Genetic Characteristics and Relationships of Climbing Perch Anabas Testudineus Populations in Thailand

FISHERIES SCIENCE 2000; 66: 840–845

Original Article

Genetic characteristics and relationships of climbing perch Anabas testudineus populations in Thailand

Masashi SEKINO,1,* AND Motoyuki HARA2

1National Research Institute of Fisheries Engineering, Fisheries Agency, Hasaki, Ibaraki 314-0421 and 2National Research Institute of Aquaculture, Fisheries Agency, Nansei, Mie 516-0193, Japan

SUMMARY: Seven natural populations of climbing perch Anabas testudineus collected from the central, eastern, and peninsular areas in Thailand were assayed by allozymic analysis to examine genetic relationships among geographic populations. Twelve out of 18 enzyme loci were polymorphic. Significant differences in allelic frequencies were observed in all pairs of populations at one or more loci, and six loci showed the regional characteristic allelic compositions. Genetic distance between populations was higher between areas, in comparison with those within an area, being significant between the peninsular and the other populations. According to a UPGMA tree based on a genetic distance matrix, the seven populations fell into three major clusters in line with the area divisions. These groupings indicate that genetic relationships among climbing perch populations are much the same as for other freshwater fishes, and would depend on geographic features such as the river systems in Thailand.

KEY WORDS: allozyme, Anabas testudineus, climbing perch, population genetics, Thailand.

INTRODUCTION land were investigated by allozymic analysis, in comparison with other species already reported. Climbing perch Anabas testudineus is widely distributed in South-East Asia, and is an important freshwater food fish.1 Artificial seed production and transportation are MATERIALS AND METHODS expected to be a solution toward increasing productivity, however, this may possibly result in disturbances in Seven geographically separated populations of climbing natural genetic population structures. For freshwater perch were collected in Thailand, from May to Novem- fishes, restriction of gene flows among populations can ber in 1996. Specimens derived from the same locality easily occur due to geographic barriers such as land were included as one population. Based on the major stretches and sea areas, and this leads to genetically river systems and the zoogeographic divisions described divergent populations.2–11 Therefore, a study of genetic by Smith,1 each population was considered as consisting relationships among geographic populations will supply of samples from: (i) the central areas including the further information for use in promoting natural stock Chiang Mai, Sukhothai and Chai Nat populations management and enhancement. Previous reports on belonging to the Chao Phraya River system; (ii) the genetic population studies for freshwater fishes in Thai- Khon Kaen and Korat populations located within the land have revealed that the genetic difference among Mekong River system in the eastern areas; and (iii) populations would depend mostly on common geo- the Narathiwat and Yala populations located in the graphic features.6,8,9 peninsular areas. The localities of samples collected, In this study, the genetic characteristics and relation- abbreviated sample names, dates of sampling, and ships for geographic climbing perch populations in Thai- numbers of individuals sampled are shown in Table1. Figure 1 shows the geographic locations of each sample with sample names and abbreviations. Each specimen in *Corresponding author: Tel: 81-479-44-5935. Fax: 81-479-44-1875. its entirety was frozen immediately after collection, and Email: [email protected] stored at -20°C until dissections and preparation of Received 29 September 1999. Accepted 8 May 2000. extracts for electrophoresis. Population structure of climbing perch in Thailand FISHERIES SCIENCE 841

Table1 Materials used in population analysis of Anabas testudineus in Thailand Locality Area Sample abbreviation Date sampled No. of individuals Chiang Mai Central CM Oct. 1996 100 Sukhothai Central SKT Oct. 1996 39 Chai Nat Central CN May–Nov. 1996 107 Khon Kaen Eastern KK June 1996 52 Korat Eastern KR Mar.–Oct. 1996 73 Yala Peninsular YL May 1996 32 Narathiwat Peninsular NW June–Oct. 1996 52

For sampling localities see Fig. 1.

drogenase (G3PDH; EC 1.1.1.8), glucose-6-phosphate isomerase (GPI; EC 5.3.1.9), isocitrate dehydrogenase (NADP+) (IDH; EC 1.1.1.42), lactate dehydrogenase (LDH; EC 1.1.1.27), malate dehydrogenase (NAD+) (MDH; EC 1.1.1.37), mannose-6-phosphate isomerase (MPI; EC 5.3.1.8), octanol dehydrogenase (ODH; EC 1.1.1.73), phosphogluconate dehydrogenase (PGDH; EC 1.1.1.44), phosphoglucomutase (PGM; EC 5.4.2.2), and superoxide dismutase (SOD; EC 1.15.1.1). Loci are shown in italics, and alleles at each locus are designated by letters in alphabetical order, starting with the allele encoding the most anodally migrating allozymes. Allelic frequencies at each locus were calculated directly from observed genotypes. The proportion of polymorphic loci (a locus was considered as polymorphic when the frequency of the most common allele did not exceed 0.95), observed average heterozygosity, and expected average heterozygosity were calculated. Heterogeneity of allelic frequencies were tested using the chi-squared method,12 and to analyze signiﬁcant departure from Hardy–Weinberg’s expectations, a test analogous to Fisher’s exact test in the Markov-chain method (Markov-chain parameters used: steps, 100 000; dememorization, 1000) was employed using Arlequin version 1.1. software.13 To examine the genetic differences and relationships between populations, Nei’s standard genetic distance14 was calculated, and a UPGMA tree based on the genetic distance matrix was constructed with 1000 bootstrap replicates using Njbafd software (provided by Dr N. Takezaki, National Institute of Genetics, Mishima, Shizuoka, Japan). Fig. 1 Sampling localities and abbreviated names for seven populations of Anabas testudineus collected in Thailand. RESULTS

For electrophoretic analysis, melting drips from frozen Table2 shows the allelic frequencies for each of the vari- skeletal muscles, and the extracts of aqueous able loci, proportion of polymorphic loci (P), and homogenates from liver tissues were used. Horizontal observed and expected average heterozygosity (Ho and starch gel electrophoresis in tris-citric acid electrode He, respectively) in each population. Twelve out of 18 buffer was conducted according to Hara et al.,9 and gel enzyme loci were judged as polymorphic in at least one staining procedures were based on the methods of Shaw population; G3PDH-1*, G3PDH-2*, GPI-1*, GPI-2*, and Prasad.11 The 12 enzymes examined were as follows: IDH-1*, LDH-2*, MDH-1*, MPI-1*, MPI-2*, ODH*, aspartate aminotransferase (AAT; EC 2.6.1.1), fumarate PGM* and SOD*. Two loci, LDH-1* and PGDH* hydratase (FH; EC 4.2.1.2), glycerol-3-phosphate dehy- showed small variations. The remaining four loci, AAT*, 842 FISHERIES SCIENCE M Sekino and M Hara

Table2 Allelic frequencies, proportion of polymorphic loci (P), and observed and expected average heterozygosity (Ho, He) in seven wild populations of Anabas testudineus in Thailand Locus Central Eastern Peninsular Chiang Mai Sukhothai Chai Nat Khon Kaen Korat Yala Narathiwat G3PDH-1* *a 0.000 0.000 0.000 0.000 0.007 0.000 0.000 *b 0.563 0.625 0.786 0.620 0.522 0.323 0.315 *c 0.437 0.375 0.214 0.380 0.471 0.677 0.685 (N)71 2491 46683146 G3PDH-2* *a 0.000 0.000 0.010 0.029 0.000 0.063 0.010 *b 0.620 0.679 0.814 0.971 1.000 0.906 0.952 *c 0.380 0.321 0.176 0.000 0.000 0.031 0.038 (N) 96 39 105 52 73 32 52 GPI-1* *a 0.000 0.000 0.005 0.000 0.000 0.000 0.077 *b 1.000 0.987 0.995 1.000 1.000 1.000 0.923 *c 0.000 0.013 0.000 0.000 0.000 0.000 0.000 (N) 55 39 107 52 73 32 52 GPI-2* *a 0.000 0.013 0.033 0.029 0.000 0.000 0.000 *b 1.000 0.961 0.967 0.971 1.000 1.000 0.904 *c 0.000 0.026 0.000 0.000 0.000 0.000 0.029 *d 0.000 0.000 0.000 0.000 0.000 0.000 0.067 (N) 55 39 107 52 73 32 52 IDH-1* *a 0.031 0.022 0.060 0.000 0.000 0.000 0.000 *b 0.969 0.978 0.940 1.000 1.000 1.000 1.000 (N)80 2391 38733051 LDH-1* *a 0.000 0.013 0.037 0.000 0.000 0.000 0.000 *b 1.000 0.987 0.963 1.000 1.000 1.000 1.000 (N)55 3981 38733252 LDH-2* *a 0.000 0.026 0.000 0.000 0.000 0.063 0.010 *b 1.000 0.974 1.000 1.000 1.000 0.937 0.990 (N)55 3956 38733251 MDH-1* *a 0.825 0.974 1.000 1.000 1.000 1.000 1.000 *b 0.175 0.026 0.000 0.000 0.000 0.000 0.000 (N) 100 38 62 52 73 32 52 MPI-1* *a 0.000 0.038 0.037 0.000 0.000 0.069 0.000 *b 1.000 0.962 0.963 1.000 1.000 0.931 1.000 (N) 74 39 107 52 73 32 51 MPI-2* *a 0.017 0.014 0.014 0.000 0.000 0.000 0.069 *b 0.983 0.986 0.977 1.000 1.000 0.547 0.549 *c 0.000 0.000 0.009 0.000 0.000 0.453 0.382 (N) 88 35 106 52 73 32 511 ODH* *a 0.040 0.000 0.000 0.156 0.120 0.313 0.039 *b 0.960 1.000 1.000 0.844 0.880 0.609 0.892 *c 0.000 0.000 0.000 0.000 0.000 0.078 0.069 (N) 100 39 107 45 71 32 51 PGDH* *a 0.000 0.000 0.000 0.000 0.000 0.000 0.010 *b 1.000 1.000 1.000 1.000 1.000 1.000 0.990 (N)54 3982 52733252 Population structure of climbing perch in Thailand FISHERIES SCIENCE 843

Table2 Continued Locus Central Eastern Peninsular Chiang Mai Sukhothai Chai Nat Khon Kaen Korat Yala Narathiwat PGM* *a 0.930 1.000 0.986 0.981 1.000 1.000 1.000 *b 0.060 0.000 0.005 0.000 0.000 0.000 0.000 *c 0.010 0.000 0.009 0.019 0.000 0.000 0.000 (N) 100 39 107 52 73 32 52 SOD* *a 0.000 0.000 0.000 0.038 0.000 0.000 0.000 *b 1.000 1.000 1.000 0.904 0.822 1.000 1.000 *c 0.000 0.000 0.000 0.058 0.178 0.000 0.000 (N)46 3962 52733238 P 0.222 0.111 0.167 0.167 0.167 0.278 0.333 Ho 0.075 0.064 0.055 0.054 0.055 0.077 0.090 He 0.087 0.072 0.058 0.060 0.057 0.106 0.092

1 A locus observed signiﬁcant departure from Hardy–Weinberg’s expectations (P < 0.01). AAT*, FH*, IDH-2* and MDH-2* showed no variations in all populations. (N) is the sample number for each locus.

Table3 Numbers of loci showing signiﬁcant difference in allelic frequency between seven populations are indicated above the diagonal, and the results for Nei’s standard genetic distance14 are shown below the diagonal Area Locality (sample abbreviation) Central Eastern Peninsular CM SKT CN KK KR YL NW Central Chiang Mai (CM) –385679 Sukhothai (SKT) 0.001 –23345 Chai Nat (CN) 0.007 0.002 – 6 8 6 8 Eastern Khon Kaen (KK) 0.011 0.007 0.005 – 1 4 7 Korat (KR) 0.013 0.009 0.009 0.001 – 6 8 Peninsular Yala (YL) 0.030 0.029 0.033 0.020 0.020 – 5 Narathiwat (NW) 0.024 0.021 0.025 0.017 0.016 0.005 –

For sampling localities see Fig. 1.

FH*, IDH-2* and MDH-2* showed no variations in all Six loci showed characteristic allelic distributions in populations. Genotype frequencies observed at each some populations, G3PDH-1*, G3PDH-2*, IDH-1*, locus were almost in agreement with Hardy–Weinberg’s MPI-2*, ODH*, and SOD* (Table2); at the G3PDH-1* expectations except for the MPI-2* locus in the Narathi- locus, the frequency of allele *b in the peninsular popu- wat population at 1% probability of worth of ﬁt. The P, lations (0.323, 0.315) was lower than in the other areas Ho, and He values ranged from 0.111 to 0.333, 0.054 to (0.522–0.786). At the G3PDH-2* locus, the frequency 0.090, and 0.056 to 0.106, respectively. These values of allele *b in the central populations (0.620–0.814) was were slightly higher in the peninsular populations than lower than in the other areas (0.906–1.000). The IDH- in the other populations. All populations showed some 1* locus showed polymorphism or small variations in measures of homozygote excess, especially in the Yala the central populations, but no variations in all other population. areas. At the MPI-2* locus, the frequency of allele *c Allelic frequencies between every pair of seven popu- in the peninsular populations (0.382, 0.453) was higher lations were signiﬁcantly different (P <0.01) for at least than in the other areas (0.000–0.009). The ODH* one locus (Table3). The number of heterogeneous loci locus was polymorphic in the eastern and the peninsular in allelic frequencies had a tendency to be small in the populations. The SOD* locus showed polymorphism in pairs of populations within an area, in comparison with the eastern populations, but no variations in all other those between areas. However, there were some excep- areas. tional combinations, for example, between the Chiang Table3 shows genetic distance matrix for all pairs of Mai and Chai Nat populations in the central areas, and populations. Genetic distance values ranged from 0.001 between the Sukhothai population in the central areas to 0.033 (mean ± SD 0.015 ± 0.010). The small values and the eastern populations. within an area (i.e. 0.001–0.007 in the central areas, 844 FISHERIES SCIENCE M Sekino and M Hara

during the Pleistocene epoch. Analogously, the high genetic distance values between the peninsular and other populations observed in this study may be caused by events such as development of mountain barriers and intensive isolation resulting from sea-level changes, although gene exchange between them may possibly have occurred during low sea-level periods. As discussed above, we were able to determine the general structure of climbing perch populations in Thai- Fig. 2 UPGMA tree based on Nei’s standard genetic dis- land. However, a significant departure from Hardy– tance14 for seven populations of Anabas testudineus in Thailand. Weinberg’s expectations regarding the Narathiwat Numbers on the nodes are percentage bootstrap values from population, and homozygote excess in each population 1000 replications of resampled loci. indicate the presence of complicated population structures. This study could not supply enough information to elucidate the factors producing these results, although 0.001 in the eastern areas, and 0.005 in the peninsular these may be caused by several multiple breeding units areas) were observed in contrast to high values between and non-random mating in each population or sampling areas (the central vs eastern areas: 0.005–0.013; the errors because of a small number of specimens analyzed. central vs peninsular areas: 0.021–0.033; the eastern vs Additional comprehensive research from the ecological peninsular areas: 0.016–0.020), and the values observed viewpoint on such factors as reproductive and migra- in combination with the peninsular and other popula- tional mechanisms in addition to genetic analysis using tions were remarkably high. According to the UPGMA other genetic markers should be studied to further tree (Fig. 2), the seven populations fell into three major account for our results. clusters: (i) the Chiang Mai, Sukhothai, and Chai Nat This study revealed that the natural climbing perch populations in the central areas; (ii) the Khon Kaen and populations in Thailand generally comprise three major Korat populations in the eastern areas; and (iii) the Yala stocks with marked genetic characteristics. The results and Narathiwat populations in the peninsular areas. described in this study correspond well with previous studies on other freshwater fishes, and indicate that geographic features such as the river systems in Thailand DISCUSSION influence freshwater fish population structures in a similar manner. This study and previous studies may The grouping based on the UPGMA tree was in line serve as a useful source of information in the preserva- with the area divisions defined in this study, and the tion and management of natural genetic resources of characteristic allelic distributions in each area were freshwater fishes in Thailand. observed. These indicate that genetic relationships of climbing perch populations would depend on geographic features in Thailand. The Chao Phraya and its tribu- ACKNOWLEDGMENTS tary streams constitute a main river system from the northern to the central part of Thailand. The Mekong We are grateful to Drs Y. Musig and P. Tabthipwon, and its tributary streams are equivalent in the eastern Kasetsart University, Dr W. Kotrpat, Narathiwat Animal part. The Bilauktung Mountains separate the peninsular Nutrition Research Center, for allowing the use of their areas from the other areas. Therefore, we suggest that facilities, and to their staff for the assistance in collec- these three groups should be considered as the Chao tion of specimens. We also thank Dr Y. Nagamine, Phraya River group, the Mekong River group, and the Tohoku National Agricultural Experiment Station, for peninsular group. These population structures are much technical advice on statistical calculations, and Dr M.N. the same as the killifish Oryzias minutillus, the catfish Wilder, Japan International Research Center for Agri- Clarias macrocephalus, and the snake-head fish Channa cultural Sciences, for critical reading of the manuscript. striatus populations.6,8,9 This study was supported by Japan International Based on the distribution of freshwater fishes popula- Research Center for Agricultural Sciences, Ministry of tions in the peninsular areas, Dodson et al.10 have studied Agriculture, Forestry, and Fisheries, Government of catfish Hemibagrus nemurus using mitochondrial DNA Japan. polymorphism analysis; these authors hypothesized that populations of the eastern part of Peninsular Malaysia REFERENCES and the Mekong River populations were separated during a high sea-level period, and faunal exchanges between 1. Smith HM. The Freshwater Fishes of Siam, or Thailand. Smithson- them would have occurred via the drainage systems on ian Institution, Washington DC. 1965. the historical Sunda dry land, after a decline in sea level 2. Billington N, Hebert PDN. Mitochondrial DNA variation in Population structure of climbing perch in Thailand FISHERIES SCIENCE 845

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