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Genetic differentiation of the northern Far East cyprinids,

Phoxinus and Rhynchocypris

YUKIMASA110,1 HARUMI SAKAI,1 SERGEI SHEDKO,2 AND SANGRN JEON3 1 NationalFisheries University,Shimonoseki 759-6595, Japer,2Institute of Biologyand Pedology,Russian Academy of Science,Yladivostok 690022, Russia, 'Sang MyungUniversity, Seoul 110-743, Korea

SUMMARY Although of the genera Phoxinus and Rhynchocyptis () are abundantly distributed in the northern Far East region, disagreements on their have persisted. Gene products of 18 allozyme or protein coding loci of 21 populations of minnows from the northern Far East were investigated in to resolve their classification and genetic relationships. Six monophyletic dusters were identified, being consistent with Phoxinus phoxinus, Rhynchocyptis oxycephalus, R. percnurus, R. czekanowskii, R. kumgangensis and R. semotilus, plus one paraphyletic group referred to as R. lagowskii.

KEY WORDS: allozyme, electrophoresis, taxonomy, Cyprinidae, phoxinin minnows

INTRODUCTION Summarizingthese studies led us to tentatively recognize seven groups of phoxinin minnows in the In thenorthern Far East region,many phoxininminnows northern Far East region: oxycephalus, lagowskii, havebeen reportedand variouslyclassified. Nakamura" pennurus, czekanowskii,kumgangensis, semotilus and used the Moroco for three Japanesespecies, M phoxinus groups, their generic classificationsbeing jouyi,M steindachneriand M percnun s sakhalinensis. ambiguous.Their morphologicaldistinctions included: On the otherhand, Nakabe inchused them in the genus oxycephalus a deeper caudal pedunclethan the other Phoxinus,regarding them as subspeciesof continental ;lagowskii a dark longitudinallateral band on the taxa,viz P. oxycephalusjouyi, P. lagouskiisteindachneri body and a blackblotch on the caudalbase; pen;nurus a and P. percmunussakhalinensis. Kim3) recognisedsix thick body and lowerjaw a littlemore prominentthan or Korean phoxinin species in two genera: Phoxinus revel withthe upperjaw, czekanowskii lateralline canal phoxinus and Rhynchocjpris oxycephalus, R. absent on body, but several pored scales above the steindachneri,R. pennons, R. kzangangensisand R. pectoral fin; kwngamgensis two- dark lateralbands on semotilus,the last two being endemic to the Korean body; semotilus a large dark spot on dorsal fin base; Peninsula.Chereslmev4) reported four species from Far phoxinus severalirregular lateral cross bands on body. East Russia,including them in the genus Phoxinus,P. However, because visual characteristics are phoxinus,P. lagawskii,P. perunurus and P. czekanowskii. somewhat subjective, they are of limited value in He treatedoxyrephalus as a subspeciesof P. lagowskii, assessingthe relationshipsof specimensfrom different and recognizedsome subspecies in both P. percnunusand localities. Accordingly,we analyzed allozyme allelic P. czekanowskii,the latter being endemic to Russia." compositionsof phoxininminnows collectedfrom the Previously,Howes' worldwide revision of phoxinin northernFar East region in an attemptto stabilizetheir minnows divided the Asian species into four genera, classification. namely,Tribolodon jouyi, Lagowskiellalagowskii, LL czekanowskii,Eupallasella perrnurus, Phoxinusphoxinus MATERIALS AND METHODS and P. semotilus. Tribolodon was appearently a misidentification,that genusbeing more correctlyapplied Twenty-one samples of tentatively-classified oxycephalus, to theFar Eastemdace, such as TThakonensis. lagowskii, percnurus, czekanowskii, kumgangensis, 76

Table 1 Sampling localities, date of collection and sample size

Fig. 1 Samplinglocalities of phoxininminnows from the northern Far East region; RhyrechocypRisaxycephalus (closed circles),R. lagowskii group (open circles),R. percnwus (closed squares),R. czekanawskii(open squares),R. kwngangensis(closed diamond),R. semotilus(open diamond),and Phoximephoxinus (closed triangles). Localitynumbers correspond to those in Table 1.

semotilusand phoxinusgroups were collectedfrom the dehydrogenase (E. C. 1.1.1.37, AIDH-1*, AIDH-2*, northernFar East region,Japan, Korea and Russia(Table AIDH-3*),phosphoglucomutase (E. C. 5.4.2.2,PGM*), 1, Fig. 1). The gene productsof 18 allozymeor protein L-iditol dehydrogenase (E. C. 1.1.1.14, IDDH*), codingloci were investigatedby stanchgel electrophoresis superoxidedismutase (E. C. 1.15.1.1,SOD*) and general (12 % gel): aspartate aminotransferase (E. C. 2.6.1.1, protein(PROT-1 *, PROT-2*). AAT-1*, AAT-2*),alcohol dehydrogenase (E. C. A neighborjoining (NJ)tree basedon Nei's genetic 1.1.1.1,ADH*), glycerol-3 phosphate dehydrogenase (E. distance(D)7) between pairs of sampleswas constructed C. 1.1.1.8, G3PDH*), glucose-6-phosphateisomerase so as to determinetheir genetic relationships.Bootstrap (E.C. 5.3.1.9,GPI-1 *, GPI-2*),isocitrate dehydrogenase probabilities(p in %) of nodeswere calculatedfrom 1000 (E. C. 1.1.1.42,IDHP*), L-lactatedehydrogenase (E.C. replications. 1.1.1.27, LDH-1*, LDH-2*, LDH-3*), malate 77

Table2 Numberof dichotomousloci in range (abovediagonal) and mean geneticdistance with standard deviation (below diagonal) between pairs of sevenspecies of Rhynchocyprisand Phaxbiur,and int a-specificgenetic distance (on diagonal)

Fig.2 Neighbor joining tree of 21 populationsof phoxinin minnowsfrom the northernFar East region based on Nei's ,emeticdistance; Rhynchxypris axycephalus (closed circles), R. lagawskiigroup (open circles), R. percmurus (closed squares),R. czekcmowskii(open squares),R. kumgangensis (closed diamond), R. semotilus (open diamond), and Phaxinusphaximrs (closed triangles). Locality numbers correspondto those in Table 1. Bootstrap probabilitiesfor 1000replications are indicatedat nodes.

RESULTS lagowskiigroup exhibited a greaterintra-specific distance (D = 0.31)than the others(D= 0.02?0.12). Thephoxinus group was the most genetically distant from In the NJ tree (Fig. 1), the phoxinus group was the others (D = 0.85?2.46, 11?16 loci being distantlyrelated to the others,which were clusteredat a dichotomous),with kumgangensrs and semotilusbeing the high bootstrapprobability (p = 90.9 %). In the latter, next distant from the remaining four groups (D = 0.71? kumgangensisand semotilus were connected as the 1.11, 8?12 loci being dichotomous) (Table 2). The latter, outermostbranches. The oxycephalus,czekanowskii and including the oxycephalus, lagowskii, percnusur and percmurusgroups formedthree monophyleticclusters of czekanowskiigroups, were relatively close to one another probabilitieshigher than 50 % (p = 54.0,60.2 and 51.5%, (D= 0.27 0.75,1. 10 loci being dichotomous). The respectively),the lattertwo groupsbeing jointed at a high 78

probability level (p = 79.6 %). On the other hand, the further research on a greater number of genetic markers lagowskii group was divided into three paraphyleticlines. from more populations representing a wider range.

DISCUSSION ACKNOWLEDGMENTS

The allozyme data indicated that two major divisions We appreciate the help of Drs. Akira Goto, Keisuke existed in the northern Far East phoxinin minnows, the Takata, Hitoshi Ida, Kazuhiko Katsura, Yuji Yamazaki, genetic distance between them (D = 0.85?2.46) Hiroshi Takahashi, Igor A. Chereshnev, Sergei V. Frolov, corresponding to the inter-generic level applied to and Sergei N. Safronov for their help in collecting Americancyprinids, D = 0.11?1.35 (mean 0.73).8) materials. Dr. Graham S. Hardy corrected the English. Therefore, the northern Far East phoxinin minnows This study was supported in part by Grants-in-Aid for should be classified into two genera, the phoxinus group Overseas Scientific Survey Nos. 06041004 and 09041138 in the present study being included in the genus Phoxinus from the Ministry of Education, Science Sports and and the others in the genus Rhynchocypris.) Culture, Japan. Because of their monophyletic nature, oxycephalus, percmuuus and czekanowskii apparently represent three REFERENCES valid species of the genus Rhynchocypris, with kungangensis and semotilus also being likely valid species, 1. Nakamu a M Cyprinidfishes of Japan. ResearchInstitute of being distantlyrelated to the former. The genetic distances NaturalResources, Tokyo, 1969. among them (D= 0.27?1.11) were a little higher than the 2. Nakabo T (ed). of Japan with pictorial keys to the inter-specificlevel determined for American cyprinids, D species,2nded TokaiUniversity Press, Tokyo, 2000. = 0.04?0.97 (mean 0.20). 8) On the other hand, the 3. Kim IS. Illustratedencyclopedia of fauna & flora of KoreaVol. lagowskii group appeared to be genetically heterogeneous 37. Freshwater fishes. Ministry of Education,Korea, Seoul, and paraphyletic, suggesting a possible subdivisionof this 1997.(in Korean). group. In fact, the genetic distance among the lagowskii 4. ChereshnevIA. Biogeographyof freshwaterfish fauna in the group (D = 0.31) corresponded to the species' level RussianFar East Dalnauka,Vladivostok,1998. (in Russian). determined for cyprinids. 8) 5. Howes GJ. A revised synonymy of the genus Consequently, the northern Far East phoxinin Pharinus Rafinesque, 1820 (Teleostei: Cyprinidae) with minnows should now be seen to represent two genera, comments on its relationshipsand distribution Bull. British including six species and one unresolved group; Phoxinus Mus.Nat Hist. (Zool.)1985;48: 57-74. phoxinus (Linnaeus), Rhynchocypris oxycephalus 6. SaitouN, Nei M The neighborjoining method:A new method (Sauvage and Dabry), R. percmurus (Pallas), R. for reconstructingphylogenetic trees. Mol. Biol. Evol. 1987;4: czekanowskii(Dybowski), R. kumgangensis (Kim) and R. 406-425. semotilus (Jordan and Starks), and the R. lagowskii 7. Nei M Genetic distancebetween populations.Am. Nat. 1972; (Dybowski) group. 106:283-292. Although there remains a possibility of subspecies 8. Buth DG, Dowling TE, Gold JR. Molecularand cytological i differentiation, such as between Japanese and Russian nvestigations.In: WinfieldU, Nelson JS (eds),Cyprinid fishes: populations of R. percumurs or between JapanKorean and systematics, biology and exploitation. Chapman & Hall, Russian populations of k oxycephalus, any such move, as London, 1991,pp. 83-126. well as classification of the lagowskii group, must follow