The existence on the territory

of Ukraine of two sympatric widespread species definitely different by their chromosome number

was proved based on the meiotic chromosome preparations. Besides the nominal species Pontastacus leptodactylus (Dana, 1852) with modal haploid chromosome number n = 93 we prove the existence of P. angulosus (Rathke, 1837) with n = 88

Гарбар, О. В.

Костюк, В. С.

Межжерін, С. В.

Костюк В. С.

Гарбар О. В.

Межжерін С. В.

Zhytomyr State University Library

UDC 595.384.1:576.316.353.7+591.492KARYOTYPES AND MORPHOLOGICAL VARIABILITY OF CRAYFISH PONTASTACUS LEPTODACTYLUS AND P. ANGULOSUS (MALACOSTRACA, DECAPODA)V. S. Kostyuk1, A. V. Garbar1, S. V. Mezhzherin21 Ivan Franko Zhytomyr State University,Velyka Berdychivska str., 40, Zhytomyr, 10008 Ukraine2 Schmalhausen Institute of Zoology, NAS of Ukraine,vul. B. Khmelnytskogo, 15, Kyiv, 01601 UkraineE-mails: 1saguaroklub@mail.ru, 2mezh@izan.kiev.uaKaryotypes and Morphological Variability of Crayfish Pontastacus leptodactylus and P. angulosus(Malacostraca, Decapoda). Kostyuk V. S., Garbar A. V., Mezhzherin S. V. – The existence on the ter-ritory of Ukraine of two sympatric widespread species definitely different by their chromosome numberwas proved based on the meiotic chromosome preparations. Besides the nominal species Pontastacus lep-todactylus (Dana, 1852) with modal haploid chromosome number n = 93 we prove the existence of P. angu-losus (Rathke, 1837) with n = 88.Ke y  wo r d s: crayfish, Astacidae, Pontastacus leptodactylus, Pontastacus angulosus, karyotype, mor-phometry.Êàðèîòèïû è ìîðôîëîãè÷åñêàÿ èçìåí÷èâîñòü äëèííîïàëûõ ðàêîâ Pontastacus leptodactylus è P. angu-losus (Malacostraca, Decapoda). Êîñòþê Â. Ñ., Ãàðáàð À. Â., Ìåææåðèí Ñ. Â. – Íà îñíîâàíèè àíàëèçàìåéîòè÷åñêèõ õðîìîñîìíûõ ïðåïàðàòîâ äîêàçàíî ñóùåñòâîâàíèå â Óêðàèíå äâóõ ñèìïàòðè÷åñêèõøèðîêî ðàñïðîñòðàí¸ííûõ âèäîâ, êîòîðûå ÷¸òêî îòëè÷àþòñÿ ïî ÷èñëó õðîìîñîì. Êðîìåíîìèíàòèâíîãî äëèííîïàëîãî ðàêà, Pontastacus leptodactylus (Dana, 1852), ñ ìîäàëüíûì ÷èñëîì õðî-ìîñîì â ãàïëîèäíîì íàáîðå íà óðîâíå n = 93 äîêàçàíî ïðèñóòñòâèå è óãëàñòîãî ðàêà, P. angulo-sus (Rathke, 1837), ñ ìîäàëüíûì çíà÷åíèåì n = 88.Êëþ÷åâûå  ñëîâà: ðå÷íûå ðàêè, Astacidae, Pontastacus leptodactylus, Pontastacus angulosus, êàðèî-òèï, ìîðôîìåòðèÿ.IntroductionThe taxonomy of European freshwater crayfish of Astacidae family has been remaining the disputable sub-ject among the scientists. One of the most intricate situations is the one concerning the long clawed crayfishcomplex where two alternative points of view were formed. On the one hand a number of authors (Brodskiy,1983; Starobogatov, 1995) assigned the long clawed crayfish to the single genus Pontastacus Bott, 1950 con-taining considerable number of species and subspecies, on the other hand the only one Astacus leptodactylusEschscholtz, 1823 species was considered (Gherardi and Holdich, 1999; Taylor, 2001; Souty-Grosset, 2006),and it could be the species complex as some authors suggested (Holdich, 2002).Such a disputable situation was caused mostly by strong morphometric variability of crayfish. As a resultthere were some attempts of using more stable characteristics in forming this group system, for instance, kary-ological and molecular genetic ones.Crayfish (and Crustaceans in general) are considered to be the group difficult for cytogenetic studies asmost of its species have relatively large diploid chromosome number (for example Pacifastacus leniskulus (Dana,1852) has 2 n = 376 (Niiyama, 1962)). As a result different authors’ data concerning the crayfish karyotypesdiffer significantly. In this connection the recent issue of Croatian scientists (Mlinarec et al., 2011) notes 2n =180 diploid chromosome number for long clawed crayfish. Consequently, the haploid number of this speciesmay come to n = 90 – the fact that does not correspond to the results of earlier studies (Silver and Cukerzis,1964) showing this species haploid number n = 184.The recent molecular-genetic research results prove the idea of long clawed crayfish being a species com-plex (Sinclair et al., 2004; Crandall and Buhay, 2007), though the taxonomic conclusions are not mentionedin these studies.Vestnik zoologii, 47(3): 205—210, 2013DOI 10.2478/vzoo-2013-0020Ôàóíà è ñèñòåìàòèêàElectrophoretic and morphological studies of long clawed crayfish on the territory of Ukraine revealedtwo groups clearly different from each other on the basis of a number of parameters. As to morphological fea-tures these forms correspond to the diagnosis of nominal species Pontastacus leptodactylus (Eschscholtz, 1823)and P. angulosus (Rathke, 1837), the taxonomic status of which is quite questionable. The allozyme analysisshowed these two forms significantly different on the basis of the trait frequency of three polymorphic loci whiletheir areals are widely overlapped (Mezhzherin et al., 2012). Thus these forms are reproductively isolated enough.The aim of our study was to carry out the morphological and karyological analysis of two studied speciesof crayfish on the territory of Ukraine.Material and methodsThe analyzed material was gathered in spring-to-autumn periods of 2010—2012 years according to thecommonly accepted methods (Brodsky, 1981). Eight samples over the territory of Ukraine were studied(table 1).The genetically marked adult males indetified according to keys (Starobogatov, 1995) as nominal speciesP. leptodactylus (65 specimens) and P. angulosus (40 specimens) were used for kariological studies.Chromosome preparations were made from the testis following the technique formerly used for investi-gations of other astacid species (Mlinarec et al., 2011; Murofushi et al., 1984). The animals were injected with0.05 % colchicine 5.5—6 hours before the dissection. Small pieces (1—2 mm3) of male gonads were placed for30 minutes in KCl (1 M) solution and fixed in the 1 : 3 mixture of glacial acetic acid and ethanol. Chromosomepreparations were obtained by the squash method with quick freezing (Sharma and Sharma, 1972). Dried slideswere stained with Giemsa-Romanovsky stain in 0. 01M phosphate buffer (pH 6.8). The chromosome spreadswere analysed with a “Delta optical genetic pro bino USB” microscope (16 x 100).Twenty-five standard quantitative indexes for morphological analysis were used (Sint et al., 2007).Statistical processing of obtained data was carried out by means of a package of applied statistical programsPAST 1.18.Results and discussionKaryological analysis showed that studied groups (defined by the morphological char-acteristics as P. leptodactylus and P. angulosus; fig. 1) differed clearly by chromosome num-ber (fig. 2).So it was established that the haploid chromosome number of P. leptodactylus wasn = 93 chromosomes (37 meiosis diakinesis stage plates were analyzed; fig. 2, b), whileP. angulosus was characterized by n = 88 haploid chromosome number (35 meiosis diaki-nesis stage plates were analyzed; fig. 2, a).The obtained results come close to the Hungarian scientists’ data (Mlinarec et al.,2011) showing the diploid chromosome number 2n = 180 for À. leptodactylus sensu lato.At the same time the studied members of this complex are characterized by different hap-loid chromosome number. Thus the further studies of the genetically marked materialappear to be important as they may allow avoiding the mistakes in studying the modalchromosome number of the individual members of this complex.We showed on the example of the previous analysis of the combined samples thatP. leptodactylus and P. àngulosus have sufficiently clear differences in a number of mor-206 V. S. Kostyuk, A. V. Garbar, S. V. MezhzherinTa b l e 1. Material used in the studyÒ à á ëèö à 1. Ìàòåðèàë, èñïîëüçîâàííûé â èññëåäîâàíèèSpecies Localities CoordinatesNumberof speci-mensP. leptodactylus r. Styr, Lutsk 50.72026î N, 25.36348î E 20Pond, Dolyna, Ivano-Frankivsk region 48.97740î N, 23.98616î E 15r. Pivdennyi Bug, Vinnytsa 49.23116î N, 28.48223î E 16r. Goryn, Zbuzh, Rivne region 50.98210î N, 26.32479î E 14P. angulosus Pond, Mali-Zagaitsi, Ternopil region 50.01337î N, 26.02979î E 21Water reservoir, Burchtyn, Ivano-Frankivsk region 49.23032î N, 24.67375î E 26Pond, Dubivtsi, Ivano-Frankivsk region 49.08016î N, 24.79717î E 19Pond, Ikva, Ternopil region 50.10880î N, 25.63917î E 17207Karyotypes and Morphological Variability of Crayfish Pontastacus leptodactylus...Fig. 1. Studied astacids species: a – P. angulosus; b – P. leptodactylus.Ðèñ. 1. Èññëåäîâàííûå âèäû: a – P. angulosus; b – P. leptodactylus.Fig. 2. Crayfish karyotypes: à – diakinesis (n = 88) of P. angulosus; b – diakinesis  (n = 93) of P. leptodacty-lus; c – haploid chromosome number distribution.Ðèñ. 2. Êàðèîòèïû äëèííîïàëûõ ðàêîâ: à – äèàêèíåç (n = 88) P. angulosus; b – äèàêèíåç (n = 93)P. leptodactylus; c – ðàñïðåäåëåíèå êîëè÷åñòâà ãàïëîèäíûõ õðîìîñîì.010203040506082 84 85 86 87 88 89 90 91 92 93 94 95 96nFrequency, %P. angulosusP. leptodactyluscphometric parameters (Mezhzherin et al., 2012). However, the nature of this charactervariability remains unclear. So we carried out the comparative morphological analysis offour largest samples of each species from geographically separated populations.The results of discriminate analysis of size characteristics and morphometric index-es combination (table 2) prove the high level discrimination of geographically separat-ed populations.Meanwhile, the populations belonging to one species form the compact groups thatare nearly not overlapped (fig. 3). Therefore in case of the analysis of these species unit-ed samples their discrimination level naturally comes to 96.62 %.It is evident from fig. 3 the first canonical function makes the greatest contributioninto the discrimination of the studied species and well-correlates with number of size param-eters and morphometric indexes (table 3). Most of these parameters are related to theclaw, carapace and abdomen proportions.The allozyme variability analysis of P. leptodactylus sensu lato (Mezhzherin et al.,2012) showed that P. leptodactylus and P. àngulosus differred apparently on the basis ofalleles’ frequencies of three polymorph loci. Here the main differentiated feature is the208 V. S. Kostyuk, A. V. Garbar, S. V. MezhzherinTa b l e 2. The reliability of the studied crayfish population discrimination on morphological parametersÒ à á ëèö à 2. Äîñòîâåðíîñòü ðàçëè÷èé èññëåäîâàííîé ïîïóëÿöèè äëèííîïàëûõ ðàêîâ ïî ìîðôîëîãè÷åñêèìïàðàìåòðàìNo t e . P. a. 1 – P. a. 4 – population of P. angulosus; P. l. 1 – P. l. 4 – population of P. leptodactylus.Population % P. a. 1 P. a. 2 P. a. 3 P. a. 4 P. l. 1 P. l. 2 P. l. 3 P. l. 4P. a. 1 95.24 20 0 0 0 0 0 1 0P. a. 2 92.31 0 24 2 0 0 0 0 0P. a. 3 94.74 0 1 18 0 0 0 0 0P. a. 4 88.24 2 0 0 15 0 0 0 0P. l. 1 90.00 0 0 0 0 18 1 0 1P. l. 2 86.67 0 0 0 1 0 13 1 0P. l. 3 100.00 0 0 0 0 0 0 16 0P. l. 4 100.00 0 0 0 0 0 0 0 14Total 93.24 22 25 20 16 18 14 18 15Fig. 3. The distribution of the studied crayfish samples in the field of first two canonical functions.Ðèñ. 3. Ðàñïðåäåëåíèå îáðàçöîâ èññëåäîâàííûõ äëèííîïàëûõ ðàêîâ â ïîëå ïåðâûõ äâóõ êàíîíè÷åñêèõôóíêöèé.P. angulosus 1P. angulosus 2P. angulosus 3P. angulosus 4P. leptodactylus 1P. leptodactylus 2P. leptodactylus 3P.Root 1Root 2leptodactylus 46420-2-4-6-8-6-4-20246allelic variants of Es-2 locus. Pontastacus leptodactylus is characterized by Es-21°° type,but P. angulosus – by Es-211°. The last case allele product is most likely overlapped withEs-3 locus product that is invariant for these species.As the areals of P. leptodactylus and P. angulosus widely overlap, the significant dif-ferences between two population groups could be observed only in case of their reliablereproductive isolation. Though it is impossible in this case to avoid the limited introgres-sive hybridization because of the genetic differences peculiarities. ConclusionsThus, the karyological, morphometric and biochemical-genetic data prove the exis-tence of two widespread and common species of the genus Pontastacus – P. leptodacty-lus and P. angulosus – on the territory of Ukraine. The scientists’ views on taxonomicstatus of the last one are disputable. So, S. Brodsky (1983) considered it only as a formof the nominative subspecies – P. leptodactylus leptodactylus f. angulosus, whileYa. Starobogatov (1995) considered it as the separate species – P. angulosus. The resultsof our study proved the last mentioned point of view. However, the status of the otherforms of long-clawed crayfish discussed by Ya. Starobogatov as the separate species remainsdisputable and requires the further studies by means of modern methods.ReferencesBrodsky S. Y. Freshwater crayfishes // Fauna Ukrainy. – Kiev : Naukova Dumka, 1981. – Vol. 26, is. 3. –211 p. – Ukrainian : Áðîäñüêèé Ñ. ß. Ð³÷êîâ³ ðàêè.Brodsky S. Y. On the systematics of palaearctic crayfishes (Crustacea, Astacidae) // Freshwater Crayfish. –1983. – N 5. – P. 464—469.Crandall K. A., Buhay J. E. Global diversity of crayfish (Astacidae, Cambaridae, and Parastacidae – Decapoda)in freshwater // Hydrobiologia. – 2008. – N 595. – P. 295—301.Holdich D. M. Distribution of crayfish in Europe and some adjoining countries // Bulletin Francais De La PecheEt De La Pisciculture. – 2002. – N 367. – P. 611—650.Gherardi F., Holdich D. M. 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Karyological study of the red swamp crayfish and the Japanese lobsterby air-drying method // Proc. Japanese Acad. – 1984. – N 60. – P. 306—309.209Karyotypes and Morphological Variability of Crayfish Pontastacus leptodactylus...Ta b l e 3. The most significant correlations (r) of the first canonical function with morphometric characteristicsÒ à áëèöà 3. Íàèáîëåå çíà÷èòåëüíûå êîððåëÿöèè (r) ïåðâûõ êàíîíè÷åñêèõ ôóíêöèé ñ ìîðôîìåòðè÷åñêèìèõàðàêòåðèñòèêàìèNo t e . ABH – abdomen height, ABL – abdomen length, ABW – abdomen width, ARW – areolarwidth, CFL – claw finger length, CLL – claw length, CLW – claw width, CPL – claw palm length, HEL –head length, ROL – rostrum length, ROW – rostrum width, TEL – telson length.Parameters CLWl ROL HEL TEL ABWr —0.29 0.35 —0.23 0.25 0.45Parameters ARW CLLr\CPLr CFLr/CPLr CLLl/CLWl CLLl/CPLlr —0.29 —0.35 —0.36 0.33 —0.27Parameters CFLl/CPLl ROL/ROW HEL/ARL ABL/ABW ABL/ABHr —0.26 0.46 —0.38 —0.50 0.28Niiyama H. On the unprecedently large number of chromosomes of the crayfish Astacus trowbridgii Stimpson //Annot. zool. Japonenses. – 1962. – N 35. – P. 229—233.Silver D., Cukerzis J. M. The number of chromosomes in Astacus leptodyctylus // Tsitologiya. – 1964. – N 6. –P. 631—633.Sinclair E. A., Fetzner J. W., Buhay J., Crandall K. A. Proposal to complete a phylogenetic taxonomy and sys-tematic revision for freshwater crayfish  (Astacidea) // Freshwater Crayfish. – 2004. – N 14. – P. 21—29.Sint D., Dalla-Via J., Füreder L. Phenotypical characterization of indigenous freshwater crayfish populations //J. Zoology. – 2007. – 273, N 2. – P. 210—219.Sharma A. K., Sharma À. Chromosome techniques – theory and practice. – London : Butterworth PublishersLtd., 1972. – 575 p.Souty-Grosset C., Holdich D. M., No¸l P. Y. et al. Atlas of Crayfish in Europe. – Paris : Muséum nationald’Histoire naturelle (Patrimoines naturels), 2006. – 64 p.Starobogatov Y. I. Taxonomy and geographical distribution of crayfishes of Asia and East Europe (Crustacea,Decapoda, Astacoidei) // Arthropoda Selecta. – 1995. – 4, N 3—4. – P. 3—25.Taylor C. A. Taxonomy and conservation of native crayfish stocks // Biology of Freshwater Crayfish /Ed. D. M. Holdich. – Oxford, 2001. – P. 236—257.Received 1 March 2013Accepted 20 May 2013210 V. S. Kostyuk, A. V. Garbar, S. V. Mezhzherin

KARYOTYPES AND MORPHOLOGICAL VARIABILITY OF CRAYFISH PONTASTACUS LEPTODACTYLUS AND P. ANGULOSUS (MALACOSTRACA, DECAPODA)

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