MORPHOLOGICAL AND GENETIC FEATURES OF STONE CRAYFISH Austropotamobius

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

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

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)

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

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)

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

Austropotamobius torrentium – morphology

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

MITOCHONDRIAN GENOME mitochondria - eucariotic organells of endosimbiontic origin mt. DNA – 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

16 S r. RNA gene - 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 mt. DNA (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

Study site – NP Žumberak-Samoborsko gorje

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

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

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

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 Gen. Bank (NCBI – National Centar for Biotechnology Information) Reagenses and laboratory material

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:

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

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

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

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

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

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

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

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

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

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

MORPHOMETRIC & MERISTIC CHARACTERISTICS - 61 individuals: 37 males, 24 females - grouping acording to molecular phylogenetic analyses: Dunav (8 m, 2 f), Žumberak/Plitvice/Bjelolasica (9 m, 10 f), Gorski Kotar (13 m, 11 f), Zeleni vir (7 m, 1 f) 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

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

Discrimination of male populations based on DA

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

Position of the thorns on the 3 rd 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

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

5. CONCLUSIONS Based on molecular- phylogenetic analyses of 16 S 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.