Morphological and Genetic Features of Stone Crayfish Austropotamobius torrentium from Žumberak-Samoborsko Gorje Nature P

1. MORPHOLOGICAL AND GENETIC FEATURES OF STONE CRAYFISH Austropotamobius torrentium (Shrank, 1803) FROM ŽUMBERAK – SAMOBORSKO GORJE NATURE PARK Nina Marn, Symbiose 2010

2. Contents: 1. INTRODUCTION Austropotamobius torrentium, family Astacidae Phylogeny, mithochondrian genom Study site – NP Žumberak-Samoborsko gorje 2. AIMS OF THE STUDY 3. MATERIALS AND METHODS 4. RESULTS AND DISCUSSION Molecular – phylogenetic and morphological analyses 5. CONCLUSION

3. 1. INTRODUCTION Familiy Astacidae - systematics Phylum: ARTHROPODA Class: CRUSTACEA Order: DECAPODA Suborder: ASTACIDA Family: PARASTACIDAE Family: CAMBARIDAE Family: ASTACIDAE Genus: ASTACUS Astacus astacus Astacus leptodactylus Astacus pachypus Genus: AUSTROPOTAMOBIUS Austropotamobius pallipes Austropotamobius torrentium (Austropotamobius italicus)

4. Astacidae in Europe • Protection of native species: A. pallipes A. torrentium A. astacus Foreign species: Pacifastacus leniusculus Orconectes limosus Procambarus clarkii Cherax sp. • European Comission Environment Directorate • IUCN • Bern Convention

5. Astacidae in Croatia • Native species: • Noble (river) crayfish (1) • Danube crayfish (2) • Whiteclawed crayfish (3) • Stone crayfish (4) Foreign species: • spiny-cheek crayfish (5) (North Am, plague) • Signal crayfish (6)

6. Austropotamobius torrentium – dispersion and species ecology • Smallest species in the Astacidae family (~11 cm) -> not for commercial exploitation • Less infected with crayfish plague (characteristic habitat) • Active at night • Reproduction in fall • Population decline due to human influence

7. Austropotamobius torrentium – morphology

8. PHYLOGENY - A scientific discipline that explores evolutionary relations between different (groups of) organisms - Analysis of phenotipic and genetic features of certain taxon • Phenotyp changes point to the beginning of diferentiation • Short-term concequences of environmental impact • genotype and fenotype changes are not necessarily connented • molecular phylogeny evolutionary theory – all organisms evolved from a single ancestor; current biodiversity is a consequence of different evolutionary mechanisms

9. MITOCHONDRIAN GENOME mitochondria - eucariotic organells of endosimbiontic origin mtDNA – circular doublestrainded molecule, size 14-42 kb(16 569 bp) • genes for 37 molecules • Advantages: • Easy to isolate • Large number of copies • No gene recombination or intrones • Mother-based inheritance • Relatively rapid evolution compared to core DNA

10. 16S rRNAgene - codes the RNA of large subunit of mitochondrian ribosome - structure more basic than core RNA, present in only one copy in the genome - conserved in the primary structure: not significally altered during the evolutionay past of mtDNA (ancient evolutionary occurences) • COI gene - codes the subunit I of citocrom-oksidase c complex (oks. fosforilation) - well studied sequence, stable universal gene primers - highly conserved aminoacid sequence among phyla: ancient evolutionary occurences - stronger phylogenetic signal than other mt genes

11. Study site – NP Žumberak-Samoborsko gorje

12. Study site – NP Žumberak-Samoborsko gorje • 90% of the area is karst – dolomites and limestone (carbonates) • Frequent rivers and streams • Vegetation: forests (beech, oak, hornbeam), grasslands, paddocks, meadows, pastures, cultivated areas • Great variety of animal and plant species

13. 2. AIMS OF THE STUDY Measuring the level of genetic diversity between populations of the stone crayfish in the area of Žumberak-Samoborsko gorje Nature Park, and corelation with other stone crayfish populations in Europe Identification of morphological (morfometric & meristic) characteristics of the populations and sorting out those morphological features that significantly distinguish crayfish of different populations Combining the results of molecular - phylogenetic and morphological analysis Analising the results in context of philogeography and protection of Autropotamobius torrentium

14. 3. MATERIALS & METHODSMaterial • Animal material - 61 individual of the stone crayfish (Austropotamobius torrentium) from 9 locations in the “Žumberak-Samoborsko gorje” NP

15. Analised sequences - isolated from captured individuals of A. torrentium (61) from NP Žumberak – Samoborsko gorje - isolated from individuals gathered in previous studies (mostly in Croatia and Bosnia and Hercegovina, Macedonia, Romania and Bulgaria) - taken from GenBank (NCBI – National Centar for Biotechnology Information) • Reagenses and laboratory material

16. Methods • DNA isolation, mark and code added to each sample • PCR of DNA fragments • DNA gel electrophoresis • DNA sequencing • Molecular-phylogenetic analyses a) Bayesian analysis b) Calculation of sequence divergences (p) and nucleotid diversity () • Morphometric and meristic measurments and analyses Početnice:

17. Morphometric & meristic measurments Continuous characteristics • 23 (30) morphometric characteristics • Statistical analyses of data(Excel, Statistica)

18. Morphometric & meristic measurments distinctive (discrete) characteristics • 5 meristic characteristics • Statistical analyses of data

19. 4. RESULTS AND DISCUSSION MOLECULAR – PHYLOGENETIC ANALYSES Alignment and analyses of sequences Grouping homologuos sequences; Bayesian analysis

20. Bayesian analysis – reconstruction of phylogenetic trees ŽPB GK ZV Dunav GK Dunav ŽPB ZV

21. Haplotype (geographical) groups of stone crayfish (A. torrentium) in NP Žumberak-Samoborsko gorje

22. Bayesian analysis – populations in Europe • six 16S haplogroupes • five COI haplogroupes

23. Haplotype (geographical) groups of stone crayfish (A. torrentium) in Europe

24. Sequence divergences (p) and nucleotid diversity () • 16S: • A. pallipes - A. italicus0,026-0,043, (Fratini et al), 0,046-0,047 (Grandjean et al) • A. torrentium - A. pallipes0,076-0,081 • A. torrentium - A. italicus0,072-0,086(Fratini et al.) COI: • A. pallipes - A. italicus0,069 - 0,07 • A. torrentium - A. pallipes • Astacus sp. (Trontelj et al)

25. Sequence divergences (p) and nucleotid diversity () • north-west Dinarids – high genetic diversity (A. pallipes, Asellus aquatica) • Dunav river basin – genetic uniformity

26. Time of divergence(COI) Possible scenarios of stone crayfish group divergences: Separation of Tethys, A.torrentium migrates to Balkan area Genesis and drainage of Panonian lake tectonic disturbance, karstification Climate oscilations, ice ages (refugiai)

27. MORPHOMETRIC & MERISTIC CHARACTERISTICS - 61 individuals: 37 males, 24 females - grouping acording to molecular phylogenetic analyses:Dunav (8m, 2f), Žumberak/Plitvice/Bjelolasica (9m, 10f), Gorski Kotar (13m, 11f), Zeleni vir (7m, 1f) • MORPHOMETRICS: - normalization (POL = HEL + ARL) - Spearman's coefficient of correlation (simmetry) - Mann – Whitney U test (M/f) : -> sexual dimorphism • Differences amnong haplogroupes (Mann –Whitney U test) • a) males: chela shape and carapax shape (some individuals)(Dunav-ZV, Dunav-GK, ŽPB-ZV, GK-ZV) • b) females (ŽPB-GK): karapax shape

28. Multivariantn discriminant analysis Sint et al. (2005, 2006) – chelae and cephalotorax shape - rostrum, apex (Maguire et al., 2003, Sint et al., 2005;) - Trontelj et al. (2005) – rostrum shape specific for individuals in upper Kupa basin

29. Discrimination of male populations based on DA

30. MERISTICS - Wald – Wolfowitz test (sex) - Spearman's coefficient of correlation (simmetry) Meristic characteristics that distinguish populations Thorns on lower egzopodit (scaphopod) side are present only in this crayfish species

31. Position of the thorns on the 3rd maxiliped • Karaman, Maguire et al. - to some extent reflects nucleotid diversity of a population: max number of combinations in GK and ŽPB - greatest diference in thorn position: between Dunav and Zeleni vir groups - number abd shape of thorns (Karaman), position of thorns

32. Number of statisticaly significant morpfological differences between populations is larger as gropus grow more geneticaly and geographicaly distant: Max nr.of differences: Dunav -Zeleni vir, Dunav - Gorski Kotar Min nr.of differences: ŽPB - ZV, ZV – GK Coincides with the results of time divergance analyses • PROTECTION GUIDELINES - genetic diversity as a part of biodiversity - haplogroups of central and northern Dinarides (GK, ŽPB, LD, ZV) could be observed as evolutionary significant units (ESU) - populations in NP Žumberak-Samoborsko gorje could be criptic species/subspecies

33. 5. CONCLUSIONS Based on molecular- phylogenetic analyses of 16S and COI mt genes, four distinct genetic groups (haplogroups) were found in the area of NP Žumberak-Samoborsko gorje. Based on molecular clock for mt COI gene, it was estimated that the first divergence occured between two larger groups (made of haplogroups) between 3,5 and 5 mya. Haplogroups than diverged a bit later (between 4,3 and 1,65 mya). Results of statistical analyses of morphological features are in accord with the results of molecular-phylogenetic analyses. Occurances of genetic and morphological diffrences between geographical groups lead to a conlusions that the species A. torrentium might be made of more than one subspecies or even species that are to be determined Biodiversity this big on a small geographical area of Nature Park Žumberak – Samoborsko gorje is a unique biological phenomenon on a European scale and will demand special care (protection and conservation-wise) in the near future.

34. Thank you for your attention!