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Home , Ahaetulla, Ahaetulla fasciolata, Ahaetulla nasuta, Ahaetulla prasina, Chrysopelea, Dendrelaphis

Turkish Journal of Zoology Turk J Zool (2020) 44: 11-21 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1904-1

First record of mycterizans (Linnaeus, 1758) (Serpentes: ) from the Lesser Sunda region, , based on molecular and morphological identification

1,2 1,3 4 1,2, Lilin Ika Nur INDAHSARI , Fatchiyah FATCHIYAH , Eric Nelson SMITH , Nia KURNIAWAN * 1 Department of Biology, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, Indonesia 2 NK Research, Department of Biology, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, Indonesia 3 Research Center of Smart Molecule of Natural Genetic Resources, Brawijaya University, Malang, Indonesia 4 Department of Biology, University of Texas at Arlington, Arlington, TX, USA

Received: 01.04.2019 Accepted/Published Online: 25.11.2019 Final Version: 03.01.2020

Abstract: Previous studies on the distribution of the Ahaetulla across Indonesia only focused on morphological characters without any molecular data. This study was aimed at analyzing phylogenetic relationships among the Ahaetulla in Indonesia based on partial mitochondrial DNA sequences of 16 specimens collected from the Sundaland and Lesser Sunda regions. The 12S-rDNA gene was PCR-amplified and sequenced to analyze phylogenetic relationships and to estimate the divergence time between the 2 Ahaetulla populations of the Sundaland and Lesser Sunda regions. Morphological characters of 3 Ahaetulla specimens from Lesser Sunda and Sundaland have also been analyzed to confirm the results based on molecular markers. Both the phylogenetic analyses and morphological characters revealed the presence of 2 Ahaetulla , A. mycterizans and A. prasina, in Sundaland as well as in the Lesser Sunda region. Previously, A. mycterizans was only known to exist in Sundaland. Its presence in the Lesser Sunda region is reported here for the first time. The divergence time estimations suggested the presence ofA. mycterizans in Lesser Sunda had occurred during the Miocene, around 12.1 Ma. Consequently, this study has added A. mycterizans to the list of that inhabit Lesser Sunda and revealed the need to initiate a survey in more detail to investigate the presence of A. mycterizans in other regions of Southeast .

Key words: Ahaetulla mycterizans, , Lesser Sunda, phylogenetic, Sundaland

1. Introduction A. mycterizans in Sumatra. A. mycterizans has also been The arboreal snake genus Ahaetulla (Link, 1807) is discovered in Java by De Rooij (1917) and Ekarini (2016). composed of 8 species distributed in most countries A. fasciolata has been found only in Sumatra (Das, 2015). of the Asian region, including Indonesia, , However, there has been no previous record about the , , , , and throughout the existence of A. mycterizans in the Lesser Sunda region. Indochinese Peninsula (Das, 2015; Mohapatra et al., 2017). De Lang (2011) reported 29 species of terrestrial and All species of this genus are characterized by an elongated semiaquatic snakes from the , but triangular head, an extremely thin and elongate body, and he found only one species of the genus Ahaetulla, i.e., horizontally-shaped (Long et al., 2012; Das, 2015; A. prasina. Due to the discovery of A. mycterizans in Lee at al., 2015). Previously, 3 species have been recorded Peninsular Malaysia, A. mycterizans has been recorded in Indonesia: (i) Ahaetulla prasina (Boie, 1827) (Oriental much farther north than its previously known distribution Vine Snake), (ii) Ahaetulla mycterizans (Linnaeus, 1758) range (Mirales and David, 2010). This discovery, as well (Malayan Vine Snake), and (iii) as the distribution of A. prasina in this region, suggested (Fischer, 1885) (Speckle-headed Vine Snake). A. prasina the possibility of the existence of A. mycterizans in the is present in much of the Sundaland region: Sumatra, Lesser Sunda Islands. Unfortunately, all of those studies Borneo (Kalimantan), Java, Bali, and (Das, 2015; on the distribution of the genus Ahaetulla in Indonesia Leo, 2015; Ekarini, 2016). Furthermore, De Lang (2011) were carried out solely on the basis of morphological reported the occurrence of A. prasina in the Lesser Sunda characters that are commonly used as the main diagnostic region, more specifically in Lombok and Sumbawa. characters for species identification. There have been no Miralles and David (2010) reported the first finding of previous studies that have used DNA sequence analysis * Correspondence: [email protected] 11

This work is licensed under a Creative Commons Attribution 4.0 International License. INDAHSARI et al. / Turk J Zool to identify Ahaetulla species in Indonesia. Furthermore, This is the first study of the genus Ahaetulla in species identification by morphological characters may Sundaland and the Lesser Sunda region using the lead to misidentifications, especially between 2 of the 12S-rDNA gene to analyze phylogenetic relationships, to Ahaetulla species, A. prasina and A. mycterizans, due to construct the haplotype network, and to estimate the time shared similar morphology, such as body color, shape, and of divergence. We also analyzed morphological characters even head shape (Miralles and David, 2010; Ekarini, 2016; to confirm the molecular identification. Lok et al., 2016). A. prasina, is much more widespread in Indonesia than A. mycterizans, which remains poorly 2. Materials and methods known. Along with morphology, molecular data is 2.1. Specimens collection and ethical clearance needed to distinguish between these 2 species accurately Ahaetulla specimens were obtained from several localities and avoid misidentification. Figueroa et al. (2016) used in Sumatra, Java, Bali, and Lombok Island as shown in mitochondrial DNA including the 12S-rDNA gene as Figure 1. A total of 16 specimens consisting of 6 specimens barcodes to determine the phylogenetic relationship from Sumatra, 7 specimens from Java, 2 specimens from among 1592 snakes of the Colubridae family. Furthermore, Bali and, 1 specimen from Lombok, were collected. the phylogenetic analysis of the snake species of the family Additional sequences were retrieved from GenBank Colubridae has not only contributed to the evaluation of (www.ncbi.nih.gov.nlm) including ornata, snake diversity, but also to the evaluation of geological pictus, and Ptyas mucosa species, as shown in history through studies on estimated genetic divergence Table 1. To collect our specimens and take ventral muscle time (Takeuchi et al., 2011; Nugraha et al., 2018). tissue, we obtained ethical clearance from the Research

Figure 1. Sample collection sites of the 16 Ahaetulla specimens used in this study, edited by QGIS software. Source of map: www.naturalearthdata.com.

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Table 1. List of specimens (column 1) of the collected in context of the present study, and outgroup samples used in the phylogenetic analysis. The localities and the GenBank accession numbers for 12S rDNA sequences and references are given in columns 2–4, respectively.

Genbank Sample name Specimens Locality Source Accession no. SUMATRA NS1 A. prasina Medan, North Sumatra MK691461 This study NS A. prasina Rau, North Sumatra MK691470 This study WS1 A. prasina Bukittinggi, West Sumatra MK691464 This study WS2 A. prasina Andalas, West Sumatra MK691460 This study LP1 A. prasina Rajabasa, Lampung MK691469 This study LP2 A. prasina Tanggamus, Lampung MK691474 This study JAVA WJ1 A. prasina Bandung, West Java MK691462 This study EJ1 A. prasina Banyuwangi, East Java MK691466 This study EJ2 A. prasina Malang, East Java MK691468 This study BA1 A. prasina Sukawati, Bali MK691472 This study BA2 A. prasina Tanah Lot, Bali MK691473 This study BN A. mycterizans Pandeglang, Banten MK691463 This study WJ2 A. mycterizans Sukabumi, West Java MK691471 This study CJ1 A. mycterizans Nusakambangan, Central Java MK691465 This study CJ2 A. mycterizans Tanjungharjo, Central Java MK691475 This study LOMBOK LO A. mycterizans Lombok, West Nusa Tenggara MK691467 This study OTHER SPECIES Chrysopeleaornata - KX694558 (Alencar et al., 2016) Dendrelaphispictus Malang, East Java KY700863 (Nugraha et al., 2018) Ptyas mucosa Malang, East Java KY700867 (Nugraha et al., 2018)

Ethics Committee of Brawijaya University in Ethical (5’-GTACGCTTACCATGTTACGACTTGCCCTG-3’) Clearance No. 1081-KEP-UB. (Jeong et al., 2013) primers. All 16 samples were sequenced 2.2. Morphological identification for the 12S rDNA fragment. The PCR amplification for Dorsal scale rows were counted at 3 places—i.e., at 1 head- 12S-rDNA was conducted in 50-μL reaction volumes with length behind the head, at midbody, and at 1 head-length 5 min denaturation at 94 °C, followed by 35 cycles at 94 anterior to the cloacal plate. Ventral and subcaudal scales °C for 30 s, 56.5 °C for 1 min, and 72 °C for 1 min, and were counted according to Dowling (1951). Measurements, then a final elongation step at 72 °C for 10 min. The PCR except for body and tail lengths, were taken with a slide- products were sequenced using the above PCR primers by caliper to the nearest 0.1 mm. All body measurements Indoseq GATC, Biosains Institute, Brawijaya University. were made to the nearest millimeter. Drawings were made 2.3. Sequence analyses using Corel Draw and Adobe® Photoshop software. 12S-rDNA sequences were aligned by using the Clustal 2.2. DNA extraction, amplification, and sequencing W (Hall, 2013) application implemented in MEGA 7.0 Genomic DNA was extracted from ventral muscle software (Kumar et al., 2016). The alignment results tissue stored in 96% ethanol using a Geneaid DNA were edited manually to trim the ambiguous sites. The isolation kit following the manufacturer’s protocol. alignment length of the 12S-rDNA sequences was 600 bp. The fragment of 12S-rDNA was amplified by The maximum likelihood (ML) tree was reconstructed polymerase chain reaction (PCR) using 12S268 using RAxML software (Stamatakis, 2006) with the (5’-GTGCCAGCGACCGCGGTTACACG-3’) and 12S916 bootstrap option. Bayesian inference (BI) was analyzed

13 INDAHSARI et al. / Turk J Zool using MrBayes software (Ronquist and Huelsenbeck, 2003) specimen of Lombok Island representing Lesser Sunda, using Markov Chain Monte Carlo (MCMC) estimated for and 2 Ahaetulla specimens of Java (Central and East 5 million generations. The most appropriate sequence Java) representing Sundaland. Based on morphological evolution models were selected using Akaike Information identification, there were 2 distinct morphological Criterion (AIC) for ML and BI. The divergence time was separations of Ahaetulla snakes found in this study. The estimated by using BEAST (Drummond et al., 2012) results of morphological identification are presented in software under the Hasegawa–Kishino–Yano model of Figure 2 and Table 2. DNA evolution with the uncorrelated log-normal relaxed Based on the morphology of the head (see Figure clock rate model (Drummond et al., 2006). The estimated 2), there were similarities between Ahaetulla of Central time divergence between C. ornata and P. mu co s a at 23.1 Java and Lombok; they both differed fromAhaetulla of Mya and between P. mu co s a and D. pictus at 37.6 Mya East Java. Ahaetulla of Central Java and Lombok have (Nugraha et al., 2018) were used for the external calibration. the following characters: 1) upper surface of snout was The estimated time divergence of West Java and Lampung convex; 2) snout was less than twice the diameter of the separation was employed for the internal calibration. There (1.8× for Ahaetulla of central Java, 1.9× for Ahaetulla has been no previous study on the estimated divergence of Lombok). However, the specimens from East Java time for Ahaetulla; thus, internal calibration for Ahaetulla had distinct characters: 1) upper surface of snout was was not included. All phylogenetic trees obtained from the flat or even depressed; 2) snout was more than twice the analyses were visualized using Figtree v1.4.2 (Drummond diameter of the eye. Based on the results of measurements and Rambaut, 2007). Finally, the haplotype input file was of the scales as given in Table 2, the following results were prepared using the software DNAsp v5 (Librado and obtained: 1) Ahaetulla of Central Java and Lombok had Rozas, 2009), and the network was constructed by using fewer than 200 ventral scales (196 and 181) while Ahaetulla the software Network 5.0.1.0 (Fluxus-engineering, Leigh of East Java had more than 200 ventral scales (206); 2) the and Bryant, 2015). loreal scale of Ahaetulla of Central Java and Lombok was in contact with the internasal scale, while that of Ahaetulla 3. Results of East Java was not. For the number of subcaudal scales, 3.1. Morphology Ahaetulla of Lombok had 163 subcaudal scales, Ahaetulla The abbreviations used for the morphological characters of Central Java had 135, and Ahaetulla from East Java had are as follows: SVL: snout–vent length; TaL: tail length; 159. TL: total body length (SVL + TL); DSR: dorsal scale rows; The other morphological observations which are not VEN: ventral scale; SC: subcaudal scale. illustrated in Figure 2 and not mentioned in Table 2 are: 1) The measurement of morphological characters The anal plate had a single scale (anal entire) inAhaetulla was carried out for 3 Ahaetulla specimens, 1 Ahaetulla specimens from Central Java and Lombok, while the anal

Figure 2. Morphological comparison between the samples from 3 localities: A) Ahaetulla of East Java (EJ2), B) Ahaetulla of Central Java (CJ1), C) Ahaetulla of Lombok. These samples represent the 2Ahaetulla species of Indonesia: A. mycterizans and A. prasina.

Table 2. The morphological comparison betweenAhaetulla specimens identified in this study based on morphometry and meristic measurement. The morphometric measurements were taken in centimeters.

Specimen Locality Sex SVL TaL TL TaL/TL VEN DSR SC A. mycterizans Lombok F 64.8 55 119.8 0.327 181 15:15:13 163 A. mycterizans Central Java F 75 35 107 0.459 196 15:16:13 135 A. prasina East Java F 43.2 22.5 65.7 0.342 206 16:16:13 159

14 INDAHSARI et al. / Turk J Zool plate in the Ahaetulla specimen of East Java had paired several localities, as illustrated in Figure 3. Based on the scales (anal divided). 2) Ahaetulla of Central Java and estimated genetic distance data, there were 2 main clades, Lombok had a white line along the outer margin of the which strongly separated the 2 Ahaetulla species (ML/ ventrals, while Ahaetulla of East Java did not. BPP = 100/1). These clades areA. prasina of Sumatra, East 3.2. Genetic distance Java, Bandung (West Java), and Bali, and A. mycterizans of The genetic distance among species of the genusAhaetulla Central Java, Sukabumi (West Java), Banten, and Lombok. was calculated and shown as p-distance: the proportion In A. prasina, 2 subclades are present (ML/BPP = 99/1) of total nucleotide differences for each site (Lemey, 2009). consisting of the subclade of Sumatra, East Java, and Bali, The results of the p-distance estimations based on the and the subclade of West Java and Lampung. Within the 12S-rDNA sequences indicated that the samples of genus A. mycterizans clade, 2 subclades can be recognized (ML/ Ahaetulla in this study were divided into 2 groups with a BPP = 90/1), consisting of the Central Java and Sukabumi high pairwise genetic distance between them, as shown in (West Java) subclade, and the Lombok and Banten clade, Table 3. The first group consisted of specimens collected respectively. A. mycterizans of Lombok and Banten forms from Sumatra, Bandung, East Java, and Bali (WS1, WS2, a clade with a significant bootstrap (ML/BPP = 99/1), NS1, NS2, LP1, LP2, WJ1, EJ1, EJ2, BA1, BA2). Within although Lombok is located in Lesser Sunda and Banten this group, the estimated pairwise genetic distances ranged in Sundaland. between 0.00% and 2.11%. The second group included 3.4. Haplotype network specimens collected from Sumatra, Bandung, East Java, The resulting haplotype network was consistent with the and Bali (BN, LO, CJ1, CJ2, WJ2). The pairwise genetic estimated genetic distances and the phylogenetic trees distances between the members of the former group and reconstructed. There were 2 main groups, consisting of the latter group ranged from about 4.69% to 6.82%. A. prasina species of Sumatra, East Java, and Bali, and 3.3. Phylogenetic analyses A. mycterizans species of Banten, West Java, Central The ML and BI trees revealed identical topology for the Java, and Lombok, as shown in Figure 4. Nucleotide base clustering of the Ahaetulla specimens collected from differences among samples were written on each branch

Table3. The uncorrected p-distances (%) of the genus Ahaetulla based on the 12S-rDNA gene sequence from different localities of Sundaland and Lesser Sunda estimated by MEGA 7.0 (Kumar et al., 2016).

No Specimen 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

1 WS1 2 WS2 0.00 3 NS1 0.34 0.34 4 NS2 0.52 0.52 0.17 5 LP1 1.04 1.04 0.69 0.87 6 LP2 1.04 1.04 0.69 0.87 0.00 7 WJ1 1.22 1.22 0.87 1.04 0.17 0.17 8 EJ1 1.39 1.39 1.04 1.21 1.39 1.39 1.57 9 EJ2 1.39 1.39 1.57 1.74 1.92 1.92 2.10 0.87 10 BA1 1.57 1.57 1.75 1.93 2.11 2.11 2.29 1.39 0.87 11 BA2 1.93 1.93 1.57 1.75 1.93 1.93 2.11 1.22 1.75 0.87 12 BN 4.90 4.90 4.70 4.89 4.90 4.90 5.08 4.69 4.69 4.89 5.28 13 LO 5.07 5.07 4.88 5.07 5.07 5.07 5.26 4.87 4.87 5.07 5.46 0.52 14 CJ1 5.26 5.26 5.26 5.26 5.45 5.45 5.64 5.24 4.69 5.26 5.84 1.04 0.87 15 CJ2 5.47 5.47 5.28 5.28 5.47 5.47 5.66 5.64 6.04 6.25 6.24 1.22 1.40 1.75 16 WJ2 6.42 6.42 6.03 6.22 6.23 6.23 6.42 6.02 6.23 6.82 6.62 3.21 2.66 2.66 3.20 17 D. pictus 9.41 9.41 9.62 9.82 9.41 9.41 9.41 10.21 10.01 10.64 10.86 9.81 10.00 10.23 10.44 11.49 18 C. ornata 10.73 10.73 10.94 11.15 10.73 10.73 10.73 11.34 11.31 11.96 12.42 11.68 11.86 11.69 12.56 13.00 9.03 19 P. mu co s a 12.54 12.54 12.35 12.56 12.16 12.16 12.36 12.13 12.13 12.58 12.81 13.18 13.36 12.95 13.41 13.38 11.26 9.44

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Figure 3. The phylogenetic tree of the genusAhaetulla in Indonesia based on 12S-rDNA and sequences. The tree was reconstructed using ML and BI analyses. Node supports represent ML bootstrap value/Bayesian posterior probability (BPP). of the haplotype network. The smallest difference between mycterizans and D. xanthozona). These 4 diagnostic the samples of A. prasina and A. mycterizans in this characters are: the number of ventral scales (186–195); study is about 37 nucleotide sites, precisely between A. anal entire; snout being less than twice the diameter of mycterizans of Banten (BN) and A. mycterizans of Malang, eye (1.8× and 1.9×); a white line along the outer margin East Java (EJ2). This estimation was in accordance with of the ventrals. The morphological characters of Ahaetulla the p-distances presented in Table 2, which suggest the samples of East Java agreed with diagnostic characters of genetic distance between Ahaetulla species of Banten (BN) A. prasina described by Smith (1943) and De Rooij (1917) and Ahaetulla species of Malang (EJ2) to be around 4.69%. (De Rooij [1917] called the species Dryophis prasinus): That is the smallest genetic distance between the 2 distinct number of ventral scales 194–235 (206); anal divided; Ahaetulla species of Indonesia obtained in this study. snout more than twice the diameter of the eye; subcaudal 3.5 The divergence time of the genus Ahaetulla scales 151–207 (159). Furthermore, we observed that the As shown in Figure 5 and Table 4, the divergence time upper surface of the snout was convex in A. mycterizans estimates suggest that the ancestral node of Ahaetulla but flat or even depressed in A. prasina, as suggested by diverged from its sister group during the Eocene (around Miralles and David (2010). These diagnostic features 37.7 Ma) and that Ahaetulla started to diversify around the strengthened our decision that Ahaetulla samples of late Oligocene to middle Miocene (24.9–14.2 Ma). During Central Java and Lombok belonged to A. mycterizans, and the Miocene until the Early Pliocene (around 18.4–1.2 Ma), Ahaetulla samples of East Java belonged to A. prasina. the species of A. prasina and A. mycterizans in Sundaland When we analyzed the number of subcaudal scales, and Lesser Sunda started to diversify. A. mycterizans of Ahaetulla samples of Central Java were in agreement with Lombok started to diversify during the Miocene (around the diagnostic character of Smith (1943); i.e., about 132– 12.1 Ma). 156 (135) for A. mycterizans, while Ahaetulla samples of Lombok had scales exceeding this range: 163. However, 4. Discussion this result was not much different from the findings of Taking the studies by Smith (1943) and De Rooij (1917) Miralles and David (2010), who discovered A. mycterizans into consideration for identifying the species based on in Sumatra, which had 168 subcaudal scales. Based on morphological characters, the Ahaetulla samples collected these morphological characters of A. mycterizans and A. from Lombok and Central Java agreed with 4 diagnostic prasina species as described by Smith (1943), De Rooij characters of A. mycterizans described in these studies (1917), and Miralles and David (2010), we were able to (De Rooij [1917] referred to the species as Dryophis group our samples as either A. mycterizans or A. prasina.

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Figure 4. Haplotype network of the Ahaetulla samples of the study collected from Sundaland and Lesser Sunda based on 12S-rDNA sequences.

As a result, Ahaetulla samples of Central Java and Lombok distinct separation among them and resulted in 2 groups were grouped as A. mycterizans, while Ahaetulla samples based on the following phenotypic characters: 1) number of East Java were grouped as A. prasina. In order to test of ventar scales, 2) upper surface of snout, 3) attachment the validity of these morphological differences at the of loreal scales, 4) anal anatomy, and 5) presence of white species level, we used the results obtained from DNA line along the outer margin. sequence analysis for comparison. As a consequence, the Our phylogenetic analysis results indicated that there morphological characterization of the samples revealed a were 2 species of the genus Ahaetulla among the samples

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Figure 5. Divergence chronogram of the genus Ahaetulla of Indonesia based on 12S-rDNA sequences. The tree was reconstructed based on a relaxed normal clock and Bayesian inference with 95% credible interval. of this study. Thus, the morphological identification of Lombok. Referring to the results, it can be determined that these 2 separate species was confirmed by the genetic Clade I, consisting of Ahaetulla samples from Sumatra, distance estimates as well as the topology and branch Bandung, East Java, and Bali, belonged to A. prasina. supports of the phylogenetic tree reconstructed using all However, Clade II, consisting of Ahaetulla samples from of the Ahaetulla samples of the study. Jeong et al. (2013) Banten, Sukabumi, Central Java, and Lombok, belonged suggested that species having a genetic distance to A. mycterizans. Consequently, both morphological and of more than 3% in analyses based on the 12S-rDNA molecular analyses results have revealed that the Ahaetulla gene should be considered as a separate species. In this snake samples collected from Lombok representing the study, the pairwise genetic distance estimates between Lesser Sunda belonged to A. mycterizans species; they are the individuals of 2 clades (one clade is composed of the the first record from this island. Therefore, this study is the samples collected from Lombok, Banten, Central Java, and first record from the Lesser Sunda region. Sukabumi, and the other clade is composed of samples The small genetic distance found between A. collected from Sumatra, Bandung, East Java, and Bali) mycterizans of Lombok and Banten (12S-rDNA: 0.52%) were above 3% based on 12S-rDNA sequences. indicates that A. mycterizans of Lombok has the closest In summary, results from both the phylogenetic kinship relationship with A. mycterizans of Banten. The analyses and the morphological analyses indicated that data suggests that the A. mycterizans of Lombok originated the Ahaetulla samples of East Java belonged to a different from Sundaland. This inference, based on our results, can species then the Ahaetulla samples of Central Java and be made referring to the study of Inger (2005), which

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Table 4. The time of divergence of the genusAhaetulla with 95% the geographical history of the Lesser Sunda Islands. Most credible interval based on the divergence chronogram. islands of the Lesser Sunda are very young (1–15 Ma) and have never been connected to the Asian mainland. Clade Divergence Time (Ma) 95% CI (Ma) This fact may indicate that their fauna began to evolve independently (De Lang, 2011). However, looking at the 1 37.7 35.43–39.3048 genetic distance between A. mycterizans specimens of 2 32.0 24.2904–36.141 Lombok and Banten, which is relatively small (12S-rDNA: 3 24.9 14.9858–34.7936 0.52%), we have assumed that A. mycterizans in Lombok 4 22.8 21.1138–24.9582 originated from the Sundaland region. We suspect that there was a diversification of A. mycterizans of Banten to 5 18.4 9.5434–27.4696 Lombok in the past. The snake diversification from one 6 14.2 6.4204–21.6003 region to another can occur due to several factors: species 7 13.8 6.9508–20.6729 migration, human-mediated introduction, and oceanic 8 12.1 3.5962–17.435 rafting (Longrich at al., 2015; Reilly et al., 2019). 9 9.4 4.052–15.7216 We considered each of these factors separately. We rejected the first factor of species migration because 10 8.7 3.4716–13.6195 there is a geographic barrier between Lombok and the 11 7.3 3.0397–12.032 Sunda mainland. The depth of the Lombok Strait kept 12 5.5 1.6292–8.8856 Lombok and the Lesser Sunda Islands in isolation from 13 4.2 0.9979–6.4754 the Sundaland mainland, as illustrated in Figure 1. 14 3.1 0.5814–6.1188 Biologists believe it was the depth of the Lombok Strait 15 2.9 1.6145–4.4118 that isolated on either side of it (Lohman et al., 2011). We also rejected the second factor of human- 16 2.3 0.3498–4.6983 mediated introduction because the earliest sea crossing 17 1.8 0.4383–2.834 by seafarers occurred in the early Pleistocene (Bednarik, 18 1.1 0.0806–2.4333 2007). Therefore, we conclude that the presence ofA. mycterizans in Lombok could be due to oceanic rafting. This is consistent with the study by Longrich et al. (2015), proposed that fauna in Wallace’s line, including the Lesser who concluded that the distribution of which had Sunda region, originated from the Sundaland region. been blocked by geographical barriers was carried out The divergence time estimates indicated that the through oceanic rafting. Similarly, Inger (2005) indicated that beyond Wallace’s Line were mainly derived genus Ahaetulla started to diverge as A. prasina and A. from Sundaland lineages via oceanic rafting. Our results mycterizans in the late Oligocene, approximately 24.9 Ma. were also in agreement with those of Reilly et al. (2019), Our results were in agreement with those of Nugraha et al. who carried out a comparative genetic analysis between (2018) that the subfamily Ahaetullinae diversified during reptiles from Lesser Sunda and Sundaland. In that study, the late Oligocene, at about 24 Ma. The late Oligocene was the authors showed that there was no significant difference the period when Sunda Shelf lakes were connected to the in genetic distance between reptiles of those archipelagos. sea and were brackish, which may have facilitated many Thus, the presence of reptiles in Lesser Sunda may be migrations of terrestrial fauna (Hall, 2013). The estimates caused by human-mediated introduction, or the reptiles by Alencar et al. (2016) suggest that a high speciation entered the archipelago relatively recently. rate occurred in the region during the Oligocene and The new record of A. mycterizans in Lombok increases Miocene. These time periods were characterized by several the number of snake species that inhabit the Lesser Sunda geographical events that occurred in Asia; in fact, species of Islands, which was reported as 29 in a previous study (De Ahaetulla are thought to have originated on this continent. Lang, 2011). This discovery also added to the distribution Our estimates indicated that the diversification ofA. area of A. mycterizans, which previously had only been prasina in the Sundaland region mostly occurred during recorded from Java, Sumatra, Malaysia, and Thailand the Pleistocene (<10 Ma). This period is known as the Ice (Sundaland region). This suggests that A. mycterizans Age, a serious cooling of the planet, which caused the sea may also be discovered in other parts of the Lesser Sunda level to significantly drop and increased the connectivity region, which are still incompletely surveyed. of landmasses (Supsup et al., 2016). Our molecular results also added evidence that The divergence time estimation based on our results also molecular analyses with mitochondrial DNA can be used suggests that A. mycterizans of Lombok started to diversify as a valid method for identifying species. Although in around the Miocene (12.4 Ma). This is in accordance with this study we did not analyze nuclear DNA, a combined

19 INDAHSARI et al. / Turk J Zool analysis of nuclear and mitochondrial DNA for species 2000005071). We thank A. M. Kadafi, B. Priambodo, and identification should provide appropriate and valid results M. Fathoni for collecting all specimens used in this study, for classifying species (Figueroa et al., 2016). D. R. Tirtosari and Y. Bare for molecular analyses, M. Fahmi for phylogenetic analyses, and F. A. D. Nugraha and Acknowledgments A. Maulidi for sharing discussions. Field sampling was funded by the NSF for Eric N. Smith (NSF Award Number: DEB-1146324) and a PEER Science Conflict of interest USAID grant to Nia Kurniawan (subgrant number: The authors declare no conflict of interest.

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