A New Subspecies Identification and Population Study of the Asian Small-Clawed Otter (Aonyx cinereus) in Malay Peninsula and Southern Thailand Based on Fecal DNA Method

Three species of otter can be found throughout Malay Peninsula: Aonyx cinereus, Lutra sumatrana, and Lutrogale perspicillata. In this study, we focused on the A. cinereus population that ranges from the southern and the east coast to the northern regions of Malay Peninsula up to southern Thailand to review the relationships between the populations based on the mitochondrial D-loop region. Forty-eight samples from six populations were recognized as Johor, Perak, Terengganu, Kelantan, Ranong, and Thale Noi. Among the 48 samples, 33 were identified as A. cinereus, seven as L. sumatrana, and eight as L. perspicillata. Phylogenetically, two subclades formed for A. cinereus. The first subclade grouped all Malay Peninsula samples except for samples from Kelantan, and the second subclade grouped Kelantan samples with Thai sample. Genetic distance analysis supported the close relationships between Thai and Kelantan samples compared to the samples from Terengganu and the other Malaysian states. A minimum-spanning network showed that Kelantan and Thailand formed a haplogroup distinct from the other populations. Our results show that Thai subspecies A. cinereus may have migrated to Kelantan from Thai mainland. We also suggest the classification of a new subspecies from Malay Peninsula, the small-clawed otter named A. cinereus kecilensis.


Introduction
Several methods for identifying species rely on DNA sequence analysis. Among them are RFLPs, AFLPs, RAPD, polymerase chain reaction, microarray, and DNA sequencing [1][2][3][4]. DNA-based methods are often used due to the reliability of DNA sequences, with the general assumption that individuals from the same species carry specific DNA (or protein) sequences that differ from those found in individuals from other species [5]. DNA is a strong tool in forensic analysis because DNA is extremely stable and lives long as a biological molecule that can be recovered from several types of forensic evidence such as bloodstains, feces, saliva, urine, and commercial products [6,7].
DNA extraction from feces is among the most complicated and high-risk processes of noninvasive samples. After DNA has been extracted, problems are often encountered in terms of relatively low DNA yields and/or recovering DNA free of inhibitory substances [8]. These issues make fecal sampling less popular among geneticists. However, fecal samples are important in studies that require DNA sources from risky or highly endangered subjects before samples are the Philippines, Taiwan, and eastern and southern China in the north [11]. However, this small species is possibly extinct in Hong Kong and Singapore [28,29]. A. cinereus inhabits coastal habitats and inland rivers, swamps, mangroves, and paddy fields up to 2000 m above sea level [30,31]. This species often inhabits areas close to human activity [31]. A. cinereus coexists with L. sumatrana and L. perspicillata in many locations, including several rivers in Thailand and Malaysia [32]. Although all three species feed on the same prey, most A. cinereus members are crab eaters, while most of the other species feed on fish [33]. Coexistence is controlled by the selection of different habitats and food. In Southeast Asia, A. cinereus is abundant in irrigation canals and rice fields where L. sumatrana is not present [16]. On Malay Peninsula, A. cinereus is limited to small rivers and irrigation canals [34].
Geographic range and population studies are important to obtain information for requirements concerning ecology and conservation efforts to preserve species threatened with extinction [35]. In this phylogeography study, we focused on the A. cinereus population ranging from the southern and eastern coast to northern regions of Malay Peninsula up to southern Thailand to review the knowledge about the relationship between populations of this species using genetic methods. Species identified from fecal samples help us determine the species in sampling locations and the coexistence of multiple species. Our region of interest was the mitochondrial DNA, D-loop control region. The mitochondrial genome has been extensively used to amplify many genes of interest for phylogenetic studies [36][37][38][39]. Sequence divergence accumulates more rapidly in mitochondrial DNA (mtDNA) than in nuclear DNA due to the faster mutation rate and lack of repair system in mtDNA, which means that it often contains high levels of information variation [40]. The D-loop is a highly variable noncoding control region that has the highest polymorphism rate among mitochondrial genes and is widely used in genetic population studies [9,[41][42][43]].  Hap 5 Penarik 5 P e n a r i k T e r e n g g a n u , M a l a y s i a A. cinereus

Materials and Methods
Hap 5 Penarik 1 P e n a r i k T e r e n g g a n u , M a l a y s i a A. cinereus Hap   . PCR conditions were as follows: 4 min denaturation at 94 ∘ C, followed by 30 cycles of 30 sec at 94 ∘ C, 30 sec at 56 ∘ C, 1 min at 72 ∘ C, and a final 7 min extension at 72 ∘ C, before cooling to 4 ∘ C for 10 min. DNA from PCR products was purified using the Vivantis G-F1 PCR Clean-up Kit and was sent directly to the sequencing service company, First Base Sdn. Bhd., to be sequenced.

Species Identification and Phylogenetic Analysis.
Sequencing results were exported as FASTA sequence files. Sequences from GenBank were obtained as positive controls for each species before the species was identified. The D-loop sequences of the studied samples were aligned using the ClustalW multiple alignment algorithm of BioEdit, together with control sequences from GenBank and an outgroup sequence of the palm civet. Identification at the species level was detected by informative polymorphic sites assigned to each species. All sequences were analyzed using PAUP 4.0b10 and MrBayes 3.1 for phylogeny reconstruction. Two methods of analysis in PAUP included the following: (1) neighbor-joining (NJ) with Kimura 2 Parameter [46], which takes into account the unequal rates of evolution of transition and transversion but assumes an equal distribution of nucleotide composition and (2) Maximum Parsimony (MP) with stepwise addition (1000 replicates) in heuristic search [47] and 50% majority rule consensus. In Maximum Parsimony (MP), gaps are treated as missing data, transitions and transversions are weighted equally, and heuristic search is performed with the TBR branch-swapping algorithm. All trees were subjected to bootstrap analysis with 1000 replicates to get bootstrap value support.

Genetic Structure of A. cinereus .
Measures of population genetic parameters such as genetic diversity, nucleotide diversity, and nucleotide divergence among populations after nucleotide diversity was accounted for within populations ( ) were estimated from the mtDNA dataset using DNASP 4.0 [48]. The demographic history was examined with Tajima's test of neutrality, test [49], and Fu's statistics test [50], to test for deviation in sequence variation from evolutionary neutrality. The tests compare the number of singleton mutations to the total number ( ) and the average number of nucleotide differences between pairs of sequences ( ), both under a neutral model [51].
Population bottlenecks and expansions, selective sweeps on the mtDNA, and mutational rate heterogeneity may all result in a Poisson distribution of substitution differences  between pairs of haplotypes [52]. Therefore, mismatch distribution analysis was performed using Arlequin version 3.0 with 1000 permutations [53] and site-frequency spectra [54] as implemented in DNASP 4.0.
The population genetic structure was analyzed for samples with five or more individuals, with an analogue of Fst, Ost, as implemented in an analysis of molecular variance AMOVA [55] in Arlequin 3.0. The statistical significance was tested using 1000 permutations. The parsimony criterion was used to reconstruct the haplotype relationships of A. cinereus, assuming that differences at any given site between two randomly drawn haplotypes were unlikely to have arisen from more than 1 mutational step [56]. A minimum-spanning network was generated using Network 4.5.0.2 [57] to illustrate this relationship.

Results
DNA from all 48 fecal samples was successfully extracted and sequenced. The partial D-loop sequences (398 bp) were converted into the FASTA format and were aligned with our control sequences of the three species L. perspicillata, A. cinereus, and L. sumatrana to identify species based on site polymorphisms. Based on the primers used in this study, several specific polymorphism sites were detected for each species including deletions and an insertion. Figure 2 shows the informative polymorphic sites for all three species used for species identification in this study. Thirty informative sites were observed from the 398 bp sequence data set of the three species. Deletions and an insertion occurred specifically in the L. sumatrana species: deletions at site numbers 46-50 (134-  The nucleotide diversity (t) among the populations was low, ranging from 0.1% to 0.6% ( Table 2). The highest (t) value was between Johor and Ranong (0.6%), while the lowest (t) value was between Kelantan and Thale Noi (0.13%). The net nucleotide divergence ( ) among the populations was also low, ranging from 0.1% to 0.9%. The highest value was between Johor and Kelantan populations (0.91%), while the lowest value was between Kelantan and Thale Noi populations (0.13%).
Phylogenetically, the NJ and MP trees shared the same tree topologies (Figures 3 and 4). From both trees, all otter species were grouped together distinct from the outgroup sample of the Eurasian otter (GenBank). In the main clade (clade A), the A. cinereus samples were grouped together in another clade (clade B) distinct from the outgroup samples of L. perspicillata (clade C). In the major clade of A. cinereus samples were two other clades (clade D and clade E) with 96% bootstrap support. CladeD consisted of samples from Kelantan (Kg Padang Salim and Tumpat) and Thailand (Ranong and Thale Noi). Samples from the two Kelantan populations formed a monophyletic clade distinct from the monophyletic clade of Ranong and Thale Noi. However, the clustering of each group was not supported by high bootstrap values (<70%). In clade E, two subclades formed, also with low bootstrap values. The first subclade consisted of samples from Terengganu (Penarik), and the second subclade consisted of two monophyletic clades from Perak (Kuala Gula) and Johor (Sg Sarang Buaya and Pontian). The tree topologies showed two cluster patterns by population: the east coast Malaysian-southern Thai population (Kelantan, Thale Noi, and Ranong) and the southern, northern, and east coast Malaysian population (Perak, Johor, and Terengganu).
Due to the limited number of haplotypes in each population, a mismatch distribution analysis was not suitable in this study except for the population involving two countries, Thailand and Malaysia. Two populations were observed for mismatch analysis after the haplotypes of each state of each country were merged ( Figure 6). The scatterplot of Malay Peninsula population indicated multimodal mismatch distribution from the observed frequencies of pairwise differences among the D-loop sequences and the expected frequencies under the sudden and spatial expansion models. However, Thai population indicated unimodal interpretation of mismatch distribution by following the sudden and spatial expansion models ( Figure 6).
Genetic distance analysis was performed using the Kimura 2 Parameter (Table 3). Results showed that the A. cinereus samples are greatly distanced from L. perspicillata (>0.0309). Among the populations, the samples from Malay Peninsula states are closer to each other and highly distant from Thai population except for Kelantan. Kelantan population was closer to Thai population (0.0044) than to the other Malay Peninsula states (0.0079-0.0100).
Differentiation between individual haplotypes within groups was low, with most separated by single base substitutions. The minimum-spanning network that describes the relationships between the population haplotypes is shown in Figure 5. Two distinct networks were observed between haplogroup A (Hap 2, Hap 3, and Hap 6) and haplogroup B (Hap 1, Hap 4, Hap 5, and Hap 7).

Discussion
DNA from all fecal samples was extracted, amplified, and sequenced successfully. Thirty-three of 48 samples were identified as A. cinereus through site polymorphisms. The primer used in this study was useful for mini-DNA barcoding for the three Malaysian otter species. The use of a fresh noninvasive sample source, feces, with the D-loop primer pairs would be suitable for forensic analysis in the future especially for identifying species. As many as 30 informative sites were observed for species-specific differentiation among the three species L. perspicillata, A. cinereus, and L. sumatrana, which represented 7.5% of the total sequence length including some deletions and an insertion in the L. sumatrana sequences. The deletion and insertion events in L. sumatrana provided the initial idea that L. sumatrana differs from the other two species based on the observed DNA barcodes, thus supporting findings by Koepfli et al. [14]. Our finding also contradicts the previous phylogeny classification based on the morphological characteristics of the grouping of L. sumatrana and L. perspicillata in one group 6 The Scientific World Journal    distinct from A. cinereus [19][20][21]. However, L. perspicillata ta and A. cinereus have significant differences in morphological characteristics and ecological aspects such as body mass and diet selection [58]. According to Koepfli et al. [14], based on continuous observation, there are some possible explanations for this grouping. (a) Hybridization between the two species may occur and is not impossible since the two species have the same chromosome number, 2 = 38. (b) The species share a similar brain structure with a bigger rear sigmoid gyrus that gives higher tactile sensitivity, influencing similarities in foraging activity for both species. (c) Based on extinct Lutrogale paleoleptonyx and Lutrogale robusta fossils found in Java, Indonesia, both Lutrogale species have teeth structures suitable for eating shelled foods, a diet similar tothat of A. cinereus. In short, the DNA barcode of the three species can be used for species identification studies. Haplotype analysis showed that there are seven haplotypes, and only Johor state has two haplotypes. The analysis showed that the A. cinereus samples from each population are highly endemic based on the partial D-loop sequences. This was supported by the low nucleotide diversity among populations valued below 0.6%. The nucleotide divergence was also observed to be low among populations with the highest , between Johor and Ranong (0.91%), which was proportional to the largest distance among other states.
Phylogenetically, all A. cinereus samples were excluded from the outgroups, the Eurasian otter and L. perspicillata. In the major A. cinereus clade, two subclades formed. The first subclade grouped all Malay Peninsula samples except for the samples from Kelantan. The second subclade grouped Kelantan samples with Thai samples. Kelantan samples were outliers in this study as Kelantan was only peninsular state grouped with Thai samples. Based on distance observation, Terengganu is closer to Kelantan, without any major natural barrier preventing gene flow between the two states. Both states are located on the east coast of Malaysia and are farther away from the other states examined in this study. However, genetic distance analysis supported the close relationships Based on the mismatch analysis for Malay Peninsula states, the observed pattern showed a disrupted flow chart and differed from the simulated pattern. However, the same analysis conducted for Thai populations showed that the observed pattern followed the simulated pattern significantly. These patterns suggested that a disruption exists among the population of Malay Peninsula, which is supported by the outlier formation of Kelantan samples in this study. No previous population study stated any close relationship within Malay Peninsular samples and with Thai populations in A. cinereus.
According to Lariviere [22], A. cinereus inhabits Bangladesh, Bhutan, Borneo, Brunei, southern China, southern India, Indonesia, Java, Karimun Island, Laos, Malay Peninsula, Myanmar, Palawan, Philippines, Sumatra, Thailand, and Vietnam. This species is likely extinct in Hong Kong and Singapore [28,29]. Based on previous studies, several subspecies of A. cinereus have been recognized. Corbet and Hill [59]  For further confirmation, another mismatch analysis was performed by excluding Kelantan population from Malay Peninsula (group 1) and combining it with the populations from Thailand (group 2). Results showed that the observed mismatch distribution pattern for both groups followed the simulated suggested patterns ( Figure 6). These patterns suggested that the otters sampled from Kelantan are closer or most likely belong to Thai population and are quite different from the samples from other states. This finding shows that Thai subspecies A. c. cinerea may have migrated to Kelantan from Thai mainland. Genetically, these results also suggested the endemic exclusion of Malay Peninsula populations and distinction from Kelantan-Thai populations.
In this study, based on several population analyses, we suggest that Malay Peninsula small-clawed otter is different from Thai subspecies A. cinereus cinerea. We also suggest the classification of the new subspecies of Malay Peninsula small-clawed otter named A. cinereus kecilensis. This new subspecies might range from the southern to the northern part of Malay Peninsula. However, there was no confirmation that Thai subspecies occurs in the two states located next to Thailand, Perlis and Kedah.
In conclusion, fecal samples were a source for identifying species, which thus supports noninvasive sampling for genetic analysis. The primer pair used in this study was also significant for DNA barcoding for the three Malaysian species L. perspicillata, L. sumatrana, and A. cinereus. This study suggested new classification of Malay Peninsula small-clawed otter, A. cinereus kecilensis. However, additional samples from other states of Malay Peninsula are needed to confirm the range of Thai subspecies in Malaysia and the range of A. cinereus kecilensis in the northern states.