applied ML using RAxML-HPC2 To estimate the age of the divergence between L. seychellensis and its on XSEDE v. 8.2.12 (Stamatakis, 2014), and Bayesian inference (BI) closest extant relative, we analysed a taxonomically reduced dataset of

3 V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152 ) page next – – – – – – – – – – – – – – – – – – – prlr on continued ( – MW711603 MW711602 – MW711601 MW711600 MW711599 MW711598 MW711597 MW711596 MW711595 MW711594 – – MW711593 MW711592 MW711591 MW711590 MW711589 rag1 bold. marked are – MW711575 MW711574 MW711573 MW711572 MW711571 MW711570 MW711569 MW711568 MW711567 MW711566 MW711565 MW711564 MW711563 MW711562 MW711561 MW711560 MW711559 MW711558 bdnf Seychelles in – MW711468 MW711467 MW711466 MW711465 MW711464 MW711463 MW711462 MW711461 MW711460 MW711459 MW711458 MW711457 MW711456 MW711455 MW711454 – MW711453 MW711452 nt3 Islands – MW711547 MW711546 MT587299 MW711545 MT587297 MT587296 MT587294 MT587292 MW711544 MW711543 MW711542 MW711541 MW711540 MW711539 MW711538 MW711537 MW711536 MW711535 cmos different data, no = – MW711530 MW711529 MT587311 MW711528 MT587309 MT587308 MT587307 MT587306 MW711527 MW711526 MW711525 – – MW711524 MW711523 MW711522 – – nd4 ) – ( endash *, MW711490 MW711489 MW711488 MT587285 MW711487 MT587284 MT587283 MT587281 MT587279 – – – – – – – MW711486 MW711485 MW711484 cytb with indicated MW699978 MW699977 MW699976 MT586821 MW699975 MT586819 MT586818 MT586817 MT586815 MW699974 MW699973 MW699972 MW699971 MW699970 MW699969 MW699968 MW699967 MW699966 MW699965 16s are Kona Kona Kona Kona Mts., Gabon Scarp of DR Sembe, Angola Guinea dataset Sashae, DR Nimba Rep. South Mamfe Angola Angola River River Gabon bridge, Mkalazi, Lunda Lunda Nguru of near Congo Congo, Congo, Congo, Congo, R., Nguru Mts., Udzungwa Uzungwa F. Tanzania Kalemie, Congo DR Moyen-Ogooue Prov., Lulele area, Norte, Lulele area, Norte, upstream Lungue-bungue River Moxico, West Cuando Cubango, Angola Dia Congo Makokou, Bafwabianga village, Congo DR DR DR DR Mount mining concession, Forestiere Region, LakeKuk, Cameroon Akwa, division, Cameroon Location 21-leaf the in used AD-37 AD-36 AI-87 AD-34 AD-33 AI-81 AI-80 AI-83 AI-82 AI-86 AI-85 AI-84 AJ-92 AJ-91 AJ-90 AI-89 AE-42 AE-41 AE-40 Sequence ID Sequences 8.39880 37.52564 35.97861 29.20000 – 11.16621 19.95686 19.95686 18.40479 23.22667 14.55000 12.83000 26.83000 22.78694 22.78694 22.78694 22.78694 10.20812 9.47322 Longitude study. this in 6.03044 8.39750 5.93333 0.05171 7.75308 7.75308 12.33012 17.57333 – 1.81000 0.54000 1.05000 2.03306 2.03306 2.03306 2.03306 7.69960 6.41072 6.05370 Latitude generated ID 75772 75491 75763 Kuk, MTSN8183 MTSN5485 WRB-636 CAS220640 CAS258133 ANG-058 ANG-056 WC-6238 ANG-438 NMP-P6V NMP-P6V NMP-P6V CRT3860 CRT3859 CRT3855 CRT3853 BMNH2008.637 Lake Cameroon NHMUK 2013:383 voucher number/ extraction sequences for codes Natriciteres sylvatica Natriciteres sylvatica* Natriciteres olivacea* Natriciteres olivacea* Natriciteres fuliginoides* Limnophis branchi Limnophis branchi Limnophis bicolor* Limnophis bangweolicus* Hydraethiops melanogaster* Hydraethiops melanogaster Hydraethiops melanogaster* Helophis schoutedeni Helophis schoutedeni Helophis schoutedeni* Helophis schoutedeni* Afronatrix anoscopus* Afronatrix anoscopus* Afronatrix anoscopus* Species accession 1 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 Sample Table GenBank

4 V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152 ) page next – MW711613 – MW711612 MW711611 – MW711610 – – – – – – – prlr – on continued ( – – – – – MW711608 – MK350256 MK350257 MW711607 MW711606 MW711605 – – rag1 MW711604 – – – MW711584 – MW711583 – MW711582 MW711581 MW711580 MW711579 MW711578 – MW711577 bdnf MW711576 – – – MW711477 – MW711476 MW711475 MW711474 MW711473 MW711472 MW711471 MW711470 – – nt3 MW711469 – – – MW711551 – MW711550 – MK350264 MK350265 MF352834 MT587301 MT587300 – MW711549 cmos MW711548 – – – – – MW711533 – MK350259 MK350258 MF352842 MT587313 MT587312 – MW711532 nd4 MW711531 – MW711498 MW711497 MW711496 MW711495 MW711494 MW711493 MK350261 MK350262 MF352838 MT587287 MT587286 – MW711492 cytb MW711491 MW699988 MW699987 MW699986 MW699985 MW699984 MW699983 MW699982 MK350254 MK350255 MF352831 MT586823 MT586822 MW699981 MW699980 16s MW699979 , , ´ ´ e e , ´ e India India Mah Mah Mt. Mt. Kerala, , , Mabu, Blanc Rouge, Rouge, Dent, Mah , , , , Aux Aux ´ ´ ´ ´ e e e e Mare Cochons, Seychelles Mah Seychelles Congo Mah Seychelles Congo Mah Seychelles Casse Mah Seychelles Mare Cochons, Seychelles Morne Trail, Seychelles Mizoram University Campus, Aizawl Mizoram, Mizoram University Campus, Aizawl Mizoram, Pookode, Wayanad District, India Myanmar Thailand Rungwe (Nkuka), Southern Highlands, Tanzania Mount Zambezi, Mozambique Location South(Pemba), Tanzania Rungwe (Nkuka), Southern Highlands, Tanzania AM-126 AC-29 AC-21 AD-39 AI-88 Sequence ID AD-38 55.41158 – 55.43765 55.43363 55.43778 55.41212 55.43744 92.93194 93.09916 76.02188 94.74683 – – 36.41244 Longitude – 4.63487 4.65043 4.64493 4.65350 4.63738 4.65894 16.28789 – 23.71083 23.76338 11.53932 20.31286 – – Latitude – ID 2369 3527 R9458 SM290 SM287 SM286 SM252 SM165 SM251 BNHS BNHS NCBS-AU164 CAS213646 FMNH250122 MUSE13726 WRB-642 voucher number/ extraction MUSE13712 * * * ) Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Smithophis bicolor Smithophis atemporalis Rhabdops olivaceous Amphiesma stolatum Fowlea flavipunctatus Natriciteres variegata Natriciteres sylvatica Species Natriciteres sylvatica* continued ( 1 34 33 32 31 30 29 28 27 26 25 24 23 22 21 Sample 20 Table

5 V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152 ) page next – MW711625 – MW711624 MW711623 MW711622 – MW711621 MW711620 MW711619 MW711618 MW711617 – MW711616 MW711615 MW711614 prlr on continued ( – MW711609 – – – – – – – – – – – – – – rag1 MW711586 MW711585 – – – – – – – – – – – – – – bdnf MW711480 MW711479 – – – – – – – – – – MW711478 – – – nt3 MW711555 MW711554 – – – – MW711553 – – – – – – MW711552 – – cmos – MW711534 – – – – – – – – – – – – – – nd4 MW711512 MW711511 MW711510 MW711509 – MW711508 MW711507 MW711506 MW711505 MW711504 MW711503 MW711502 MW711501 MW711500 MW711499 – cytb MW700004 MW700003 MW700002 MW700001 MW700000 MW699999 MW699998 MW699997 MW699996 MW699995 MW699994 MW699993 MW699992 MW699991 MW699990 MW699989 16s , , ´ ´ e e , , , , , Dent, Dent, ´ e Mai, Mai, Mai, Mai, Mah Mah Mare Mare Jardin Jardin Morne , , , , , Rouge de de de de Mahe Praslin Hilton , , , Aux Aux Kerlan Mah to to to ´ ´ ´ e e e Cochons Cochons Casse Casse ee ee ee ee ´ ´ ´ ´ Reserve, Trail Marron, Hotel LeBreeze, Silhouette Seychelles Trail Marron, Silhouette Seychelles Vall Praslin Seychelles Praslin Seychelles Anse River, Seychelles Vall Praslin Seychelles Vall Praslin Seychelles Vall Praslin Seychelles Congo trail, Seychelles Between Aux and Mah Seychelles Between Aux and Mah Seychelles Mare Cochons, Seychelles Mare Cochons, Seychelles Trail Blanc, Seychelles La Mah Seychelles Location AM-127 Sequence ID 55.23967 55.24621 55.23781 55.73762 55.73384 55.69084 55.73763 55.73885 55.73885 55.43450 55.41194 55.42858 55.41139 55.41602 55.43421 55.50081 Longitude 4.48474 4.47890 4.48528 4.33143 4.33475 4.30359 4.33077 4.33180 4.33180 4.64675 4.65114 4.64883 4.62853 4.64007 4.65716 4.70760 Latitude ID SM225 SM213 SM206 R9469 R9459 SM112 SM89 SM88 SM87 SM385 SM340 SM339 SM293 SM292 R6909 R6716 voucher number/ extraction ) Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Species continued ( 1 50 49 48 47 46 45 44 43 42 41 40 39 38 37 36 35 Sample Table

6 V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152 MW711630 MW711629 – MW711628 – – – MW711627 MW711626 prlr – – – – – – – – – – rag1 – – – – MW711588 MW711587 – – – bdnf – – – – MW711483 MW711482 MW711481 – – – nt3 – – – – – MW711557 MW711556 – – – cmos – – – – – – – – – – nd4 – MW711521 MW711520 MW711519 MW711518 MW711517 MW711516 MW711515 MW711514 MW711513 cytb – – – – MW700011 MW700010 MW700009 MW700008 MW700007 MW700006 16s MW700005 , , , , La La Jardin Jardin , , , , , , , Vue, Source to to ´ ´ ´ ´ ´ egate egate egate egate egate Argent, ’ Fr Seychelles Fr Seychelles Fr Seychelles Fr Seychelles Fr Seychelles Anse d Digue Seychelles Belle Digue Seychelles Trail Marron, Silhouette Seychelles Trail Marron, Silhouette Seychelles Location Silhouette Seychelles Silhouette Seychelles Sequence ID 55.93317 55.93317 55.93317 55.94387 55.94387 55.82741 55.84062 55.23800 55.23544 Longitude 55.24260 4.58475 4.58475 4.58475 4.58581 4.58581 4.37145 4.35872 4.48497 4.48704 4.48450 Latitude ID SM507 SM506 SM505 R9466 R9465 R9464 R9460 R6723 R6680 voucher number/ extraction R9467 ) Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Lycognathophis seychellensis Species Lycognathophis seychellensis continued ( 1 60 59 58 57 56 55 54 53 52 Sample 51 Table

7 V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152 ) ) ’ b ’ page (part next on – HM234061 – – EU402833 KC347416 AY988072 – – – KC347438 – EU402836 KC330012 KC347423 EU402849 – – – – EU402839 – AY988065 – EU402840 AY988076 KC330027 – KC750047 AY988071 EU402843 JQ073200 – DQ465571 AY988074 HQ399516 – – – – – – – – – – – – – – – – – – rag1 ) continued ’ ( a ’ (part – – MW711590 MW711591 – – – – – – – – – – – KM519732 – – – JF357954 – – – – – – – – – AY662613 – – – – – – – – MW711592 – MK621524 MW711594 – MW711597 MW711600 – – – MW711608 – – – – MW711601 rag1 FJ433981 AY988036 MW711559 MW711560 EU402623 – EU402625 – – FJ433984 – – EU402628 – KC330044 EU402646 – – – FJ433989 EU402631 FJ434005 FJ433974 KX694704 EU402632 FJ433978 KC330056 GU112346 JX576167 AY988037 EU402636 JQ073079 KC330073 EU402639 – KC330076 GU902394 FJ434002 MW711561 – JQ599029 MW711565 FJ433959 MW711568 MW711571 – EU402650 EU402651 MW711583 – JQ073081 – – MW711572 bdnf FJ434082 KX694991 MW711453 – – – FJ434066 – – FJ434085 – – KX694977 – – FJ434094 – – – KX694998 – KX694999 FJ434077 KX695001 FJ434069 FJ434079 DQ465554 – – – – – JX576186 DQ465569 – – GU902566 KX695019 MW711454 – KX695021 MW711458 FJ434075 MW711461 MW711464 – – FJ434072 MW711476 – KX695030 – – MW711465 nt3 HM234057 HM234058 MW711536 MW711537 – KC347378 AF544722 – AY187967 JF827696 KC347400 KX660336 AF544695 AY187970 AF544676 FJ404270 FJ404347 AY611951 AY611910 AY058924 AF544682 AF471103 AY099961 – AF544731 AY099971 KC329991 AF471134 KC750007 AF471133 AF471156 – KC330008 AF544716 – HQ399536 – AF544684 MW711538 MG458766 AY611901 MW711542 AF544726 MT587292 MT587297 AY187985 AF544727 AY444035 MW711550 DQ486173 AY187992 AY611905 AY611904 MW711545 cmos HM234054 HM234055 – MW711522 AF156577 KC347512 FJ755180 – FJ404331 JF827650 KC347527 KX660597 AY352808 DQ305475 AB177354 FJ404365 AY611950 – DQ486328 EU579523 – – – EF395922 – AF302959 KC329966 GU112419 KC750018 AB179619 EU913478 – KC329975 – GQ225672 KC329978 AM236345 AF544663 MW711523 – FJ404338 MW711525 – MT587306 MT587309 FJ404373 – – MW711533 – DQ897723 KX130765 MW711528 nd4 AF217841 KX694897 MW711485 MW711486 EU483383 KC347454 U69738 – AY188006 JF827673 KC347477 KX660469 AY352747 DQ305457 AB177354 AF471060 AY612041 AY612042 AY612001 EU579523 AY099985 AF471081 AY099984 EF395897 U69755 AY099986 – GU112384 KC750012 AB179619 EU913478 – HQ399501 U69811 GQ225658 HQ399499 AM236345 AY612048 – MG458758 AY611992 – U69839 MT587279 MT587284 DQ979985 AF544672 AY099993 MW711494 DQ486349 DQ486461 AY611996 AY611995 MW711487 cytb data. no = – AF544786 AF512745 MW699966 MW699967 AF156566 KC347339 FJ755180 AY953431 AY188045 AF544787 KC347361 KX660197 AF057234 AY188048 AB177354 AY188079 AY611859 AY611860 AY611818 EU579523 Z46494 KX694624 EU419850 KX694627 AF544827 AF544816 – AY577030 – AB179619 EU913478 – – AF544819 AF512743 AM236347 AM236345 AY611866 MW699968 – AY611809 MW699972 EF545051 MT586815 MT586819 AY188063 AF366762 AF544828 MW699983 – AY188070 AY611813 AY611812 MW699975 16s ID analyses. 126 33 41 42 89 84 82 81 AE AE AI AI AI AI AM AD Sequence dating molecular in sedis sedis used Anomochiliidae Anomochilidae + + (Aparallactinae) (Lamprophiinae) (Lamprophiinae) incertae incertae (Cyclocorinae) (Atractaspidinae) (Pseudoxyrhophiinae) (Aparallactinae) (Psammophiinae) (Natricinae) (Natricinae) (Ahaetuliinae) (Natricinae) (Calamariinae) (Dipsadinae) (Grayiinae) (Natricinae) (Natricinae) (Natricinae) (Natricinae) (Natricinae) (Natricinae) (Charininae) (Crotalinae) (Azemiopinae) (Viperinae) (Viperinae) (Pareinae) (Pareinae) sequences gene Acrochordidae Acrochordidae Colubridae Colubridae Viperidae Colubridae Aniliidae Cylindrophiidae Atractaspididae Pareidae Colubridae Pareidae Viperidae Viperidae Boidae Lamprophiidae Lamprophiidae Lamprophiidae Lamprophiidae Elapidae Calabariidae Colubridae Candoiidae Homalopsidae Bolyeridae Charinidae Boidae Colubridae Boidae Cylindrophiidae Viperidae Lamprophiidae Boidae Erycidae Erycidae Boidae Gerrhopilidae Colubridae Colubridae Lamprophiidae Lamprophiidae Colubridae Pythonidae Colubridae Colubridae Lamprophiidae Anomalepididae Loxocemidae Colubridae Lamprophiidae Lamprophiidae Elapidae Elapidae Colubridae Family for numbers melanoleuca mossambica seychellensis sexlineatus malaccanus lacteus voucher melanogaster philippinum leonardi striatus capensis granulatus javanicus ruffus contortrix ater boa mirus fuliginoides albirostris bicolor pavimentata anoscopus anoscopus vivax bangweolicus branchi reinhardtii mahfalensis pulverulenta bicoloratus feae fasciatus ceylonensis cenchria violacea fuliginosus notaeus schoutedeni carinata depressiceps procterae annulatus and dussumieri bottae russellii scytale ornata tenuis mackloti (Afronaja) (Boulengerina) colubrinus conicus nasicornis constrictor Acrochordus Acrochordus Afronatrix Afronatrix Agkistrodon Ahaetulla Anilius Anomochilus Aparallactus Aplopeltura Aspidura Asthenodipsas Azemiops Bitis Boa Boaedon Bothrolycus Buhoma Buhoma Bungarus Calabaria Calamaria Candoia Cantoria Casarea Charina Chilabothrus Contia Corallus Cylindrophis Daboia Ditypophis Epicrates Eryx Eryx Eunectes Gerrhopilus Grayia Helophis Hologerrhum Homoroselaps Hydraethiops Liasis Limnophis Limnophis Liopholidophis Liotyphlops Loxocemus Lycognathophis Micrelaps Mimophis Naja Naja Natriciteres Species accession 2 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 1 Sample Table GenBank

8 V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152

) 73 leaves to decrease computational time required for analyses ’ b ’ (Table 2). This includes 12 natricines, including one L. seychellensis, and nine mainland sub-Saharan African natricines (eight currently recog­ (part nised species). In addition, we included 61 non-natricines in the dataset – – – – – – – – – – AY988067 KC347449 EU402867 – EU402869 DQ465564 – MK340913 rag1 in order to make use of some of the best fossil calibrations available for snakes (see Head, 2015, Head et al., 2016; Zaher et al., 2019). Beyond )

’ the seven genes for which we generated new data, we also included an a ’ additional region in rag1 (Wiens et al., 2008) because sequences of this 007441886 (part marker were available for almost 40% of the samples. This dataset – included in total 5,021 base pairs (Table 2). The data were newly aligned MW711602 MW711603 – EU546877 – – – – XM – – – – – MW711605 – – – – rag1 and the best-fit gene and codon partitioning scheme (Table S3) was selected using PartitionFinder2 as reported above. We applied seven fossil calibrations, largely those recommended by Head (2015) and Head et al. (2016): (1) oldest divergence within crown MW711574 MW711575 – – FJ433985 – – JQ599045 XM7433022 FJ433966 AY988033 – KF811074 EU402665 MW711578 EU402667 EU402668 – MK344197 bdnf Alethinophidia based on Hassiophis terrasanctus (Tchernov et al., 2000); minimum age 93.9 Ma; (2) divergence between non-xenodermid colu­ brids and their closest living relative (Xenodermidae in our tree) based on Procerophis sahnii (Rage et al., 2008); minimum age 50.5 Ma; (3) divergence between Boinae and its sister taxon (Erycinae + Candoiinae MW711467 MW711468 – – FJ434086 – – KX695042 FJ434071 – – – FJ434081 MW711470 – FJ434073 – – nt3 in our tree) based on Titanoboa cerrejonensis (Head et al., 2009); mini­ mum age 58 Ma; (4) divergence between Corallus and (Chilabothrus, (Epicrates,Eunectes)); Corallus priscus (Rage, 2001); minimum 50.2 Ma; (5) divergence between Viperinae and Crotalinae based on Vipera aspis MW711546 MW711547 DQ112081 EU546916 JF827702 FJ404293 DQ486167 AF471102 AF435016 AF544719 EU403580 KC347411 KF811110 AF544724 MT587300 AF544711 AF544689 – MK344193 cmos complex (Szyndlar and Rage, 1999); minimum age 20.0 Ma; (6) diver­ gence between Acrochordus javanicus and (A. arafurae, A. granulatus) based on Acrochordus dehmi (Hoffstetter, 1964); minimum age 18.1 Ma and (7) oldest divergence between Naja (Afronaja) and Naja (Bou­ MW711529 MW711530 – EF210827 JF827653 FJ404389 DQ486319 – – – – KC347516 – – MT587312 U49320 AB179620 – MK340910 nd4 lengerina) based on Naja romani (Hoffstetter, 1939) (A.B. Quadros pers. comm. 2019, based on work presented by Quadros et al., 2019); mini­ mum age 17 Ma. Input values applied to each calibration prior are re­ ported in Table S4. Dating analyses were carried out using the CIPRES Science Gateway MW711488 MW711489 AF471029 EU547051 JF827677 FJ404319 AY612080 AF471080 JX401131 AF544673 U69866 KC347471 KF811124 U69870 MT587286 – AB179620 AY574279 – cytb v3.3 (Miller et al., 2010). We estimated divergence times using BEAST2 on XSEDE v2.6 (Bouckaert et al., 2019) under a Yule tree prior. A relaxed lognormal clock was assigned for each partition of the concatenated BEAST2 analyses. We set up two independent runs, the Markov Chain MW699976 MW699977 – EU547149 AF544802 FJ404222 AY611898 JF697330 KF010492 AY701028 AY336066 KC347373 AF512733 AF544833 MT586822 AF544810 AB179620 – MK340908 16s Monte Carlo (MCMC) was run for 200,000,000 generations sampling every 10,000 trees, and effective sample size (ESS) values were evalu­ ID ated using Tracer v. 1.7 (Rambaut et al., 2014). Initial runs applied the 36 21 best-fitpartitioning scheme and models, but this resulted in poor mixing 87 & AI AD AC Sequence of the GTR model parameters for partitions 1, 2, 3 4. We therefore repeated BEAST2 analyses implementing the less complex HKY model for these partitions. Additionally, we estimated divergence dates using Penalized Likelihood (Sanderson, 2002) as implemented in TreePL (Smith and O’Meara, 2012), applying the fossil dates above for cali­ brations 1–7 as hard minimum and maximum ages. Penalized likelihood uses a tree with branch lengths and calibrations without prior para­

(Cyclocorinae) (Prosymninae) (Pseudaspidinae) metric distributions. We ran TreePL with 10,000 iterations and a smoothing parameter of 100. (Natricinae) (Natricinae) (Pseudoxenodontinae) (Sibynophiinae) (Natricinae) (Ungaliophiinae)

(Pareinae) (Xylophiinae) Historical biogeography was investigated by estimating ancestral areas for the dated phylogeny using dispersal-extinction-cladogenesis (DEC; Ree et al., 2005, 2008) and dispersal-vicariance analysis (DIVA­ Colubridae Colubridae Lamprophiidae Elapidae Pareidae Lamprophiidae Lamprophiidae Colubridae Pythonidae Uropeltidae Sanziniidae Colubridae Tropidophiidae Charinidae Colubridae Xenodermidae Xenopeltidae Xenophidiidae Pareidae Family LIKE; Ronquist, 1997; Ronquist and Sanmartín, 2011) models imple­ mented in BioGeoBEARS v.1.1 (Matzke, 2013) in R v 4.0 (R Core Team, 2020). We also implemented these models using the peripatric specia­ tion “j” (‘jump’) parameter, which although criticised by Ree and San­ martín (2018), is often the most biologically realistic scenario for

karlschmidti colonization and subsequent divergence on remote islands (Tsang et al., leporinum schaeferi javanicus feicki continentalis olivacea sylvatica 2020). We defined three areas: Asia, Seychelles, and mainland sub- scutellatus subpunctatus cana unicolor janii drummondhayi madagascariensis perroteti

) Saharan Africa, and set the maximum number of ancestral areas to flavipunctatus bivittatus carinatus three, because this was the maximum number of areas occupied by any Natriciteres Natriciteres Oxyrhabdium Oxyuranus Pareas Prosymna Pseudaspis Pseudoxenodon Python Rhinophis Sanzinia Sibynophis Tropidophis Ungaliophis Fowlea Xenodermus Xenopeltis Xenophidion Xylophis Species clade in our analysis. We compared the fit of these four models (DEC, + + continued DEC j, DIVALIKE and DIVALIKE j) using Akaike Information Cri­ (

2 terion (AIC) scores and weights. 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 Sample Table

9 V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152

2.4. Genetic diversity within Lycognathophis seychellensis mainland sub-Saharan African genera with the first two letters of their name, the two sets of relationships (Li,Na) and (Af,(He,Hy)) are maxi­ PopArt (Leigh and Bryant, 2015) was used to generate haplotype mally supported (Fig. 2B). The relationship of L. seychellensis to these networks to display intraspecificvariation for L. seychellensis for 16s and two lineages is less clearly resolved in analyses of the concatenated nu cytb under the median-joining algorithm (Bandelt et al., 1999). and mt data. In the 157-leaf dataset, L. seychellensis is sister to (Af,(He, Hy)), with ML bootstrap and Bayesian posterior probability support of 3. Results 63 and 0.97, respectively (Fig. S1). The same relationship was recovered in ML(RAxML) analysis of the 21-leaf dataset, with lower support (37%), 3.1. Molecular phylogenetics but in the BI (MrBayes) analysis of this dataset, L. seychellensis is instead sister to all mainland sub-Saharan African natricines (support for The best-fitpartitioning scheme for the 21-leaf dataset comprised six monophyly of the latter clade 0.59) (Fig. 2; Table 3). partitions, by gene and by codon position (Table S5). Mainland sub- Examination of majority rule partition tables in PAUP* (Swofford, Saharan African natricines plus L. seychellensis are strongly supported 2000) for the 500 RAxML ML bootstrap trees and 2,000 post-burnin as monophyletic (Fig. 2A), and almost all intergeneric relationships MrBayes BI trees for the 21-leaf dataset shows that support is lowest within this lineage are well supported. In particular, and denoting the for the third alternative resolution of the position of L. seychellensis with

Fig. 2. Phylogenetic relationships of Seychelles Lycog­ nathophis seychellensis and other (mainland) sub- Saharan African natricines based on analyses of DNA sequence data for three mitochondrial and four nuclear loci. (A) Majority rule consensus of post burn-in BI trees, and (B) ML tree. Codes in parentheses correspond to samples in Tables 1 and 2. Bayesian posterior prob­ abilities and bootstrap support values shown at internal branches. Coloured squares indicate regions from where vouchers were collected.

10 V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152

Table 3 mt and nu data analyses) of the relationships among L. seychellensis and Quantitative support (ML bootstrap and Bayesian posterior probability values) (Af,(He,Hy)) and (Li,Na) yields similar time estimates for the split be­ for three incompatible clades, representing the three best supported resolutions tween L. seychellensis and its closest extant (mainland sub-Saharan Af­ of the relationships of Lycognathophis seychellensis under ML and BI analyses of rican) relative (Table 5). For BEAST2 runs applying GTR, as suggested the 21-leaf, concatenated mt and nu dataset. Values in bold indicate best- by PartitionFinder2 results, ESS values were low for posterior (46) and supported resolution for each analysis. Abbreviations of genera: Af = Afrona­ prior (37) distributions, though the divergence date in question was trix; He = Helophis; Hy = Hydraethiops; Li = Limnophis; Ly = Lycognathophis; Na = similarly estimated (Fig. S3). Natriciteres. All of the BioGeoBEARS analyses agree with each other and with Analysis (Ly(Hy,He,Af)) ((Na,Li)(Hy,He,Af)) (Ly(Na,Li)) parsimony (performed manually; results not shown) in their estimation RAxML (gene & codon position) 37 35 27 of all ancestral areas. Asia is the estimated ancestral area for the sampled RAxML (gene) 40 36 24 taxa, with a dispersal/vicariance event to sub-Saharan Africa. Dispersal MrBayes 0.28 0.59 0.12 rather than vicariance is the most likely explanation for origin of the BEAST 0.88 0.08 0.04 sub-Saharan African clade because the phylogenetic split with Asian natricines is estimated to be much younger (48.7 Ma; HPD: 34.1–56.6 respect to (Li,Na) and to (Af,(He,Hy)), that is, as sister to the former clade Ma) than the Gondwanan breakup. However, the route of dispersal to (Li,Na), which receives support of only 27% (RAxML) and 0.12 sub-Saharan Africa and whether this was via land or sea is open to (MrBayes) (Table 3). For the ML analysis, these support values are very further investigation. A single dispersal event from mainland Africa to similar when the same dataset is partitioned by gene instead of by gene the Seychelles occurred relatively early on in the history of the group and codon (Table 3). Both the AU and SH tests indicate that differences (Fig. 3). The divergence between the Seychelles natricine and its closest in likelihood between the fit of the three alternative topologies to the mainland African relative (35.7 Ma; HPD: 43.2–24.6 Ma) is much data are not significant (Table 4). Bayes factors indicate that the dif­ younger than the break-up between Seychelles and other Gondwanan ferences in fitto the data among the three alternative topologies are also landmasses (63 Ma or older, see Section 2.3), strongly supporting not substantial, but differences in fit to data between the trees with L overseas dispersal. Among the four models implemented, DEC + j was seychellensis as sister to (Af,(He,Hy)) and as sister to either (Li,Na) or to the best fitting, closely followed by DIVALIKE + j and DEC (Table 6). ((Li,Na)(Af,(He,Hy))) are greater than differences in fitto data between the trees with the latter two sets of relationships (Table 4). 3.3. Intraspecific genetic diversity Analysis of the 21-leaf dataset for the nu and the mt data separately reveals that incongruence between these partitions explains a substan­ Intraspecific genetic diversity across the range of Lycognathophis tial part of the lack of compelling resolution of the relationships of seychellensis is low. Except for five single-site ambiguities (possible L. seychellensis in analyses of the concatenated data. For the mt data, heterozygosity) in prlr and one in rag1 there was no variation for any of L. seychellensis is most strongly supported as sister to (Af,(He,Hy)), the five nuclear gene sequences generated. For 16 s and cytb there are whereas for the nu data this is the least well supported of the three only two and four segregating sites, and zero and one parsimony alternative resolutions, with instead L seychellensis as sister to (Af,(He, informative sites, respectively. Therefore, we generated haplotype net­ Hy)) receiving the most support (Table S6). The Bayesian multispecies works but not phylogenetic trees to examine spatial patterns in genetic coalescent analysis using StarBEAST yielded 17,998 trees with 43 variation (Fig. 4). There is only one instance of a single island having a different topologies. Among these trees, 98% included L. seychellensis as haplotype not found on other islands, though this single cytb haplotype sister to (Af,(He,Hy)). from Silhouette differs from those found on other islands by only a single step.

3.2. Divergence dating & historical biogeography 4. Discussion When applying the HKY model in BEAST2 analyses, to those parti­ 4.1. Molecular phylogenetics and genetic diversity tions for which good mixing was not achieved when applying GTR, good mixing was achieved for all parameters, for posteriors and priors (ESS > Our molecular phylogenetic analyses are the first to comprehen­ 200). Highest posterior density (HPD) intervals are wide for divergence sively sample extant sub-Saharan African (including Seychelles) natri­ times estimated in BEAST2 analyses. In unconstrained BEAST2 analysis, cine genera (all six genera, and 11 out of 14 species), and we find L. seychellensis is recovered as sister to the mainland sub-Saharan African compelling support for their monophyly. Our results are consistent with (Af,(He,Hy)), with the split between these two lineages estimated to previous work based on morphology that proposed that Helophis is a have occurred with a 95% HPD interval of 43.2–24.6 Ma (mean 35.3 natricine and is closely related to Hydraethiops (de Witte, 1922; Broad­ Ma) (Fig. 3; Fig. S2), which is similar to the TreePL estimate of this ley, 1998; Nagy et al., 2014), and that Limnophis and Natriciteres are divergence time for this node, at 35.7 Ma. closely related (Dowling and Duellman, 1978; Zaher, 1999). We were Enforcing the two alternative competing resolutions (from separate unable to identify with precision and reasonable support the closest living relatives of the sub-Saharan African (including Seychelles) natri­ Table 4 cine radiation. However, our results point to this radiation arising from Comparisons of fit of data to three trees with alternative best-supported reso­ within one of the Asian radiations (the clade excluding New World lutions of the sister-group relationships of Lycognathophis seychellensis for the 21- natricines plus (Eur)Asian Natrix, Sinonatrix, Smithophis, and leaf concatenated mt and nu data. Above the diagonal are SH and AU test p values, respectively, for differences between ML trees. See Table 3 for support Opisthotropis). for each phylogenetic resolution. Below the diagonal are Bayes Factor scores for The phylogenetic results regarding the identity of the sister group of path (and stepping stone) sampling results under Bayesian analysis. * indicates L. seychellensis are not compelling from the analyses of the concatenated best-supported resolution under MrBayes analysis; ** best-supported resolution nu and mt sequence data. However, partitioned analyses provide evi­ under BEAST analysis. See Table 3 for abbreviations of genera. dence of substantial nu-mt incongruence, and analysis using a multi­ (Ly((Na,Li)(Hy,He,Af))) (Ly(Na,Li)) (Ly(Hy,He,Af)) species coalescent approach that is designed to cope with incomplete lineage sorting strongly supports the hypothesis that L. seychellensis is (Ly((Na,Li)(Hy,He,Af)))* 0.418; 0.298 0.501; 0.401 + + (Ly(Na,Li)) 1.2 (1.1) 0.772; 0.682 sister to Hydraethiops Helophis Afronatrix. (Ly(Hy,He,Af))** 4.0 (3.9) 2.8 (2.8) The almost complete lack of molecular genetic variation within L. seychellensis across five Seychelles islands is consistent with there

11 V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152

Fig. 3. BEAST chronogram showing divergence times for Seychelles and other (mainland) sub-Saharan Af­ rican natricine snakes inferred from the 73-leaf, concatenated mt and nu dataset. Numbers at inter­ nal branches indicate mean divergence ages, with blue bars showing 95% highest posterior density in­ tervals. Seychelles-Africa divergence is indicated with a *. Numbers in parentheses are sample codes (see Tables 1 and 2). See Fig. S2 for complete tree, including non-natricine alethinophidians, and values of 95% highest posterior density intervals. Ancestral area estimations (DEC + j model) at nodes represent areas before an inferred instantaneous speciation event, coloured boxes at tips indicate the current distribution of extant species. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 5 Estimates of age (in Ma) of divergence between Lycognathophis seychellensis and its closest living (mainland sub-Saharan African) relative, for trees consistent with the three best-supported resolutions of the sister group of L. seychellensis. Results reported for TreePL and BEAST2 analyses of 73-leaf dataset. Values are means, with 95% highest posterior density interval in square brackets; values in and not in parentheses are from analyses implementing GTR and HKY models of sequence evolution, respectively. L. seychellensis sister group

Analysis (Hy,He,Af) ((Na,Li)(Hy,He,Af)) (Na,Li)

TreePL 35.68 (38.57) 39.31 (39.31) 41.27 (41.53) BEAST2 35.31 [24.57–43.19] 43.34 [27.83–47.18] 29.35 [24.11–37.84] (43.09 (42.41 (29.42 [28.14–47.06]) [27.06–47.82]) [25.51–39.28])

Table 6 Comparison of four models of ancestral area estimations implemented using BioGeoBEARS. Abbreviations: Ln L, log-likelihood; d, dispersal; e extinction; j jump parameter; k, number of parameters. AIC scores (sc) and weights (wt) are reported.

Model Ln L K d e j AIC sc AIC_wt

DEC 8.55 2 0.0000 0.0000 0.00 21.110 0.210 = = DEC + j 7.08 3 0.0000 0.0000 0.05 20.170 0.330 Fig. 4. Haplotype networks for mitochondrial cytb (n 29) and 16s (n 30) DIVALIKE 11.14 2 0.0033 0.0029 0.00 26.280 0.016 for Lycognathophis seychellensis from five Seychelles islands using the median- DIVALIKE + j 7.3 3 0.0000 0.0000 0.06 20.600 0.270 joining (MJ) network algorithm. See Table 1 for sample and locality details.

no record of this taxon occurring beyond Seychelles, a very recent, being only a single extant species of natricine occurring on the human mediated dispersal to Seychelles is highly unlikely and can be Seychelles. The very low intraspecific variation contrasts with that discounted with confidence. The lack of genetic variation among pop­ found for other native Seychelles squamate reptiles and other taxa (e.g., ulations of L. seychellensis from different islands is consistent with this frogs: Labisko et al., 2019; caecilians: Maddock et al., 2020; crabs: species being a relatively good disperser among the major granitic Daniels, 2011), which have substantial and strongly spatially structured, islands of the Seychelles (perhaps the best of Seychelles squamates intraspecificmolecular genetic variation. This structured variation often studied thus far), across relatively shallow, narrow seas and/or during includes a distinct genetic lineage on Silhouette, a hint of which is also multiple low sea level stands during the Pleistocene, when these islands found here for L. seychellensis cytb. would have been repeatedly dis- and reconnected (Warren et al., 2010). The lack of genetic variation suggests that the current genetic di­ A closer examination of L. seychellensis intraspecific genetic variation versity within L. seychellensis is the product of a strong founder effect for within and among islands using rapidly evolving nuclear markers and a a relatively recent overseas arrival (not supported by our dating results) denser sampling would be worthwhile. and/or within-Seychelles dispersal from a substantially contracted ref­ ugial population representing some form of secondary founder effect. Due to the relatively deep phylogenetic divergence of L. seychellensis and

12 V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152

4.2. Historical biogeography editing. Werner Conradie: Conceptualization, Resources, Data cura­ tion, Writing - review & editing. Sara Rocha: Resources, Formal anal­ Our results are consistent with the L. seychellensis lineage reaching ysis, Visualisation, Writing - review & editing. D. James Harris: the Seychelles via overseas dispersal from Africa, approximately 43–25 Resources, Writing - review & editing. Ana Perera: Resources, Writing - Ma. Probably this is a similar mode and timing of origin to all of the review & editing. Vaclav´ Gvozdík:ˇ Conceptualization, Resources, Data other Seychelles native squamate reptiles for which data are available. curation, Writing - review & editing. Thomas M. Doherty-Bone: Re­ For example, the endemic Seychelles chameleon Archaius tigris is most sources, Data curation, Writing - review & editing. Rachunliu G. closely related to East African Rieppeleon spp., with the two diverging an Kamei: Resources, Investigation, Data curation, Writing - review & estimated 43–27 Ma (Tolley et al., 2013: Table S4). The Seychelles editing. Michele Menegon: Resources, Writing - review & editing. Jim skinks Trachylepis sechellensis and T. wrightii are estimated to have Labisko: Resources, Writing - review & editing. Charles Morel: Re­ diverged from their closest extant relatives, mostly occurring in Africa, sources, Writing - review & editing. Natalie Cooper: Conceptualization, approximately 41–24 Ma (Lima et al., 2013; Weinell et al., 2019). Dating Formal analysis, Visualisation, Writing - original draft, Writing - review estimates are not yet available for other Seychelles squamates, but & editing. Julia J. Day: Conceptualization, Resources, Formal analysis, native and endemic Seychelles species of geckos of the genera Phelsuma, Visualisation, Writing - review & editing. David J. Gower: Conceptu­ Urocotyledon and Ailuronyx have closest extant relatives in Africa and/or alization, Resources, Writing - original draft, Writing - review & editing. Madagascar (Rocha et al., 2010; Gamble et al., 2012). The monotypic Seychelles fossorial skinks Janetaescincus spp. and Pamelaescincus gar­ Declaration of Competing Interest dineri are sister to a large clade of extant African, Arabian and Indian Ocean island taxa (Brandley et al., 2005). The authors declare that they have no known competing financial The origins of Seychelles squamates contrast with those for most of interests or personal relationships that could have appeared to influence the native amphibians. Although the hyperoliid frog Tachycnemis sey­ the work reported in this paper. chellensis arrived from overseas dispersal from Madagascar approxi­ mately 35.3–9.8 Ma (Crottini et al., 2012; Maddock et al., 2014; ca. Acknowledgements 20–12 Ma based on Portik et al. (2019: Fig. S2)), the two ancient Seychelles amphibian lineages that comprise eight indotyphlid caecilian VD was funded by EU Marie Skłodowska-Curie Fellowship 751567. species (Maddock et al., 2017, 2018) and at least four sooglossid frog STM was funded by an NHM-UCL IMPACT PhD studentship. STM and JL species have their closest extant relatives in India, and are Gondwanan each received awards from the Systematics Research Fund of the Sys­ relics (Wilkinson et al., 2002; Biju and Bossuyt, 2003; Gower et al., tematics Association and Linnean Society of London, and the Moham­ 2011, 2016; Kamei et al., 2012; Janani et al., 2017; Labisko et al., 2019). med bin Zayed Conservation Fund (Projects 172515128 and Globally, natricines are ecologically diverse, utilising ground, shrub 162513749). Seychelles research was also funded by the BBSRC’s Syn­ and tree, soil, semiaquatic and/or aquatic habitats. Mainland sub- Tax scheme (awarded to M. Wilkinson, JJD and DJG). VG was supported Saharan African natricines, as well as being relatively species poor, by the IVB CAS institutional support (RVO: 68081766), and the Ministry are seemingly ecologically more conservative despite being the product of Culture of the Czech Republic (DKRVO 2019–2023/6.VII.c, National of approximately 30 million years of evolution, with perhaps all species Museum, 00023272). TDB’s fieldwork was funded by the Royal being predominantly semiaquatic or even aquatic (Gibbons and Dorcas, Geographical Society, and Golder Associates. RGK received EU Marie 2004; Vitt and Caldwell, 2009). Additional and more detailed ecological Skłodowska-Curie Fellowship (PIIF-GA-2013-625870). This work was data would be useful, but L. seychellensis appears to be the only member also supported by Darwin Initiative grant 19-002 (to J.J. Groombridge of the sub-Saharan African (including Seychelles) radiation that is pre­ and colleagues). For the Seychelles component we thank Aaron Bauer, dominantly terrestrial. It is typically encountered on the ground, though Berthilde Belle, Rachel Bristol, Lyndsay Chong-Seng, Beryl Ondiek, Jeff can be observed in vegetation at heights of at least 5 m as well as on the Streicher and Mark Wilkinson for help with logistics and sampling, and margins of pools and streams (STM, JL, RGK & DJG pers. obs.). It is not companionship in the field, the Seychelles Bureau of Standards for clear whether this disparate ecology is ancestral to the L. seychellensis providing required field collection permits, and our Seychelles Darwin lineage and a factor that might have promoted its overseas dispersal to Initiative grant project partners: Seychelles National Park Authority, Seychelles, or whether it is a derived feature acquired since the lineage Seychelles Islands Foundation, Island Conservation Society, National reached Seychelles. All mainland sub-Saharan African natricines are Museum of Seychelles, and the Ministry of Environment. For help with oviparous. Lycognathophis seychellensis is probably also oviparous the Praslin fieldwork, we especially thank Nancy Bunbury, Wilna (Nussbaum, 1984) but direct observations have yet to be reported Accouche, Marc Jean-Baptiste and other staff at the Vallée de Mai. We (Gerlach and Ineich, 2006). thank the late Bill Branch for contributing some African samples. WC Extant mainland sub-Saharan African natricines are relatively thanks Brian Huntley for organising the Lagoa Carumbo Expedition in species-poor, comprising only ca. 5% of global natricine diversity. 2011(National Geographic Society grant number 891-11), Chris Brooks Natricines are unknown from Madagascar or Indian Ocean islands other for organising the Southern Africa Regional Environmental Program’s than Seychelles. As argued for other Indian Ocean and Malagasy taxa, (SAREP) Aquatic Biodiversity Survey of the lower Cuito and Cuando the overseas dispersal of the L. seychellensis lineage from mainland Africa Rivers in Angola in 2013, the National Geographic Okavango Wilderness eastwards was perhaps assisted by sporadic but strong eastward oceanic Project (National Geographic Society grant number EC0715–15) for currents and large, continental freshwater outflows during the Paleo­ funding fieldwork to Angola 2015–2019, and the Angolan Ministry of gene (65.5–23 Ma: Ali and Huber, 2010; Townsend et al., 2010). Environment (MINAMB) for issuing permits. VD thanks Gabriela Bittencourt-Silva, Ana Serra Silva and Gontran Sonet for practical CRediT authorship contribution statement assistance, GBIF for open access distribution data, and Maria Chiara Deflorian (Museo delle Scienze, Trento, Italy) for loaning specimens of V. Deepak: Conceptualization, Investigation, Formal analysis, Vis­ Natriciteres to London. We thank Alan Resetar (Field Museum (FMNH), ualisation, Data curation, Writing - original draft, Writing - review & Chicago) and Lauren Scheinberg (California Academy of Sciences (CAS), editing. Simon T. Maddock: Conceptualization, Resources, Investiga­ California) for making tissue samples available for this study. VD thanks tion, Formal analysis, Visualisation, Data curation, Writing - original Jeff Streicher and Ana Serra Silva for their help with the TreePL analysis draft, Writing - review & editing. Rhiannon Williams: Investigation, and discussions on molecular dating analysis. Formal analysis, Visualisation, Writing - review & editing. Zoltan´ T. Nagy: Conceptualization, Resources, Data curation, Writing - review &

13 V. Deepak et al. Molecular Phylogenetics and Evolution 161 (2021) 107152

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Table S1. Primers used in this study for amplifying and sequencing target genes.

Gene Primers Annealing temperature(s) References 16s 16Sar-L & 16Sar-H 48-50°C Palumbi et al . (1991) cytb Gludg & H16064 46-58°C Palumbi (1996) & Burbrink et al . (2000) & de Queiroz et al. (2002) nd4 ND4 & Leu 42-48°C Arévalo et al . (1994) nt3 nt3f & nt3r 48-51°C Townsend et al . (2008) bdnf BDNF-F & BDNF-R 57°C Noonan & Chippindale (2006) rag1 R13 & R18 50-60°C Groth & Barrowclough (1999) cmos S77 & S78 52-56°C Lawson et al . (2005) prlr PRLRF1 & PRLRR3 46°C Townsend et al. (2008)

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Sample Species voucher number/extractionID 16s cytb nd4 cmos nt3 bdnf rag1a Location 1 Natriciteres fuliginoides CAS258133 MW699975 MW711487 MW711528 MW711545 MW711465 MW711572 MW711601 Moyen-Ogooue Prov., Gabon 2 Natriciteres olivacea CAS220640 MT586821 MT587285 MT587311 _ MW711466 MW711573 _ DR Congo 3 Natriciteres sylvatica MTSN5485 MW699977 MW711489 MW711530 MW711547 MW711468 MW711575 MW711603 Udzungwa Mts., Uzungwa Scarp F. R., Mkalazi, Tanzania 4 Natriciteres sylvatica MTSN8183 MW699978 MW711490 _ _ _ _ _ Nguru South Mts., Nguru South (Pemba), Tanzania 5 Natriciteres sylvatica MUSE13712 MW699979 MW711491 MW711531 MW711548 MW711469 MW711576 MW711604 Rungwe Mt. (Nkuka), Southern Highlands, Tanzania 6 Natriciteres variegata MUSE13726 MW699981 ______Rungwe Mt. (Nkuka), Southern Highlands, Tanzania 7 Afronatrix anoscopus 1 NHMUK 2013:383 MW699965 MW711484 _ MW711535 MW711452 MW711558 MW711589 Akwa, Mamfe division, Cameroon 8 Afronatrix anoscopus 2 Lake Kuk, Cameroon MW699966 MW711485 _ MW711536 MW711453 MW711559 MW711590 LakeKuk, Cameroon 9 Afronatrix anoscopus 3 MW08018/BMNH2008.637 MW699967 MW711486 MW711522 MW711537 _ MW711560 MW711591 Mount Nimba mining concession, Forestiere Region, Guinea 10 Limnophis branchi ANG-056 MT586818 MT587283 MT587308 MT587296 MW711463 MW711570 MW711599 Lugela river area, Lunda Norte, Angola 11 Limnophis branchi ANG-058 MT586819 MT587284 MT587309 MT587297 MW711464 MW711571 MW711600 Lugela river area, Lunda Norte, Angola 12 Limnophis bangweolicus ANG-438 MT586815 MT587279 MT587306 MT587292 MW711461 MW711568 MW711597 Camp site 6km west of SashaE, Cuando Cubango, Angola 13 Limnophis bicolor WC-6238 MT586817 MT587281 MT587307 MT587294 MW711462 MW711569 MW711598 upstream of Lungue Bungu Bridge, Moxico, Angola 14 Hydraethiops melanogaster 1 NMP-P6V 75763 MW699972 _ MW711525 MW711542 MW711458 MW711565 MW711594 Bafwabianga village, DR Congo 15 Hydraethiops melanogaster 2 NMP-P6V 75491 MW699973 _ MW711526 MW711543 MW711459 MW711566 MW711595 Gabon 16 Hydraethiops melanogaster 3 NMP-P6V 75772 MW699974 _ MW711527 MW711544 MW711460 MW711567 MW711596 Congo Rep., Sangha 17 Natriciteres olivacea WRB-636 MW699976 MW711488 MW711529 MW711546 MW711467 MW711574 MW711602 Kalemie, DR Congo 18 Natriciteres sylvatica WRB-642 MW699980 MW711492 MW711532 MW711549 _ MW711577 _ Mount Mabu, Zambezi, Mozambique 19 Helophis schoutedeni 1 CRT3853 MW699968 _ MW711523 MW711538 MW711454 MW711561 MW711592 DR Congo; Kona 20 Helophis schoutedeni 2 CRT3855 MW699969 _ MW711524 MW711539 MW711455 MW711562 MW711593 DR Congo; Kona 21 Helophis schoutedeni 3 CRT3859 MW699970 _ _ MW711540 MW711456 MW711563 _ DR Congo; Kona 22 Helophis schoutedeni 4 CRT3860 MW699971 _ _ MW711541 MW711457 MW711564 _ DR Congo; Kona 23 Smithophis bicolor BNHS 2369 MK350254 MK350261 MK350259 MK350264 MW711474 MW711582 MK350256 Mizoram University Campus, Aizawl , Mizoram, India 24 Smithophis atemporalis BNHS 3527 MK350255 MK350262 MK350258 MK350265 MW711473 MW711581 MK350257 Mizoram University Campus, Aizawl , Mizoram, India 25 Rhabdops olivaceous NCBS-AU164 MF352831 MF352838 MF352842 MF352834 MW711472 MW711580 MW711607 Pookode, Wayanad District, Kerala, India 26 Lycognathophis seychellensis SM165 MW699983 MW711494 MW711533 MW711550 MW711476 MW711583 MW711608 Mahé, Seychelles 27 Lycognathophis seychellensis SM213 MW700003 MW711511 MW711534 MW711554 MW711479 MW711585 MW711609 Silhouette, Seychelles 28 Adelophis foxi LSUMZ 40846, LSUMZ H08272 _ AF420069 AF420072 _ KF234031 KF258601 _ Mexico, Durango 29 Afronatrix anoscopus * ROM 19842 (ATOL A111) _ AF420073 _ AF471123 _ EU402622 _ Liberia 30 Aspidura ceylonensis RS-145 KC347361 KC347477 KC347527 KC347400 _ _ _ Sri Lanka 31 Aspidura drummondhayi RS-M KC347340 KC347455 KC347519 KC347379 _ _ _ Sri Lanka, Nuwara Eliya Dist, Maskeliya 32 Aspidura guentheri RAP0437 KC347341 KC347472 KC347507 KC347380 _ _ _ Sri Lanka, Galle Dist, Kanneliya Forest Reserve 33 Aspidura trachyprocta RS-134 KC347343 KC347473 KC347523 KC347382 _ _ _ Sri Lanka, Nuwara Eliya Dist, Horton Plains National Park 34 Atretium schistosum RS-R _ KC347487 KC347525 KC347383 _ _ _ Sri Lanka, Nuwara Eliya Dist, Norton Bridge 35 Atretium yunnanensis * GP842 _ GQ281787 JQ687423 JQ687448 KJ685764 _ KJ685602 China, Yunnan 36 Rhabdophis ceylonensis * RS-D, HT0785 KC347344 KC347474 KC347520 KC347384 _ LC326026 Sri Lanka, Galle Dist, Kanneliya Forest Reserve 37 Clonophis kirtlandii LSUMZ H08189 _ KF258647 KF258630 _ KF234026 KF258596 _ USA, Illinois 38 Haldea striatula LSUMZ:H18238 _ KF258657 KF258640 _ KF234036 KF258606 _ USA, Louisiana 39 Hebius atemporale GP2318 _ KJ685695 _ KJ685645 KJ685747 _ KJ685587 China, Yunnan 40 Hebius atemporale GP1626 _ KJ685680 _ KJ685630 KJ685732 _ KJ685572 China, Guangdong 41 Hebius bitaeniatum GP1940 _ KJ685688 _ KJ685648 KJ685740 _ KJ685580 China, Guangxi 42 Hebius bitaeniatum GP1863 _ KJ685686 _ KJ685636 KJ685738 _ KJ685578 China, Guizhou 43 Hebius boulengeri GP2433 _ KJ685699 _ KJ685649 KJ685751 _ KJ685589 China, Fujian 44 Hebius boulengeri GP2134 _ KJ685691 _ KJ685641 KJ685743 _ KJ685583 China, Hainan 45 Hebius boulengeri GP1789 _ KJ685684 _ KJ685634 KJ685736 _ KJ685576 China, Guangdong 46 Hebius craspedogaster GP455 _ KJ685704 _ KJ685654 KJ685759 _ KJ685597 China, Sichuan 47 Hebius craspedogaster GP1712 _ KJ685682 _ KJ685632 KJ685734 _ KJ685574 China, Sichuan 48 Hebius craspedogaster GP1636 _ KJ685681 _ KJ685631 KJ685733 _ KJ685573 49 Hebius craspedogaster GP1240 _ KJ685672 _ KJ685622 KJ685723 _ KJ685565 China, Guizhou 50 Hebius craspedogaster GP139 _ JQ687429 JQ687412 JQ687437 KJ685730 _ KJ685569 China 51 Hebius concelarum KUZ: R20253 & KUZ:R18557 _ AB989268 AB989270 AB989271 LC047778 _ LC047774 Japan:Okinawa, Miyakojima shi, Miyakojima 52 Hebius deschauenseei AMNH 148575 _ KJ685665 KJ685614 KJ685715 _ KJ685558 Vietnam, Ha Giang 53 Hebius ishigakiensis KUZ:R48405 _ AB989284 AB989286 AB989287 LC047781 _ LC047772 Okinawa, Japan 54 Hebius sp. KUZ: R67949 _ AB989298 AB989300 AB989301 LC047780 _ LC047773 Taiwan 55 Hebius johannis GP1569 _ KJ685678 _ KJ685628 KJ685731 _ KJ685571 China, Yunnan 56 Hebius khasiense CAS 221525 _ KJ685669 _ KJ685618 KJ685719 _ KJ685562 Myanmar, KaChin 57 Hebius khasiense CAS 221504 _ KJ685668 _ KJ685617 KJ685718 _ KJ685561 Myanmar, KaChin 58 Hebius khasiense AMNH 148552 _ KJ685663 _ KJ685612 KJ685713 _ KJ685556 Vietnam, Quang Nam 59 Hebius metusium GP871 _ KJ685707 _ KJ685657 KJ685766 _ _ China, Sichuan 60 Hebius modestum CAS 234262 _ KJ685671 _ KJ685620 KJ685721 _ KJ685564 China, Yunnan 61 Hebius modestum MVZ 226514 _ KJ685709 _ KJ685659 KJ685769 _ KJ685608 Vietnam, Vinh Phu 62 Hebius octolineatum GP1242 _ KJ685673 _ KJ685623 KJ685724 _ _ China, Guizhou 63 Hebius octolineatum HT0586 _ LC325321 _ LC325767 _ _ _ China 64 Hebius optatum GP1885 _ KJ685687 _ KJ685637 KJ685739 _ KJ685579 China, Guizhou 65 Hebius optatum AMNH 147155 _ KJ685662 _ KJ685611 KJ685712 _ KJ685555 Vietnam, Vinh Phu 66 Hebius parallelum CAS 215036 _ KJ685666 _ KJ685615 KJ685716 _ KJ685559 Vietnam, Ha Giang 67 Hebius popei GP2386 _ KJ685697 _ KJ685647 KJ685749 _ KJ685588 China, Guizhou 68 Hebius popei GP2169 _ KJ685692 _ KJ685642 KJ685744 _ KJ685584 China, Hainan 69 Hebius pryeri KUZ: R67983 _ AB989102 AB989104 AB989105 LC047779 _ LC047776 Mt. Yuwan, Naon, Yamatoson 70 Hebius sauteri GP864 _ KJ685706 _ KJ685656 KJ685765 _ KJ685603 China, Sichuan 71 Hebius sauteri GP2549 _ KJ685701 _ KJ685651 KJ685754 _ KJ685592 China, Taiwan 72 Hebius sauteri GP2382 _ KJ685696 _ KJ685646 KJ685748 _ _ China, Sichuan 73 Hebius venningi CAS:233206 _ KJ685670 _ KJ685619 KJ685720 _ KJ685563 Myanmar, KaChin 74 Hebius venningi GP2468 _ KJ685700 _ KJ685650 KJ685752 _ KJ685590 Thailand 75 Hebius venningi GP1300 _ KJ685675 _ KJ685625 KJ685727 _ _ China, Yunnan 76 Hebius vibakari GP1352 _ KJ685677 _ KJ685627 KJ685729 _ KJ685568 China, Heilongjiang 77 Hebius vibakari KUZ: R21587 _ AB989302 AB989304 AB989305 _ _ _ Japan:Kyoto 78 Herpetoreas burbrinki YBU 071128/GP600 _ GQ281781 JQ687418 JQ687443 KJ685761 _ KJ685599 China 79 Herpetoreas platyceps GP2096 _ KJ685690 _ KJ685640 KJ685742 _ KJ685582 China, Xizang 80 Liodytes alleni LSUMZ:H08565 _ KF258653 KF258636 _ KF234032 KF258602 _ USA, Florida 81 Liodytes pygaea LSUMZ 42686, SJA 7787 _ KF258654 KF258637 _ KF234033 KF258603 _ USA, Georgia, USA, Florida, Alachua Co. 82 Liodytes rigida LSUMZ:H20703 _ KF258659 KF258642 _ KF234038 KF258608 _ USA, Louisiana 83 Lycognathophis seychellensis COG _ _ _ FJ387220 _ _ _ Seychelles 84 Lycognathophis seychellensis _ _ _ _ FJ404294 _ _ _ Seychelles 85 Rhabdophis plumbicolor HT0782 _ LC325336 _ LC325782 _ _ LC326025 Sri Lanka 86 Rhabdophis rhodomelas ADM 0003 KX660258 KX660528 _ KX660399 KX652069 _ _ 87 Macropisthodon rudis USNM 539454 KX694651 KX694875 _ _ KX695027 KX694735 _ Indonesia, Java: Central Java 88 Macropisthodon rudis GP384 _ GQ281780 JQ687417 JQ687442 KJ685758 _ KJ685596 China, Guangdong 89 Natriciteres olivacea PEM:R15463 AY611836 AY612019 _ AY611928 _ _ _ Mozambique, Zambezi delta 90 Natrix natrix helvatica LSUMZ:41506 _ AY866544 AY873710 AF471121 _ _ _ England, Kent, Sheppey Island 91 Natrix natrix persa ROM 26841 & 42 _ KX694876 AY873723 KX694827 _ JQ599036 _ Armenia, Ankavan 92 Nerodia clarkii clarkii LSUMZ 43426 _ _ KT884436 _ KT884208 KT884149 _ USA, Alabama 93 Nerodia cyclopion H08217 _ KF258649 KF258632 _ _ KF258598 _ USA, Florida 94 Nerodia erythrogaster SJA 6512, UTA R-59349, JDM 1004 _ AF402912 KT884443 JN090137 KT884211 KT884157 _ USA, AR, Lonoke Co., USA, Texas 95 Nerodia fasciata _ _ KF258648 _ _ KF234027 KF258597 _ 96 Nerodia floridana LSUMZ 40090 _ _ KT884425 _ KT884197 KT884136 _ USA, Florida 97 Nerodia sipedon LSUMZ 40906 _ _ KT884432 _ KT884204 KT884144 _ USA, Georgia 98 Nerodia sipedon ROM 22671, MEA 503 KX694654 KX694873 _ KX694812 KX695033 KX694741 _ Canada, Ontario, USA, Illinois, Cook Co. 99 Nerodia taxispilota LSUMZ 40308 _ _ KT884427 _ KT884199 KT884138 _ USA, Florida 100 Opisthotropis cheni GP383 _ GQ281779 JQ687416 JQ687441 _ _ _ China, Guangdong 101 Opisthotropis guangxiensis GP746 _ GQ281776 JQ687422 JQ687447 _ _ _ China, Guangxi 102 Opisthotropis lateralis GP646 _ GQ281782 JQ687420 JQ687445 _ _ _ China 103 Opisthotropis latouchii GP647 _ GQ281783 JQ687421 JQ687446 _ _ _ China 104 Opisthotropis typica HT0794 _ LC325343 _ LC325789 _ _ LC326028 Malaysia 105 Regina grahami H08535, MEA 505 _ KF258650 KF258633 _ KF234029 KF258599 USA, Louisiana, USA, Texas 106 Regina septemvittata LSUMZ 40101 _ KF258646 KF258629 _ KF234025 KF258595 USA 107 Rhabdops aquaticus NCBS-AU163 MF352830 MF352835 MF352840 MF352833 _ _ 108 Rhabdophis conspicillatus HT0791 _ LC325342 _ LC325788 _ _ LC326027 Malaysia 109 Rhabdophis callichromus HT0674 _ LC325325 _ LC325771 _ _ LC326020 Vietnam 110 Rhabdophis chrysargos HT0342 _ LC325313 _ LC325759 _ _ LC326017 Malaysia 111 Rhabdophis himalayanus HT0847 _ LC325299 _ LC325746 _ _ LC326011 China 112 Rhabdophis leonardi HT0851 _ LC325300 _ LC325747 _ _ LC326012 China 113 Rhabdophis nigrocinctus HT0253 _ LC325307 _ LC325753 _ _ LC326015 Thailand 114 Rhabdophis nuchalis HKV 36838, HT0701 _ AF402907 _ LC325779 _ _ LC326022 China, Sichuan, Hongya Xian 115 Rhabdophis nuchalis GP251 _ GQ281786 JQ687413 JQ687438 _ _ China, Sichuan 116 Rhabdophis swinhonis HT0717 _ LC325334 _ LC325780 _ _ LC326023 Taiwan 117 Rhabdophis tigrinus lateralis GP613 _ GQ281785 JQ687419 JQ687444 _ _ China 118 Rhabdophis tigrinus HT0098 _ LC325305 _ LC325751 _ _ LC326013 Japan 119 Sinonatrix aequifasciata GP357 _ JQ687430 JQ687415 JQ687440 _ _ _ China 120 Sinonatrix annularis GP889 _ JQ687431 JQ687424 JQ687449 _ _ _ China 121 Sinonatrix percarinata GP324 _ GQ281784 JQ687414 JQ687439 _ _ _ China, Sichuan 122 Sinonatrix percarinata ROM 35669 KX694667 KX694872 _ KX694819 KX695050 KX694763 _ Vietnam, Cao Bang 123 Storeria dekayi H07700 _ AF471050 KF258627 AF471154 KF234023 KF258593 _ USA, Pennsylvania, USA, Illinois, Cook Co. 124 Storeria dekayi ROM 41891 KX694669 KX694874 _ KX694821 _ Canada, Ontario 125 Storeria occipitomaculata LSUMZ 80970 _ _ KT884438 _ KT884210 KT884151 _ USA, Louisiana 126 Storeria storerioides LSUMZ 08265 _ KF258651 KF258634 _ KF234030 KF258600 _ Mexico 127 Thamnophis atratus atratus LSUMZ 44386 _ _ KT884437 _ KT884209 KT884150 _ USA, California 128 Thamnophis brachystoma CU 12379, LSUMZ 58447 _ AF420089 AF420092 _ KT884191 KT884131 _ USA, Pennsylvania, McKean Co. and USA, New York, Cattaraugus Co. 129 Thamnophis butleri H07718, TBUT1 _ KF258645 KF258628 _ KF234024 KF258594 _ Canada, Ontario 130 Thamnophis chrysocephalus isolate 7310B & MVZ 164784 _ _ AF420098 _ KT884187 KT884127 _ Mexico, Oaxaca 131 Thamnophis couchii H08146 _ _ KT884426 _ KT884198 KT884137 _ 132 Thamnophis cyrtopsis LSUMZ 40426 _ _ KT884431 _ KT884203 KT884143 _ USA, New Mexico 133 Thamnophis elegans LSUMZ39641 _ _ KT884424 _ KT884196 KT884135 _ USA, New Mexico 134 Thamnophis eques LSUMZ 40752 _ _ KT884429 _ KT884202 KT884141 _ Mexico, Durango 135 Thamnophis fulvus LSUMZ 57127 _ _ KT884420 _ KT884190 KT884130 _ Guatemala, Jalapa 136 Thamnophis godmani MZFC:10202 _ AF420135 AF420138 AF471165 _ _ _ Mexico, Oaxaca 137 Thamnophis hammondii LSUMZ 37179 _ _ KT884422 _ KT884194 KT884133 _ USA, California 138 Thamnophis marcianus LSUMZ 48745 _ _ KT884418 _ KT884188 KT884128 _ USA, Texas 139 Thamnophis melanogaster LSUMZ 37429 _ _ KT884423 _ KT884195 KT884134 _ Mexico, Michoac√°n 140 Thamnophis nigronuchalis LSUMZ 40849 _ _ KT884428 _ KT884201 KT884140 _ Mexico, Durango 141 Thamnophis proximus LSUMZ:H19835 _ KF258658 KF258641 _ KF234037 KF258607 _ USA, Louisiana, USA, Texas, San Saba Co. 142 Thamnophis pulchrilatus LSUMZ 35379 _ _ KT884421 _ KT884193 KT884132 _ Mexico, Durango 143 Thamnophis radix LSUMZ H02935 _ _ KT884419 _ KT884189 KT884129 _ USA, Wisconsin 144 Thamnophis rufipunctatus CU 12457 _ AF420173 AF420176 _ _ USA, New Mexico, Catron Co. 145 Thamnophis sauritus LSUMZ 41508 _ _ KT884434 _ KT884206 KT884146 _ USA, Florida 146 Thamnophis sirtalis LSUMZ 41181 _ _ KT884433 _ KT884205 KT884145 _ USA, Maine 147 Thamnophis sirtalis ROM 26937 KX694670 _ _ _ KX695054 KX694767 _ 148 Trachischium monticola GP1487 _ JQ687435 JQ687428 JQ687453 _ China 149 Tropidoclonion lineatum H13044 _ KF258655 KF258638 KF234034 KF258604 _ USA 150 Virginia valeriae LSUMZ:H16063 _ KF258656 KF258639 KF234035 KF258606 _ USA, Louisiana 151 Virginia valeriae RAP 0572 KR814654 KR814700 KR814721 KR814673 _ _ _ 152 Fowlea asperrimus RS-J KC347376 KC347480 _ KC347413 _ _ _ Sri Lanka, Kandy Dist, Nawalapitiya 153 Xenochrophis maculatus HT0720 _ LC325335 _ LC325781 _ _ LC326024 Malaysia 154 Fowlea piscator HT0347 _ LC325317 _ LC325763 _ _ LC326018 Thailand 155 Fowlea punctulatus CAS HERP 206594 _ AF471079 AY487074 AF471106 _ _ Myanmar, Ayeyarwade Div., vic Mwe Hauk Village 156 Xenochrophis trianguligerus HT0795 _ LC325344 _ LC325790 _ _ LC326029 Malaysia 157 Xenochrophis vittatus FMNH 257460, HT0615 EF395846 EF395895 _ EF395920 _ _ LC326019 Indonesia 158 Grayia ornata † MNHN 1997.6517 AY611866 AY612048 AF544663 AF544684 KX695019 FJ434002 _ Ogooue, Gabon 159 Sibynophis subpunctatus † RAP0491 KC347373 KC347471 KC347516 KC347411 _ _ _ Hambantota Dist., Yala Natl. Park, Sri Lanka Table S3. Partitions and models of sequence evolution employed in analyses of the 73-leaf dataset. 1st, 2nd and 3rd denote codon position for protein-coding genes.

Partition Sites BI P1 16s GTR+I+G P2 cytb 1st, nd4 1st GTR+I+G P3 cytb 2nd, nd4 2nd GTR+I+G P4 cytb 3rd , nd4 3rd GTR+G P5 cmos 1st K80+G P6 nt3 2nd, rag1a 1st, cmos 2nd, rag1b 1st HKY+G P7 rag1a 3rd, cmos 3rd, rag1b 3rd HKY+G P8 nt3 1st HKY+G P9 nt3 3rd HKY+G P10 bdnf 1st K80+G P11 bdnf 2nd & 3rd, rag1b 2nd, rag1a 2nd HKY+I Table S4. Parameter values for fossil calibrations used in the BEAST divergence dating analysis. Ages in Ma. All maximum ages soft, except hard maximum for calibration 6.

Maximum Standard Calibration Node calibrations Offset Mean 5% 95% age deviation 1 Oldest divergence within crown Alethinophidia 93.9 100.5 1.5 1.25 94 99.3 Oldest divergence between non-xenodermid colubroids and their closest living 2 relative (Xenodermidae) 50.5 72.1 6.1 1.25 50.9 72.3 3 Divergence between Boinae and its sister taxon (Erycinae + Candoiinae) 58 64 1.8 1.25 58.1 64.4 4 Divergence between Corallus and (Chilabothrus + (Epicrates + Eunectes)) 50.2 64.0 4 1.25 50.4 64.5 5 Divergence between Viperinae and Crotalinae 20.0 23.8 1 1.25 20.1 23.6 6 Divergence between Acrochordus javanicus and (A. ararfurae + A. granulatus ) 18.1 23.1 1.5 1.25 18.2 23.5 7 Oldest divergence between Naja (Afronaja) and Naja (Boulengerina) 17.0 20.0 1 1.25 17.1 20.6 Table S5. Partitions and models of sequence evolution employed in analyses of the 21-leaf dataset. 1st, 2nd and 3rd denote codon position for protein-coding genes.

Partition Sites ML BI P1 16s , cytb 1st, nd4 1st GTR+G GTR+I+G P2 cmos 3rd, cytb 2nd, nd4 2nd GTR+G GTR+I+G P3 nd4 3rd, cytb 3rd, GTR+G GTR+I+G P4 bdnf 1st, cmos 1st GTR+G K80 P5 nt3 1st & 2nd, bdnf 2nd & 3rd, cmos 2nd, rag1 2nd & 3rd, cmos 2nd GTR+G HKY+I P6 nt3 3rd, rag1 1st GTR+G K80+G Table S6. Support values for three incompatible clades representing the three competing resolutions of the relationships of L. seychellensis and Sub-Saharan African natricines for the 21- leaf dataset. Values in each cell are given separately for analyses of mt - nu - mt+nu data (in that order). Highest support values for each analysis are highlighted in bold.

Ly ,(Hy,He,Af ) ((Na,Li )(Hy,He,Af )) Ly, (Na ,Li ) RaxML bootstrap (gene & codon position) 9 - 53 - 37 73 - 6 - 35 18 - 41 - 27 RaxML bootstrap (gene) 16 - 62 - 40 70 - 3 - 36 14 - 35 - 24 MrBayes posterior probability x100 6 - 47 - 28 89 - 23 - 59 5 - 30 - 12 BEAST posterior probability x100 37 - 95 - 88 58 - <1 - 8 5 - 4 - 4