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http://journals.tubitak.gov.tr/zoology/

Turkish Journal of Zoology Turk J Zool(2020) 44: 11-21© TÜBİTAKdoi:10.3906/zoo-1904-1

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

Lilin Ika Nur INDAHSARI1,2, Fatchiyah FATCHIYAH1,3

, Eric Nelson SMITH4, Nia KURNIAWAN1,2,*

1Department of Biology, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, Indonesia2NK Research, Department of Biology, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, Indonesia

3Research Center of Smart Molecule of Natural Genetic Resources, Brawijaya University, Malang, Indonesia4Department of Biology, University of Texas at Arlington, Arlington, TX, USA

* Correspondence: wawan@ub.ac.id

1. IntroductionThe arboreal snake genus Ahaetulla (Link, 1807) is composed of 8 species distributed in most countries of the Asian region, including Indonesia, Malaysia, Thailand, Myanmar, India, Sri Lanka, and throughout the Indochinese Peninsula (Das, 2015; Mohapatra et al., 2017). All species of this genus are characterized by an elongated triangular head, an extremely thin and elongate body, and horizontally-shaped pupils (Long et al., 2012; Das, 2015; Lee at al., 2015). Previously, 3 species have been recorded in Indonesia: (i) Ahaetulla prasina (Boie, 1827) (Oriental Vine Snake), (ii) Ahaetulla mycterizans (Linnaeus, 1758) (Malayan Vine Snake), and (iii) Ahaetulla fasciolata (Fischer, 1885) (Speckle-headed Vine Snake). A. prasina is present in much of the Sundaland region: Sumatra, Borneo (Kalimantan), Java, Bali, and Sulawesi (Das, 2015; Leo, 2015; Ekarini, 2016). Furthermore, De Lang (2011) reported the occurrence of A. prasina in the Lesser Sunda region, more specifically in Lombok and Sumbawa. Miralles and David (2010) reported the first finding of

A. mycterizans in Sumatra. A. mycterizans has also been discovered in Java by De Rooij (1917) and Ekarini (2016). A. fasciolata has been found only in Sumatra (Das, 2015). However, there has been no previous record about the existence of A. mycterizans in the Lesser Sunda region.

De Lang (2011) reported 29 species of terrestrial and semiaquatic snakes from the Lesser Sunda Islands, but he found only one species of the genus Ahaetulla, i.e., A. prasina. Due to the discovery of A. mycterizans in Peninsular Malaysia, A. mycterizans has been recorded much farther north than its previously known distribution range (Mirales and David, 2010). This discovery, as well as the distribution of A. prasina in this region, suggested the possibility of the existence of A. mycterizans in the Lesser Sunda Islands. Unfortunately, all of those studies on the distribution of the genus Ahaetulla in Indonesia were carried out solely on the basis of morphological characters that are commonly used as the main diagnostic characters for species identification. There have been no previous studies that have used DNA sequence analysis

Abstract: Previous studies on the distribution of the Ahaetulla snake across Indonesia only focused on morphological characters without any molecular data. This study was aimed at analyzing phylogenetic relationships among the genus 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 species, 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 of A. mycterizans in Lesser Sunda had occurred during the Miocene, around 12.1 Ma. Consequently, this study has added A. mycterizans to the list of snakes 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 Asia.

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

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

Research Article

This work is licensed under a Creative Commons Attribution 4.0 International License.

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to identify Ahaetulla species in Indonesia. Furthermore, species identification by morphological characters may lead to misidentifications, especially between 2 of the Ahaetulla species, A. prasina and A. mycterizans, due to shared similar morphology, such as body color, shape, and even head shape (Miralles and David, 2010; Ekarini, 2016; Lok et al., 2016). A. prasina, is much more widespread in Indonesia than A. mycterizans, which remains poorly known. Along with morphology, molecular data is needed to distinguish between these 2 species accurately and avoid misidentification. Figueroa et al. (2016) used mitochondrial DNA including the 12S-rDNA gene as barcodes to determine the phylogenetic relationship among 1592 snakes of the Colubridae family. Furthermore, the phylogenetic analysis of the snake species of the family Colubridae has not only contributed to the evaluation of snake diversity, but also to the evaluation of geological history through studies on estimated genetic divergence time (Takeuchi et al., 2011; Nugraha et al., 2018).

This is the first study of the genus Ahaetulla in Sundaland and the Lesser Sunda region using the 12S-rDNA gene to analyze phylogenetic relationships, to construct the haplotype network, and to estimate the time of divergence. We also analyzed morphological characters to confirm the molecular identification.

2. Materials and methods2.1. Specimens collection and ethical clearanceAhaetulla specimens were obtained from several localities in Sumatra, Java, Bali, and Lombok Island as shown in Figure 1. A total of 16 specimens consisting of 6 specimens from Sumatra, 7 specimens from Java, 2 specimens from Bali and, 1 specimen from Lombok, were collected. Additional sequences were retrieved from GenBank (www.ncbi.nih.gov.nlm) including Chrysopelea ornata, Dendrelaphis pictus, and Ptyas mucosa species, as shown in Table 1. To collect our specimens and take ventral muscle 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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Ethics Committee of Brawijaya University in Ethical Clearance No. 1081-KEP-UB.2.2. Morphological identificationDorsal scale rows were counted at 3 places—i.e., at 1 head-length behind the head, at midbody, and at 1 head-length anterior to the cloacal plate. Ventral and subcaudal scales were counted according to Dowling (1951). Measurements, except for body and tail lengths, were taken with a slide-caliper to the nearest 0.1 mm. All body measurements were made to the nearest millimeter. Drawings were made using Corel Draw and Adobe® Photoshop software. 2.2. DNA extraction, amplification, and sequencingGenomic DNA was extracted from ventral muscle tissue stored in 96% ethanol using a Geneaid DNA isolation kit following the manufacturer’s protocol. The fragment of 12S-rDNA was amplified by polymerase chain reaction (PCR) using 12S268 (5’-GTGCCAGCGACCGCGGTTACACG-3’) and 12S916

(5’-GTACGCTTACCATGTTACGACTTGCCCTG-3’) (Jeong et al., 2013) primers. All 16 samples were sequenced for the 12S rDNA fragment. The PCR amplification for 12S-rDNA was conducted in 50-μL reaction volumes with 5 min denaturation at 94 °C, followed by 35 cycles at 94 °C for 30 s, 56.5 °C for 1 min, and 72 °C for 1 min, and then a final elongation step at 72 °C for 10 min. The PCR products were sequenced using the above PCR primers by Indoseq GATC, Biosains Institute, Brawijaya University.2.3. Sequence analyses12S-rDNA sequences were aligned by using the Clustal W (Hall, 2013) application implemented in MEGA 7.0 software (Kumar et al., 2016). The alignment results were edited manually to trim the ambiguous sites. The alignment length of the 12S-rDNA sequences was 600 bp. The maximum likelihood (ML) tree was reconstructed using RAxML software (Stamatakis, 2006) with the bootstrap option. Bayesian inference (BI) was analyzed

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.

Sample name Specimens Locality Genbank Accession no. Source

SUMATRANS1 A. prasina Medan, North Sumatra MK691461 This studyNS A. prasina Rau, North Sumatra MK691470 This studyWS1 A. prasina Bukittinggi, West Sumatra MK691464 This studyWS2 A. prasina Andalas, West Sumatra MK691460 This studyLP1 A. prasina Rajabasa, Lampung MK691469 This studyLP2 A. prasina Tanggamus, Lampung MK691474 This studyJAVAWJ1 A. prasina Bandung, West Java MK691462 This studyEJ1 A. prasina Banyuwangi, East Java MK691466 This studyEJ2 A. prasina Malang, East Java MK691468 This studyBA1 A. prasina Sukawati, Bali MK691472 This studyBA2 A. prasina Tanah Lot, Bali MK691473 This studyBN A. mycterizans Pandeglang, Banten MK691463 This studyWJ2 A. mycterizans Sukabumi, West Java MK691471 This studyCJ1 A. mycterizans Nusakambangan, Central Java MK691465 This studyCJ2 A. mycterizans Tanjungharjo, Central Java MK691475 This studyLOMBOKLO A. mycterizans Lombok, West Nusa Tenggara MK691467 This studyOTHER 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)

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using MrBayes software (Ronquist and Huelsenbeck, 2003) using Markov Chain Monte Carlo (MCMC) estimated for 5 million generations. The most appropriate sequence evolution models were selected using Akaike Information Criterion (AIC) for ML and BI. The divergence time was estimated by using BEAST (Drummond et al., 2012) software under the Hasegawa–Kishino–Yano model of DNA evolution with the uncorrelated log-normal relaxed clock rate model (Drummond et al., 2006). The estimated time divergence between C. ornata and P. mucosa at 23.1 Mya and between P. mucosa and D. pictus at 37.6 Mya (Nugraha et al., 2018) were used for the external calibration. The estimated time divergence of West Java and Lampung separation was employed for the internal calibration. There has been no previous study on the estimated divergence time for Ahaetulla; thus, internal calibration for Ahaetulla was not included. All phylogenetic trees obtained from the analyses were visualized using Figtree v1.4.2 (Drummond and Rambaut, 2007). Finally, the haplotype input file was prepared using the software DNAsp v5 (Librado and Rozas, 2009), and the network was constructed by using the software Network 5.0.1.0 (Fluxus-engineering, Leigh and Bryant, 2015).

3. Results3.1. MorphologyThe abbreviations used for the morphological characters are as follows: SVL: snout–vent length; TaL: tail length; TL: total body length (SVL + TL); DSR: dorsal scale rows; VEN: ventral scale; SC: subcaudal scale.

The measurement of morphological characters was carried out for 3 Ahaetulla specimens, 1 Ahaetulla

specimen of Lombok Island representing Lesser Sunda, and 2 Ahaetulla specimens of Java (Central and East Java) representing Sundaland. Based on morphological identification, there were 2 distinct morphological separations of Ahaetulla snakes found in this study. The results of morphological identification are presented in Figure 2 and Table 2.

Based on the morphology of the head (see Figure 2), there were similarities between Ahaetulla of Central Java and Lombok; they both differed from Ahaetulla of East Java. Ahaetulla of Central Java and Lombok have the following characters: 1) upper surface of snout was convex; 2) snout was less than twice the diameter of the eye (1.8× for Ahaetulla of central Java, 1.9× for Ahaetulla of Lombok). However, the specimens from East Java had distinct characters: 1) upper surface of snout was flat or even depressed; 2) snout was more than twice the diameter of the eye. Based on the results of measurements of the scales as given in Table 2, the following results were obtained: 1) Ahaetulla of Central Java and Lombok had fewer than 200 ventral scales (196 and 181) while Ahaetulla of East Java had more than 200 ventral scales (206); 2) the loreal scale of Ahaetulla of Central Java and Lombok was in contact with the internasal scale, while that of Ahaetulla of East Java was not. For the number of subcaudal scales, Ahaetulla of Lombok had 163 subcaudal scales, Ahaetulla of Central Java had 135, and Ahaetulla from East Java had 159.

The other morphological observations which are not illustrated in Figure 2 and not mentioned in Table 2 are: 1) The anal plate had a single scale (anal entire) in 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 2 Ahaetulla species of Indonesia: A. mycterizans and A. prasina.

Table 2. The morphological comparison between Ahaetulla 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 163A. mycterizans Central Java F 75 35 107 0.459 196 15:16:13 135A. prasina East Java F 43.2 22.5 65.7 0.342 206 16:16:13 159

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plate in the Ahaetulla specimen of East Java had paired scales (anal divided). 2) Ahaetulla of Central Java and Lombok had a white line along the outer margin of the ventrals, while Ahaetulla of East Java did not.3.2. Genetic distanceThe genetic distance among species of the genus Ahaetulla was calculated and shown as p-distance: the proportion of total nucleotide differences for each site (Lemey, 2009). The results of the p-distance estimations based on the 12S-rDNA sequences indicated that the samples of genus Ahaetulla in this study were divided into 2 groups with a high pairwise genetic distance between them, as shown in Table 3. The first group consisted of specimens collected from Sumatra, Bandung, East Java, and Bali (WS1, WS2, NS1, NS2, LP1, LP2, WJ1, EJ1, EJ2, BA1, BA2). Within this group, the estimated pairwise genetic distances ranged between 0.00% and 2.11%. The second group included specimens collected from Sumatra, Bandung, East Java, and Bali (BN, LO, CJ1, CJ2, WJ2). The pairwise genetic distances between the members of the former group and the latter group ranged from about 4.69% to 6.82%.3.3. Phylogenetic analyses The ML and BI trees revealed identical topology for the clustering of the Ahaetulla specimens collected from

several localities, as illustrated in Figure 3. Based on the estimated genetic distance data, there were 2 main clades, which strongly separated the 2 Ahaetulla species (ML/BPP = 100/1). These clades are A. prasina of Sumatra, East Java, Bandung (West Java), and Bali, and A. mycterizans of Central Java, Sukabumi (West Java), Banten, and Lombok. In A. prasina, 2 subclades are present (ML/BPP = 99/1) consisting of the subclade of Sumatra, East Java, and Bali, and the subclade of West Java and Lampung. Within the A. mycterizans clade, 2 subclades can be recognized (ML/BPP = 90/1), consisting of the Central Java and Sukabumi (West Java) subclade, and the Lombok and Banten clade, respectively. A. mycterizans of Lombok and Banten forms a clade with a significant bootstrap (ML/BPP = 99/1), although Lombok is located in Lesser Sunda and Banten in Sundaland.3.4. Haplotype networkThe resulting haplotype network was consistent with the estimated genetic distances and the phylogenetic trees reconstructed. There were 2 main groups, consisting of A. prasina species of Sumatra, East Java, and Bali, and A. mycterizans species of Banten, West Java, Central Java, and Lombok, as shown in Figure 4. Nucleotide base 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. mucosa 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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of the haplotype network. The smallest difference between the samples of A. prasina and A. mycterizans in this study is about 37 nucleotide sites, precisely between A. mycterizans of Banten (BN) and A. mycterizans of Malang, East Java (EJ2). This estimation was in accordance with the p-distances presented in Table 2, which suggest the genetic distance between Ahaetulla species of Banten (BN) and Ahaetulla species of Malang (EJ2) to be around 4.69%. That is the smallest genetic distance between the 2 distinct Ahaetulla species of Indonesia obtained in this study. 3.5 The divergence time of the genus AhaetullaAs shown in Figure 5 and Table 4, the divergence time estimates suggest that the ancestral node of Ahaetulla diverged from its sister group during the Eocene (around 37.7 Ma) and that Ahaetulla started to diversify around the late Oligocene to middle Miocene (24.9–14.2 Ma). During the Miocene until the Early Pliocene (around 18.4–1.2 Ma), the species of A. prasina and A. mycterizans in Sundaland and Lesser Sunda started to diversify. A. mycterizans of Lombok started to diversify during the Miocene (around 12.1 Ma).

4. DiscussionTaking the studies by Smith (1943) and De Rooij (1917) into consideration for identifying the species based on morphological characters, the Ahaetulla samples collected from Lombok and Central Java agreed with 4 diagnostic characters of A. mycterizans described in these studies (De Rooij [1917] referred to the species as Dryophis

mycterizans and D. xanthozona). These 4 diagnostic characters are: the number of ventral scales (186–195); anal entire; snout being less than twice the diameter of eye (1.8× and 1.9×); a white line along the outer margin of the ventrals. The morphological characters of Ahaetulla samples of East Java agreed with diagnostic characters of A. prasina described by Smith (1943) and De Rooij (1917) (De Rooij [1917] called the species Dryophis prasinus): number of ventral scales 194–235 (206); anal divided; snout more than twice the diameter of the eye; subcaudal scales 151–207 (159). Furthermore, we observed that the upper surface of the snout was convex in A. mycterizans but flat or even depressed in A. prasina, as suggested by Miralles and David (2010). These diagnostic features strengthened our decision that Ahaetulla samples of Central Java and Lombok belonged to A. mycterizans, and Ahaetulla samples of East Java belonged to A. prasina.

When we analyzed the number of subcaudal scales, Ahaetulla samples of Central Java were in agreement with the diagnostic character of Smith (1943); i.e., about 132–156 (135) for A. mycterizans, while Ahaetulla samples of Lombok had scales exceeding this range: 163. However, this result was not much different from the findings of Miralles and David (2010), who discovered A. mycterizans in Sumatra, which had 168 subcaudal scales. Based on these morphological characters of A. mycterizans and A. prasina species as described by Smith (1943), De Rooij (1917), and Miralles and David (2010), we were able to group our samples as either A. mycterizans or A. prasina.

Figure 3. The phylogenetic tree of the genus Ahaetulla 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).

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As a result, Ahaetulla samples of Central Java and Lombok were grouped as A. mycterizans, while Ahaetulla samples of East Java were grouped as A. prasina. In order to test the validity of these morphological differences at the species level, we used the results obtained from DNA sequence analysis for comparison. As a consequence, the morphological characterization of the samples revealed a

distinct separation among them and resulted in 2 groups based on the following phenotypic characters: 1) number of ventar scales, 2) upper surface of snout, 3) attachment of loreal scales, 4) anal anatomy, and 5) presence of white line along the outer margin.

Our phylogenetic analysis results indicated that there were 2 species of the genus Ahaetulla among the samples

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

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of this study. Thus, the morphological identification of these 2 separate species was confirmed by the genetic distance estimates as well as the topology and branch supports of the phylogenetic tree reconstructed using all of the Ahaetulla samples of the study. Jeong et al. (2013) suggested that reptile species having a genetic distance of more than 3% in analyses based on the 12S-rDNA gene should be considered as a separate species. In this study, the pairwise genetic distance estimates between the individuals of 2 clades (one clade is composed of the samples collected from Lombok, Banten, Central Java, and Sukabumi, and the other clade is composed of samples collected from Sumatra, Bandung, East Java, and Bali) were above 3% based on 12S-rDNA sequences.

In summary, results from both the phylogenetic analyses and the morphological analyses indicated that the Ahaetulla samples of East Java belonged to a different species then the Ahaetulla samples of Central Java and

Lombok. Referring to the results, it can be determined that Clade I, consisting of Ahaetulla samples from Sumatra, Bandung, East Java, and Bali, belonged to A. prasina. However, Clade II, consisting of Ahaetulla samples from Banten, Sukabumi, Central Java, and Lombok, belonged to A. mycterizans. Consequently, both morphological and molecular analyses results have revealed that the Ahaetulla snake samples collected from Lombok representing the Lesser Sunda belonged to A. mycterizans species; they are the first record from this island. Therefore, this study is the first record from the Lesser Sunda region.

The small genetic distance found between A. mycterizans of Lombok and Banten (12S-rDNA: 0.52%) indicates that A. mycterizans of Lombok has the closest kinship relationship with A. mycterizans of Banten. The data suggests that the A. mycterizans of Lombok originated from Sundaland. This inference, based on our results, can be made referring to the study of Inger (2005), which

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.

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proposed that fauna in Wallace’s line, including the Lesser Sunda region, originated from the Sundaland region.

The divergence time estimates indicated that the genus Ahaetulla started to diverge as A. prasina and A. mycterizans in the late Oligocene, approximately 24.9 Ma. Our results were in agreement with those of Nugraha et al. (2018) that the subfamily Ahaetullinae diversified during the late Oligocene, at about 24 Ma. The late Oligocene was the period when Sunda Shelf lakes were connected to the sea and were brackish, which may have facilitated many migrations of terrestrial fauna (Hall, 2013). The estimates by Alencar et al. (2016) suggest that a high speciation rate occurred in the region during the Oligocene and Miocene. These time periods were characterized by several geographical events that occurred in Asia; in fact, species of Ahaetulla are thought to have originated on this continent. Our estimates indicated that the diversification of A. prasina in the Sundaland region mostly occurred during the Pleistocene (<10 Ma). This period is known as the Ice Age, a serious cooling of the planet, which caused the sea level to significantly drop and increased the connectivity of landmasses (Supsup et al., 2016).

The divergence time estimation based on our results also suggests that A. mycterizans of Lombok started to diversify around the Miocene (12.4 Ma). This is in accordance with

the geographical history of the Lesser Sunda Islands. Most islands of the Lesser Sunda are very young (1–15 Ma) and have never been connected to the Asian mainland. This fact may indicate that their fauna began to evolve independently (De Lang, 2011). However, looking at the genetic distance between A. mycterizans specimens of Lombok and Banten, which is relatively small (12S-rDNA: 0.52%), we have assumed that A. mycterizans in Lombok originated from the Sundaland region. We suspect that there was a diversification of A. mycterizans of Banten to Lombok in the past. The snake diversification from one region to another can occur due to several factors: species migration, human-mediated introduction, and oceanic rafting (Longrich at al., 2015; Reilly et al., 2019).

We considered each of these factors separately. We rejected the first factor of species migration because there is a geographic barrier between Lombok and the Sunda mainland. The depth of the Lombok Strait kept Lombok and the Lesser Sunda Islands in isolation from the Sundaland mainland, as illustrated in Figure 1. Biologists believe it was the depth of the Lombok Strait that isolated animals on either side of it (Lohman et al., 2011). We also rejected the second factor of human-mediated introduction because the earliest sea crossing by seafarers occurred in the early Pleistocene (Bednarik, 2007). Therefore, we conclude that the presence of A. mycterizans in Lombok could be due to oceanic rafting. This is consistent with the study by Longrich et al. (2015), who concluded that the distribution of reptiles which had been blocked by geographical barriers was carried out through oceanic rafting. Similarly, Inger (2005) indicated that frogs beyond Wallace’s Line were mainly derived from Sundaland lineages via oceanic rafting. Our results were also in agreement with those of Reilly et al. (2019), who carried out a comparative genetic analysis between reptiles from Lesser Sunda and Sundaland. In that study, the authors showed that there was no significant difference in genetic distance between reptiles of those archipelagos. Thus, the presence of reptiles in Lesser Sunda may be caused by human-mediated introduction, or the reptiles entered the archipelago relatively recently.

The new record of A. mycterizans in Lombok increases the number of snake species that inhabit the Lesser Sunda Islands, which was reported as 29 in a previous study (De Lang, 2011). This discovery also added to the distribution area of A. mycterizans, which previously had only been recorded from Java, Sumatra, Malaysia, and Thailand (Sundaland region). This suggests that A. mycterizans may also be discovered in other parts of the Lesser Sunda region, which are still incompletely surveyed.

Our molecular results also added evidence that molecular analyses with mitochondrial DNA can be used as a valid method for identifying species. Although in this study we did not analyze nuclear DNA, a combined

Table 4. The time of divergence of the genus Ahaetulla with 95% credible interval based on the divergence chronogram.

Clade Divergence Time (Ma) 95% CI (Ma)

1 37.7 35.43–39.30482 32.0 24.2904–36.1413 24.9 14.9858–34.79364 22.8 21.1138–24.95825 18.4 9.5434–27.46966 14.2 6.4204–21.60037 13.8 6.9508–20.67298 12.1 3.5962–17.4359 9.4 4.052–15.721610 8.7 3.4716–13.619511 7.3 3.0397–12.03212 5.5 1.6292–8.885613 4.2 0.9979–6.475414 3.1 0.5814–6.118815 2.9 1.6145–4.411816 2.3 0.3498–4.698317 1.8 0.4383–2.83418 1.1 0.0806–2.4333

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analysis of nuclear and mitochondrial DNA for species identification should provide appropriate and valid results for classifying species (Figueroa et al., 2016).

AcknowledgmentsField sampling was funded by the NSF for Eric N. Smith (NSF Award Number: DEB-1146324) and a PEER Science USAID grant to Nia Kurniawan (subgrant number:

2000005071). We thank A. M. Kadafi, B. Priambodo, and M. Fathoni for collecting all specimens used in this study, D. R. Tirtosari and Y. Bare for molecular analyses, M. Fahmi for phylogenetic analyses, and F. A. D. Nugraha and A. Maulidi for sharing discussions.

Conflict of interestThe authors declare no conflict of interest.

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