FISHERIES SCIENCE 2001; 67: 1175–1177

Short Paper

Clarification of the origin of landlocked ayu, Plecoglossus altivelis, populations in the Kasumigaura Lake system, ,

K KAEWSANGK,1 K- HAYASHIZAKI,1 T ASAHIDA,1 T NEMOTO2 AND H IDA1,*

1School of Fisheries Sciences, Kitasato University, Kesen, Iwate 022-0101 and 2Ibaraki Prefectural Freshwater Fisheries Experimental Station, Namekata, Ibaraki 311-3512, Japan

KEY WORDS: allozyme, Kasumigaura Lake system, landlocked ayu, population origin.

The Kasumigaura Lake system, including Nishiura, extreme water temperature, below 4°C in winter Kitaura, and adjacent tributaries, is the second and over 30°C in summer, due to the shallowness largest lake system in Japan and connects to the of the lake (4 m in average depth).5 On the other lowermost part of the system flowing hand, landlocked populations of ayu are known to into the Pacific Ocean. Since the full operation of a exist in Japan, typically the population native to water gate at the junction with Tone River in 1973, Lake Biwa that is the primary source of juveniles the fish fauna in the lake system has gradually which were released for stock supplementation changed in that the number of marine and brack- into rivers in Japan. But there are no records of ayu ish water fish species has fallen due to a decline of introduction in the Kasumigaura Lake system.5 salinity of the lake system and also the obstacle of By successive ecological surveys since the upstream migration from the sea.1 winter in 1992, all stages of ayu in its life history In 1992, ayu had been caught in unexpectedly were observed to exist, both in Kitaura and large amounts in the lake system, and the high Nishiura, suggesting ayu lives in the lake system level catch has continued ever since with some throughout in its entire annual life cycle.2–5 More- annual fluctuation in amount.2,3 Ayu Plecoglossus over, it was reported that the population was altivelis is an osmeroid fish of commercial im- uniquely adapted to the extreme environment of portance for freshwater fisheries in Japan. Its the lake system, by temporal and also spatial seg- amphidromous populations commonly occur in regation of juvenile to young from competitors in the rivers throughout Japan, and also in the rivers winter, utilization of abundant insects and resul- near the lake system including the main stream tant rapid growth in spring, over-summering in the and upper branches of the Tone River. Although inlet streams, which was supposed to enable main- the occurrence of this fish in the lake system and tenance of ayu abundance in the lake system.5 inlet streams was known before and even after the Therefore, the determination of origin of ayu pop- water gate construction,1 its abundance in the lake ulation restricted to the lake system as a land- system had never been large enough as a fishery locked population is necessary for undertaking target.4 Also, the limited timing of occurrence of stock management of this species in the Kasumi- ayu in the lake system suggested that the small gaura Lake system. amphidromous population spawning in the inlet On the possible sources of the origin of the ayu streams only partly used the lake as a passage population in the lake system, four hypotheses between streams and the ocean because of the were set out as follows. (i) Amphidromous popula- tion native to Kasumigaura Lake system; (ii) nearby amphidromous population; (iii) Lake Biwa ayu *Corresponding author: Tel: 81-192-44-1900. Fax: 81-192-44- unintentionally introduced with other fish; and 2125. Email: [email protected] (iv) Gunma hatchery stock, which has been the Received 10 July 2000. Accepted 5 February 2001. primary source of juveniles used to supplement 1176 FISHERIES SCIENCE K Kaewsangk et al.

ayu stocks in the upper branch of the Tone River, The frequencies of variant alleles observed in restricted only in Gunma Pref. Fish samples were the present study are shown in Table1. Among collected from the Kasumigaura Lake system 12 loci examined, five loci were polymorphic including both Nishiura and Kitaura, and also from among populations that were GPI-1*, MDH-1*, populations corresponding to the above hypothe- MPI*, PGDH* and PGM*. Within the samples ses except (i) as listed in Table 1. Sampling sites in from Kasumigaura Lake system, departure from the Kasumigaura Lake system and also the nearby Hardy–Weinberg expectations occurred in six out rivers are shown in Fig. 1. After capture, the white of a possible 36 (without four monomorphic cases) muscle and eye tissues were dissected and stored tests. Four of the six significant values were at - 80°C until used. We conducted allozyme analy- recorded at the MPI* locus (Station (Stn.) 2 in 1998, sis to yield the genetic characteristics of each Stns. 1, 2 and 3 in 1999), and the remaining were at assumed source. Allele frequencies was estimated the PGM* locus (Stn. 2 in 1998) and at the GPI-1* at the 12 loci, from eight enzymes activities, which locus (Stn. 4 in 1998), and all were due to an excess were GPI-1*, GPI-2*, IDHP-1*, IDHP-2*, LDH-1*, of homozygotes. On the other hand, for the rest of LDH-2*, MDH-1*, MDH-3*, MPI*, PGDH*, PGM*, the samples including amphidromous, landlocked SOD*, followed the horizontal starch gel elec- and hatchery populations, departure from Hardy– trophoresis method described in Kaewsangk et al.6 Weinberg equilibrium occurred in only one out of For data analysis, additional genetic data of four a possible 43 (without two monomorphic cases) distant amphidromous populations in Japan were tests, and was due to a excess of homozygotes at included, namely Urahama River (R.) (Iwate Pref.), the PGDH* locus of samples at Kuji R. in 1998. Yoshihama R. (Iwate Pref.), Sanze R. (Yamagata When allele frequencies at each locus in the Pref.), and Hiwasa R. (Tokushima Pref.) popula- Kasumigaura Lake system are compared among tions.6 Departure from Hardy–Weinberg expecta- sampling years and sites, a significant difference tion was examined with c2 goodness-of-fit tests. was observed at the PGDH* locus (P < 0.01). A tem- Homogeneity of samples in the Kasumigaura Lake poral or spatial genetic homogeneity was not nec- system among sampling sites and years was tested essarily ensured for ayu inside the lake system. with log-likelihood ratio analysis (G-tests). For The allele frequencies at each locus of amphidro- both tests 2000 times Monte Carlo resampling pro- mous, Lake Biwa, and Gunma hatchery popula- cedures with replacement were conducted to infer tions were agreeable with the genetic data reported the probability of the test. The genetic relation- previously.8,9 ships among ayu populations in the lake system The dendrogram from UPGMA clustering of and possible source populations were evaluated Nei’s genetic distances among populations is by cluster analysis. The UPGMA dendrogram was shown in Fig. 1. The genetic relationships showed constructed by PHYLIP version 3.51c based on that six amphidromous samples first clustered Nei’s genetic distances.7 together (D = 0.0017 on average) and then grouped

Table 1 Frequencies of 100 common alleles of five polymorphic loci observed in the ayu populations studied Populations Map Year of No. SL GPI-1* MDH-1* MPI* PGDH* PGM* codea sampling fish (mm) 100 100 100 100 100 Lake Nishiura Station 1 1 1998 60 63–116 0.580 1.000 0.742 0.960 0.925 1999 77 56–108 0.552 0.906 0.733 0.988 0.956 Station 2 2 1998 60 110–165 0.634 0.958 0.767 0.950 0.933 1999 77 70–102 0.617 0.969 0.722 0.900 0.950 Station 3 3 1998 60 72–155 0.640 0.975 0.780 0.960 0.950 1999 80 52–106 0.631 0.968 0.783 0.974 0.948 Lake Kitaura Station 4 4 1998 40 60–155 0.663 0.963 0.700 0.950 0.938 1999 78 75–147 0.532 0.963 0.720 0.981 0.943 Amphidromous fish 5 1998 80 58–86 0.675 0.982 0.857 0.925 1.000 1999 77 52–78 0.591 0.994 0.875 0.956 0.994 Naka River 6 1999 80 48–80 0.619 0.981 0.861 0.938 0.963 Landlocked fish Lake Biwa 7 1998 60 60–106 0.444 1.000 0.370 1.000 0.962 Hatchery fish 8 1997 60 138–183 0.195 0.892 0.675 1.000 0.875

a Site numbers correspond to those shown in Fig. 1. SL, standard length. Origin of ayu in Kasumigaura FISHERIES SCIENCE 1177

with a cluster of the Kasumigaura Lake system (D = 0.0025 on average) with an average D of 0.0051. As shown in Fig. 1, Lake Biwa fish grouped in one single cluster with Gunma hatchery stock (D = 0.0221), which is distinctly distanced from a cluster of two former groups with an average D of 0.056. The results obtained in the present study revealed the genetic similarity of ayu in the Kasum- igaura Lake system, despite its landlocked life form, to amphidromous populations including Kuji R. and Naka R. Nevertheless, Lake Biwa and Gunma hatchery populations were obviously dis- similar to the ayu population in the lake system, which should reject the hypotheses (iii) and (iv) as a primary source of origin. Between the remaining two hypotheses of amphidromous origin, although neither of them could be rejected by this result, the Fig. 1 Unweighted Pair Group Method with Arithmetic suddenness and unpredictability of the large catch mean (UPGMA) dendrogram using Nei’s genetic dis- in 1992 was supposed to support the origin from tance7 based on allele frequencies at five polymorphic outside the lake system hypothesis (hypothesis loci for ayu populations studied. Asterisks indicate (ii)), rather than the inner origin hypothesis the four amphidromous populations from Hiwasa R. (hypothesis (i)). Therefore, it was suggested to basi- (Tokushima Pref.), Sanze R. (Yamagata Pref.), Urahama cally support the hypothesis5 that abundant ayu R. and Yoshihama R. (Iwate Pref.), which their genetic data corresponding to Kaewsangk et al.6 had been supplied from outside of the lake system in 1992, which were probably the upstream migrants from the ocean passing through the water Pref. Freshwater Fish. Exp. Stn. 1993; 29: 39–45 (in gate from the main stream of the Tone River, and Japanese). its offspring had acclimatized and proliferated in 3. Nemoto T, Kawasaki T, Kubota J. Ecological study on the the freshwater lake system. However, the differ- Ayu, Plecoglossus altivelis, in Kasumigaura waters-I: Geo- ence of allele frequencies among sites and years graphical distribution and reproductive success. Bull. inside the lake system was observed, although only Ibaraki Pref. Freshwater Fish. Exp. Stn. 1996; 32: 21–35 (in Japanese). at the PGDH* locus, and the difference itself was 4. Nemoto T, Kubota J, Nakamura M, Sugiura M. Ecological small. Also, the observed homozygote excesses of study on the Ayu in Kasumigaura waters-II: Life cycle of Ayu ayu inside the lake system might indicate a mixture brood class in 1995, from Tomoe River, a tributary of Lake of genetically different groups. But these should be Kasumigaura. Bull. Ibaraki Pref. Freshwater Fish. Exp. Stn. evaluated with such methods as microsatellite 1997; 33: 1–16 (in Japanese). DNA analysis in future, which would be expected 5. Nemoto T, Nakamura M, Sugiura M. Ecological study on the to supply genetic information with higher resolu- Ayu in Kasumigaura waters: Completion of total life cycle tion than the allozyme analysis we used.10 The of Ayu in freshwater system in Kasumigaura Lake. Bull. addition of ayu samples from Tone River to be Ibaraki Pref. Freshwater Fish. Exp. Stn. 1998; 34: 1–21 (in included in the analysis should also be required to Japanese). 6. Kaewsangk K, Hayashizaki K, Asahida T, Ida H. An evalua- compare the allele frequencies directly between tion of the contribution of stocks in the supplementation of the assumed origin and ayu in the lake system. ayu, Plecoglossus altivelis, in the Tohoku area, using Such additional samples and new methods might allozyme markers. Fisheries Sci. 2000; 66: 915–923. also enable an evaluation of the hypothesis 7. Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package) remaining unrejected by the present study, such as version 3.5c. Distributed by the author. Department of partial contribution of unintentionally trans- Genetics, University of Washington, Seattle. http://evolu- planted ayu from Lake Biwa and/or ayu native to tion.genetics.washington.edu/phylip.html the Kasumigaura Lake system. 8. Taniguchi N, Seki S, Inada Y. Genetic variability and dif- ferentiation of amphidromous, landlocked, and hatchery populations of ayu Plecoglossus altivelis. Nippon Suisan REFERENCES Gakkaishi 1983; 49: 1655–1663. 9. Seki S, Taniguchi N, Jeon S. Genetic divergence among 1. Nakamura M. Species composition of fish in Lake Kasumi- natural populations of ayu from Japan and Korea. Nippon gaura and Kitaura. Bull. Ibaraki Pref. Freshwater Fish. Exp. Suisan Gakkaishi 1988; 54: 559–568. Stn. 1986; 23: 61–66 (in Japanese). 10. Takagi M, Shoji E, Taniguchi N. Microsatellite DNA poly- 2. Ishikawa H. Unusual occurrence of larvae of ayu, Plecoglos- morphism to reveal genetic divergence in ayu, Plecoglossus sus altivelis, from . Bull. Ibaraki altivelis. Fisheries Sci. 1999; 65: 507–512.