Distribution Ranges of Snail-Eating Turtle Species (Malayemys spp.) in Northeastern and Eastern Thailand
Abstract
Snail-eating turtles (Malayemys spp.) are freshwater turtles native to mainland Southeast Asia. Three snail-eating turtle species (Malayemys khoratensis, Malayemys macrocephala, and Malayemys subtrijuga) were previously reported in Thailand. Before the present study, information on the distribution of snail-eating turtles in northeastern and eastern Thailand was limited, and a question arose regarding the distribution range of the new species, Malayemys khoratensis. From 2018 to 2020, we surveyed and collected data on the morphological traits of 109 snail-eating turtles along with 60 blood samples from 23 out of 27 provinces across northeastern and eastern Thailand. We found that both M. khoratensis and M. subtrijuga occur in northeastern Thailand. The distribution of M. khoratensis is in the Chi River Basin and upper Mun River Basin to the Mekong River. while the distribution of M. subtrijuga is from the lower Mun River to the Mekong River. Malayemys macrocephala occur in eastern Thailand and were found in 2 large provinces (Nakhon Ratchasima and Udon Thani) in northeastern Thailand. Based on morphological characteristics known to distinguish these species, 28 of the sampled individuals from areas in lower Mun River basin could not be assigned to any of the 3 species. Therefore, we reconstructed the phylogenetic relationships of 26 of these samples as well as other samples from known species based on 2 mitochondrial genes (Cyt b and ND4). Results based on mtDNA data indicated that the turtles in the unknown group belonged to 1 of the 3 clades corresponding to M. khoratensis, M. macrocephala, and M. subtrijuga. This result confirms that the unknown group does not represent a novel species within the genus Malayemys. However, it also indicates a discordance between the genetic and morphological characteristics of the turtles, highlighting the complexity of species identification within the genus Malayemys.
Malayemys is an endemic turtle genus mostly found in freshwater wetland habitats on the mainland of Southeast Asia and Java (Srinarumol 1995; Brophy 2004, 2005; Bonin et al. 2006; Dawson et al. 2018, 2020; Platt et al. 2022). Before 2016, the genus Malayemys was subdivided into 2 species, Malayemys subtrijuga and Malayemys macrocephala (Brophy 2004). The former species was assumed to be restricted to the Mekong River Basin, while the latter was believed to be distributed widely from the Chao Phraya River Basin to the Malay Peninsula (Brophy 2005).
In 2016, a new species of snail-eating turtle, the Khorat snail-eating turtle Malayemys khoratensis, was formally described from northeastern Thailand (Ihlow et al. 2016). Malayemys khoratensis was reported to be found in the Khorat Basin of northeastern Thailand (Sikhio District, Nakhon Ratchasima Province, Mun River and Chi River drainage, and Udon Thani and Nong Bua Lamphu provinces, according to Ihlow et al. 2016, and the Nam Phong River system in Nong Khai Province according to Sumontha et al. 2016) and Lao PDR (Vientiane Province, Vientiane Prefecture, Khammouan Province, and Savannakhet Province, according to Sumontha et al. 2016 and Platt et al. 2022).
The discovery of M. khoratensis changed the current view of the distribution ranges of the other 2 species: Malayemys subtrijuga was reported to be found in Cambodia, Indonesia (island of Java), Lao PDR (Champasak Province), and Vietnam (Ihlow et al. 2016; Dawson et al. 2020; Platt et al. 2022), while Malayemys macrocephala was reported to occur from northern Thailand through northern Peninsular Malaysia (Ihlow et al. 2016; Dawson et al. 2018) with some uncertain reports in northeastern Thailand (Sakon Nakhon Province) and Lao PDR (Vientiane Province; Ihlow et al. 2016).
The distribution map of the 3 Malayemys species in previous studies was based on only a few locations in northeastern Thailand (4 out of 20 provinces; Ihlow et al. 2016; Sumontha et al. 2016) and in eastern Thailand (3 out of 7 provinces; Brophy 2005; Ihlow et al. 2016). Therefore, the distribution boundary of the Khorat snail-eating turtle within northeastern Thailand is still unknown, and the distribution ranges of M. subtrijuga and M. macrocephala in northeastern and eastern Thailand remain uncertain.
Although Malayemys are relatively common and widespread in Southeast Asia, population sizes and trends of each Malayemys species remain completely unknown due to a lack of direct studies for estimating population sizes (Dawson et al. 2018, 2020). In addition, in Thailand and in other countries in mainland Southeast Asia, these turtles are commonly traded in fresh markets or temples for merit releases in Buddhist ceremonies, and turtles and turtle eggs are also consumed by local people or used as medicine (Dawson et al. 2018, 2020; Platt et al. 2022). In Thailand, M. subtrijuga and M. macrocephala are protected under Thailand’s Wild Animal Conservation and Protection Act, B.E. 2562 (2019), while the Khorat snail-eating turtle is presently not protected even though it faces the same threats. For this reason, assessing the distribution range and population status of M. khoratensis accurately is crucial to evaluate the potential threats faced by the species.
We surveyed the area in northeastern (20 provinces) and eastern Thailand (7 provinces) to determine the distribution ranges of turtles in the genus Malayemys to provide some insights about the population status of M. khoratensis. We identified the turtles by morphological characteristics according to Brophy (2004), Sumontha et al. (2016), and Ihlow et al. (2016). Interestingly, we found 28 turtles from the Mun River Basin showing different morphological characteristics from all other Malayemys species previously described. We thus collected blood samples from these turtles and other known turtle samples and sequenced 2 mitochondrial DNA fragments (Cyt b and ND4 genes) for species identification. The result will improve the understanding of the distribution ranges of the genus Malayemys in northeastern and eastern Thailand.
METHODS
Study Area, Survey, and Sampling. —
Northeastern Thailand, or Isan, shares borders with Lao PDR to the northeast and with Cambodia to the southeast, while the western side is separated from central Thailand by the Sankamphaeng Range and the Phetchabun Range. The primary forests in these areas consist of both deciduous forest (comprising dipterocarp and mixed deciduous forest) and dry evergreen forest. The area of northeastern Thailand covers 170,000 km2 (Keyes 1967). Previous studies reported that M. khoratensis occurred in at least 4 out of 20 provinces (Nakhon Ratchasima, Udon Thani, Nong Bua Lamphu, and Nong Khai) in northeastern Thailand (Ihlow et al. 2016; Sumontha et al. 2016), while M. macrocephala was reported in Sakon Nakhon Province (Ihlow et al. 2016), even though Sakon Nakhon Province does not directly connect to native distribution range of M. macrocephala (northern, central, eastern, and southern Thailand to northern Malaysia; Dawson et al. 2018).
Eastern Thailand is separated from northeastern Thailand by the Sankamphaeng Range Basin and hills. It shares borders with Cambodia to the east and central Thailand to the west. The area of eastern Thailand is approximately 34,380 km2. Brophy (2005) and Ihlow et al. (2016) reported that M. macrocephala occurred in Chonburi, Prachin Buri, and Trat provinces (3 out of 7 provinces). Generally, Malayemys turtles are common in these 2 regions of Thailand.
We performed multiple field surveys in 2018–2020 in northeastern Thailand (Chi River Basin, Mun River Basin, and Mekong River Basin) and in eastern Thailand (Prachin Buri River Basin, Bang Pakong River Basin, and East Coast Gulf River Basin) during rainy seasons (June to October) and cool dry seasons (November to February) where Malayemys turtles are active (they might be inactive and bury into mud during the hot dry season between March and May; Dawson et al. 2018, 2020). In total, 18 surveys were conducted across 27 provinces in northeastern and eastern Thailand (20 provinces in northeastern Thailand and 7 provinces in eastern Thailand). We visited each province and contacted local fishermen and local markets. We collected samples from fishermen or local residents if they had them on hand, or we were notified by them when they caught the turtles later. All turtles were captured by hand or fish traps by local people from rice fields, ponds, natural wetlands, or irrigation canals, the habitats of turtles in rainy season.
Each turtle was photographed for morphological analysis as described below. Blood sample collection was performed by drawing approximately 0.5 ml of blood from the dorsocervical sinus of each living turtle (less than 0.5% of body mass; the range of turtle body mass in the present study was between 150 g and 1.5 kg). Before drawing blood, the skin of each turtle was cleaned using 70% ethyl alcohol. Blood was collected from turtles using a 23-gauge needle and a 3-ml syringe. Then the blood sample was stored in EDTA K3 disposable vacuum blood collection tubes (Zhejiang Gongdong Medical Technology Co., Ltd.). The blood collection tubes were then stored on ice until returned to the laboratory. If freshly dead turtles were found, they were preserved in 70% ethanol. Blood and turtle samples were then ultimately kept in a freezer at −20°C in the Department of Environmental Science, Faculty of Science, Khon Kaen University, Khon Kaen Province, Thailand.
Morphological Analysis. —
We recorded characteristics used to identify Malayemys species according to Brophy (2004), Dawson et al. (2018, 2020), Ihlow et al. (2016), and Sumontha et al. (2016) from 109 snail-eating turtles in the field (Table 1). Each specimen was photographed from the head to the plastron positions, and the adult males and females were separated according to sexually dimorphic characteristics (body size, carapace ratio, tail size, and cloaca position) according to Srinarumol (1995). The turtle samples that could not be identified by using morphology were classified as unknown. Blood samples of some of these turtles were collected for further sequence analyses.
DNA Extraction, Fragment Amplification, and Sequencing. —
Genomic DNA of a total of 60 fresh blood samples of Malayemys turtles (identified by morphology as 8 M. macrocephala, 19 M. khoratensis, 7 M. subtrijuga, and 26 of the unknown group) were extracted using a GF-1 Blood DNA Extraction Kit (Vivantis Technologies Sdn Bhd, Malaysia) according to the manufacturer’s protocol. The DNA was qualitatively and quantitatively measured by a Nanodrop Spectrophotometer (Thermo Scientific™). Two mtDNA genes (Cyt b and ND4) were amplified separately using the following primers: a forward primer called “CytbG” (5′-AACCATCGTTGTAATCAACTAC-3′) and a reverse primer called “mt-f-na” (5′-AGGGTGGAGTCTTCAGTTTTTGGTTTACAAGACCAATG-3′) are specific for the Cyt b region (1200 bp), and a forward primer called “L-ND4” (5′-GTAGAAGCCCCAATCGCAG-3′) and a reverse primer called “H-Leu” (5′-ATTACTTTTACTTGGATTTGCACCA-3′) are specific for the ND4 region (892 bp; Spinks et al. 2004; Stuart and Parham 2004; Fritz et al. 2006). We prepared PCR Master Mix in a total volume of 25 µl containing 1 µl of DNA sample, 1 µl of each primer (5 µM), 12.5 µl PCR Master Mix (Vivantis Technologies Sdn Bhd, Malaysia), and 9.5 µl distilled water. The reaction conditions were performed as follows: the initial denaturation was for 5 minutes (at 95°C for Cyt b and at 94°C for ND4), followed by 35 cycles for 45 seconds (denaturation temperature at 95°C for Cyt b and at 94°C for ND4), annealing for 30 seconds (annealing temperature at 56°C for Cyt b and at 55°C for ND4), and extension at 72°C for 60 seconds, with final extension at 72°C for 8 minutes for Cyt b and 10 minutes for ND4. The PCR products were checked by 1% agarose gel electrophoresis. Then they were sent for purification and sequencing at Macrogen (South Korea) or Bio Basic (Canada). The sequences were manually checked and edited using BioEdit 7.0 (Hall 1999). All sequences have been submitted to GenBank, with accession numbers provided in Supplemental Tables S3 and S4 (all supplemental material is available at http://dx.doi.org/10.2744/CCB-1515.1).
Sequence Analyses. —
We concatenated the sequences of Cyt b and ND4 from the 60 samples from the present study and 65 samples for Cyt b and ND4 of Ihlow et al. (2016) obtained from GenBank (for accession numbers of these samples, see Supplemental Tables S1 and S2) and aligned using MUSCLE (Edgar 2004) in MEGAX (Kumar et al. 2018). The final concatenated sequences consisted of 1459 bp (753 bp for Cyt b and 706 bp of ND4 plus adjacent tRNAs). A homologous sequence of Mauremys reevesii was downloaded from GenBank (accession number AY676201) and used as an outgroup. The phylogenetic trees for the sequences were constructed using MrBayes 3.2.6 (Ronquist et al. 2012). A Markov chain Monte Carlo (MCMC) approach with 10,000,000 generations starting with random trees and sampled every 1000 generations was applied. The analysis was, however, stopped when the average standard deviation of split frequencies was below 0.01. The initial 450 generations were discarded as burn-in samples.
The sequences of Cyt b and ND4 were further collapsed into haplotypes separately and further analyzed for number of polymorphic sites, number of haplotypes, haplotype diversity, and nucleotide diversity using DnaSP 6.12 (Rozas et al. 2017). The haplotype network based on each gene was constructed by median-joining networks (Bandelt et al. 1999) using the program PopART (Population Analysis with Reticulate Trees; Leigh and Bryant 2015).
RESULTS
Morphological Analysis. —
We found Malayemys turtles in 17 out of 20 provinces (all except Loei, Maha Sarakham, and Sakon Nakhon provinces) in northeastern provinces and in 6 out of 7 provinces (all except Chonburi Province) in eastern Thailand (Fig. 1). In total, we examined 109 snail-eating turtles. Of these, we were able to assign 81 to morphologically recognized Malayemys species based on face, head, first vertebral scute, and lower marginal scute characteristics (Table 1 and Fig. 2, Supplement Table S3) as described by Brophy (2004), Dawson et al. (2018, 2020), and Ihlow et al. (2016), as follows: 15 M. macrocephala (5 males and 10 females), 43 M. khoratensis (18 males and 25 females), and 23 M. subtrijuga (13 males and 10 females). Among these, we used the molecular technique (see below) to confirm the species of 34 individuals (19 M. khoratensis, 8 M. macrocephala, and 7 M. subtrijuga), and the results agreed with the morphological identification. Even though the majority of characteristics of each turtle species matched the previous description (position of infraorbital stripe, shape of infraorbital stripe, orbital ring, and postocular stripe), we observed variations in certain characteristics in the identifiable group (Table 2) as follows.
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World's Turtle and Tortoise Journal 23, 2; 10.2744/CCB-1515.1
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World's Turtle and Tortoise Journal 23, 2; 10.2744/CCB-1515.1
The first vertebral scute was roughly square and not tapered posteriorly in some M. macrocephala (7%) and M. subtrijuga (13%), and the first vertebral scute was tapered posteriorly in 7% of M. khoratensis. Moreover, in our observations, a proportion of M. macrocephala (13%) and M. subtrijuga (9%) turtles exhibited an hourglass shape in their first vertebral scute (equal anterior and posterior sizes with a narrower midsection in the scute) that was not described in previous studies (Fig. 3a).
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World's Turtle and Tortoise Journal 23, 2; 10.2744/CCB-1515.1
In some individuals, most parts of lower marginal scutes 8–12 were brown or black (Fig. 3b), a characteristic not previously described in any Malayemys species. This pattern was found in all 3 species: in 10 out of 43 individuals of M. khoratensis (23%), 1 out of 15 individuals of M. macrocephala (7%), and 9 out of 23 individuals of M. subtrijuga (39%).
Ihlow et al. (2016) stated that M. khoratensis has 2 nasal stripes, but in the present study, we observed that 13 out of 43 individuals (30%) of M. khoratensis had 4 nasal stripes instead of 2 (mtDNA of 9 out of these 13 individuals were sequenced and they were grouped with M. khoratensis).
Twenty-eight snail-eating turtles (10 males and 18 females) in the lower Mun River basin (Fig. 1; 25 individuals from the area between Lam Chakkarat Stream and Lam Plai Mat Stream, tributaries of the Mun River at the border between Nakhon Ratchasima and Buriram Province, 2 individuals from an area under the middle of the Mun River in Surin Province, and 1 individual from Sisaket Province) could not be assigned into any of those species because they had mixed characteristics of M. khoratensis, M. macrocephala, and/or M. subtrijuga (Table 3, Fig. 2 and Supplemental Fig. S1, Supplemental Table S4). Therefore, we classified this group as an unknown group. We thus collected blood from 26 turtles in these group (blood was not successfully drawn from 2 turtles) for DNA sequencing and analysis as described above.
Phylogenetic Analysis of Cyt b and ND4. —
Analysis of the concatenated mtDNA (Cyt b and ND4) of 125 turtle samples (60 samples from the present study and 65 samples from Ihlow et al. (2016) showed 3 clades (Fig. 4): M. khoratensis, M. macrocephala, and M. subtrijuga clades (identified according to Ihlow et al. 2016). The unknown turtles were grouped within one of 3 known clades: 15 individuals (KMmsp 01–04, 07, 09, 12–15,17, 18, 19, 20, and 22) were grouped within the M. khoratensis clade, five individuals (KMmsp 06, 08, 11, 16, and 23) within the M. macrocephala clade, and 6 individuals (KMmsp 05, 10, 21, and 24–26) within the M. subtrijuga clade.
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World's Turtle and Tortoise Journal 23, 2; 10.2744/CCB-1515.1
Based on the phylogenetic tree, we observed discrepancies in certain characteristics that do not match the species’ characteristic descriptions and that we did not observe in individuals of known species. For example, turtles identified by mtDNA as M. khoratensis (KMmsp_0401, 03, 04, 07, 09, 13–17, 19, and 20) showed some characteristics similar to those of M. macrocephala or M. subtrijuga, e.g., an infraorbital stripe that reached the loreal seam and that was distinctly curved or angled. Three individuals had 5 (KMmsp_0415) or 6 (KMmsp_0412 and KMmsp_0422) nasal stripes, and 7 out of 15 individuals possessed 2 orbital rings. In contrast, all turtles identified as M. subtrijuga in this unknown group possessed only 2–4 nasal stripes (KMmsp 05, 10, 21, 24–26) and 1 individual (KMmsp_0426) had only 1 orbital ring. One M. macrocephala (KMmsp_0408) had 7 nasal stripes, and other individuals had infraorbital stripes that extended across the loreal seam and did not broaden at the suture.
Haplotype analysis of Cyt b and ND4. —
We further analyzed the haplotypes of 2 genes separately of all species and the unknown individuals were assigned to one of the Malayemys species according to the phylogenetic tree (Supplemental Tables S3 and S4). The result shows that M. macrocephala had the highest haplotype diversity in both genes, followed by M. khoratensis in Cyt b and M. subtrijuga in ND4 (see Table 4). Interestingly, in M. khoratensis, the unknown group seem to contribute much haplotype diversity. Fifteen individuals of M. khoratensis from the unknown group possessed 10 haplotypes (7 private haplotypes) in Cyt b and 5 haplotypes (3 private haplotypes) in ND4. Five haplotypes (4 private haplotypes) in Cyt b and 2 haplotypes occurred in 5 individuals of M. macrocephala and 5 haplotypes (4 private haplotypes) in Cyt b and 2 haplotypes (1 private haplotype) occurred in 6 individuals of M. subtrijuga. We constructed haplotype networks for each gene (Fig. 5). In general, the results agreed with the phylogenetic tree except that M. macrocephala was separated into 2 groups by M. subtrijuga in ND4. The haplotypes of the unknown individuals were grouped into each species, just as in the phylogenetic tree. One haplotype of Cyt b (Hap_21 from sample KMmsp_0419 of the unknown group, which grouped with M. khoratensis) was, however, quite distinct from those of all other species.
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World's Turtle and Tortoise Journal 23, 2; 10.2744/CCB-1515.1
DISCUSSION
During the survey from 2018–2020, a total of 109 snail-eating turtles were collected from northeastern and eastern Thailand. Based on the morphological characteristics, 81 snail-eating turtles could be distinctly identified as M. macrocephala, M. subtrijuga, or M. khoratensis. The remaining 28 individuals could not be assigned to 1 of those species because some characteristics (nasal stripes and infraorbital stripe) differed from the species descriptions of Brophy (2004), Dawson et al. (2018, 2020), and Ihlow et al. (2016). Twenty-six of these unknown turtles were then subjected to phylogenetic analysis of mtDNA (Cyt b, ND4), and they were each assigned into 1 of 3 Malayemys species.
For the known group, we identified 5 characteristics described by Ihlow et al. (2016) that can be used for species identification with accuracy. These characteristics include the position of the infraorbital stripe, the shape of the infraorbital stripe, the number of nasal stripes, the number of orbital rings, and the postocular stripe. However, we discovered that M. khoratensis can have 2–4 nasal stripes rather than only 2 as described by Ihlow et al. (2016). In addition, we observed variations in the first vertebral scute and lower marginal scutes 8–12 that were not described in Ihlow et al. (2016). These variations were quite common, accounting for 7%–39% of observed cases depending on the species (Table 2, Fig. 3). Furthermore, there is a similarity in these characteristics among species. These variations might pose challenges in the identification of turtles when only a turtle shell is available for examination, as all the other 5 characteristics require consideration of the turtle's face and head.
The 5 characteristics that can be used to identify Malayemys turtle with accuracy cannot be used to identify the unknown group because the morphological characteristics of the unknown species were an admixture of 3 known species, and these characteristics of snail-eating turtles have never been reported in other species. Thus, we performed phylogenetic analysis and haplotype analysis with DNA sequences of Cyt b and ND4. The results indicate that 58% of the unknown Malayemys group belongs to M. khoratensis, 23% to M. subtrijuga, and 19% to M. macrocephala. This result confirms that the unknown group does not represent a novel species within the Malayemys genus. However, the facial characteristics of most samples did not match with morphological description by Ihlow et al. (2016). We hypothesize that the unknown group might represent variations of each Malayemys species that have not been described before. If this hypothesis is correct, it might lead to difficulties in identification based on morphological characteristics, as some turtles exhibit characteristic admixtures of 2 or 3 Malayemys species. Interestingly, the unknown group of each species exhibited high haplotype diversity and possessed some private haplotypes, suggesting that some genetic events might occur in these populations. However, our data are limited in indicating specific genetic events because mtDNA is only maternally inherited. A future study should investigate this unknown group using other genetic markers to provide more information about the population structure and evolutionary history of the unknown snail-eating turtles.
Distribution of the Genus Malayemys.—
In the present study, we found 3 species of snail-eating turtles in northeastern and eastern Thailand, including M. macrocephala, M. subtrijuga, and M. khoratensis, as well as the unknown group, individuals of which can be assigned into one of those 3 species only by using molecular methods. The results showed that native snail-eating turtles in northeastern Thailand are M. subtrijuga (Mekong snail-eating turtle) and M. khoratensis (Khorat snail-eating turtle). The distribution of M. khoratensis is in the Chi River Basin and upper Mun River Basin to the Mekong River (Fig. 6). The species is abundant in the middle of the Chi River Basin between Khon Kaen Province and Yasothon Province. The distribution of M. subtrijuga is at the lower Mun River to the Mekong River. Malayemys subtrijuga has a main population in southern northeastern Thailand close to Cambodia, including Buriram, Surin, Sisaket, and Ubon Ratchathani provinces. The field survey indicated that the 2 species were separated by the Mun River. We believe that the main stem of the Mun River is a natural barrier for M. subtrijuga and M. khoratensis. However, the 2 species overlapped in the joint area of the Chi River and Mun River in Sisaket and Ubon Ratchathani provinces (Fig. 6).
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World's Turtle and Tortoise Journal 23, 2; 10.2744/CCB-1515.1
We found M. macrocephala only in eastern Thailand and in 2 large provinces in northeastern Thailand (Nakhon Ratchasima and Udon Thani provinces). We assume that M. macrocephala in northeastern Thailand might have been introduced by turtle trade as reported by Mokarat (2018). The distribution range of M. macrocephala in eastern Thailand and not in northeastern Thailand aligns with the report by Dawson et al. (2018). Malayemys macrocephala is separated from other snail-eating turtle species by the Sankamphaeng Range between eastern and northeastern Thailand and the Phetchabun Range between central and northeastern Thailand.
CONCLUSION
In 2016, a new species, M. khoratensis, was discovered in northeastern Thailand and Lao PDR (Ihlow et al. 2016; Sumontha et al. 2016; Platt et al. 2022). Before the present study, the information on the distribution range of the new species of the snail-eating turtle in eastern and northeastern Thailand, especially in the Mun River Basin, was limited. The distribution range of the other snail-eating turtle species also was in question for this region. Here we report that the distribution of M. khoratensis is in the Chi River Basin and the upper Mun River Basin, reaching the Mekong River. In contrast, M. subtrijuga is found in the lower Mun River region to the Mekong River, while M. macrocephala is located exclusively in eastern Thailand. During our survey, we found some samples could not be assigned by their morphological characteristics in lower northeastern Thailand. The morphological characteristics of the unknown group are obviously an admixture of 2 or 3 Malayemys species. However, by using mtDNA genes (Cyt b and ND4), they could be assigned to M. khoratensis, M. subtrijuga, or M. macrocephala. However, the existence of the unknown group indicates a discordance between the genetic and morphological characteristics of the turtles, highlighting the complexity of species identification within the genus Malayemys.
Map of northeastern and eastern Thailand and the sampling sites. The numbers on the map show districts and provinces where the turtles were sampled in the present study. The symbols indicate the turtle species found in the present study: gray triangles: Malayemys khoratensis, black circles: Malayemys macrocephala, light gray diamonds: Malayemys subtrijuga, dark gray squares: unknown.
Distinctive morphological characteristics of the unknown individuals compared with those of 3 snail-eating turtle species (Malayemys macrocephala, Malayemys subtrijuga, and Malayemys khoratensis). For the unknown group, the identification of species was based on the molecular techniques: A = Nasal stripe, B = Infraorbital stripe, C = Supraorbital stripe, D = Postorbital stripe, E = Loreal seam.
The characteristic variation of Malayemys observed in the present study: (a) an hourglass shape in their first vertebral scute (equal anterior and posterior sizes with a narrower midsection in the scute); (b) the brown or black color in most parts of lower marginal scutes 8–12.
Phylogenetic tree from Bayesian inference of the concatenated mtDNA (Cyt b and ND4) of 125 turtle samples (60 samples from the present study and 65 samples from Ihlow et al [2016]; for the samples of Ihlow et al. [2016], the isolate numbers are shown in the figure). The homologous sequence of Mauremys reevesii (accession number AY676201) was used as an outgroup. Species on the clades were identified according to Ihlow et al. (2016): gray triangles: Malayemys khoratensis, black circles: Malayemys macrocephala, light gray diamonds: Malayemys subtrijuga, dark gray squares: unknown.
Unrooted haplotype networks for (a) Cyt b and (b) ND4 sequences showing relationships among haplotypes of Malayemys. Circle size is relative to haplotype frequency and dashes represent the mutation events between haplotypes. Coloration indicates species identified by Ihlow et al. (2016) or according to the clades in Fig. 4.
(a) Putative distribution map of the genus Malayemys in central, northern, eastern, and northeastern Thailand, Lao PDR, Cambodia, and southern Vietnam (dark gray with diagonal lines = Malayemys macrocephala, gray with dots = Malayemys subtrijuga, light gray with grids = Malayemys khoratensis) according to Ihlow et al. (2016), Sumontha et al. (2016), Dawson et al. (2018, 2020), and Platt et al. (2022), drawn based on sampling sites of Ihlow et al. (2016). (b) Proposed distribution of the genus Malayemys based on the present study and the location of unknown (black patches).
Contributor Notes
Handling Editor: Peter V. Lindeman
