Molecular identification •Species, individual, sex http://upload.wikimedia.org/wikipedia/commons/thumb/2/23/Female.svg/120px-Female.svg.png http://upload.wikimedia.org/wikipedia/commons/thumb/c/c1/Male.svg/100px-Male.svg.png http://upload.wikimedia.org/wikipedia/commons/thumb/2/23/Female.svg/120px-Female.svg.png http://upload.wikimedia.org/wikipedia/commons/thumb/c/c1/Male.svg/100px-Male.svg.png http://upload.wikimedia.org/wikipedia/commons/thumb/c/c1/Male.svg/100px-Male.svg.png http://www.nhbs.com/images/jackets_resizer_xlarge/19/196496_3.jpg Species identification DNA barcoding Definition of species •Biological species concept (Mayr, 1942) – RIM = post- or prezygotic barriers of gene flow (not always so simple - hybrid zones, alopatric speciation, asexual species atd.) •Other concepts (e.g. typological = morphological, genetic, phylogenetic aj.) • •Why it is necessary to identify species (genetically)? •Conservation biology needs names – it is necessary to decide which taxonomic unit (species) requires conservation attention •Forensic genetics, developemental stages without identification traits, identification of prey items, etc. Barcoding is a standardized approach to identifying plants and animals by minimal sequences of DNA, called DNA barcodes DNA Barcode: A short DNA sequence, from a uniform locality on the genome, used for identifying species The Barcoding Pipeline first idea in 2003 CBOL CBOL in 2005 iBol iBOL 2010-2015 500 000 species barcoded in 2015 world species known and unknown 26dec2004 Why barcode animal and plant species? Crisis of biodiversity and classical taxonomy DNA barcoding is important part of „integrative taxonomy“ Integrative taxonomy What are the benefits of standardization? why barcode standardization Suitable standard for animals → mtDNA Why barcode animals with mitochondrial DNA? Mitonuclear coevolution as the genesis of speciation (?) Rand et al. 2004 red = mtDNA blue = nDNA Hill 2016 Why barcode animals with mitochondrial DNA? •Four properties make mitochondrial genomes especially suitable for identifying species why mitochondria 1. Greater differences among species, on average 5- to 10-fold higher in mitochondrial than in nuclear genes (lower Ne for mtDNA). Thus shorter segments distinguish among species, and because shorter, less expensively. •2. Copy number There are 100-10,000 more copies of mitochondrial than nuclear DNA per cell, making recovery, especially from small or partially degraded samples, easier and cheaper. • •3. Relatively few differences within species in most cases. Small intraspecific and large interspecific differences signal distinct genetic boundaries between most species, enabling precise identification with a barcode. • •4. Introns, which are non-coding regions interspersed between coding regions of a gene, are absent from mitochondrial DNA of most animal species, making amplification straightforward. Nuclear genes are often interrupted by introns, making amplification difficult or unpredictable. •For animals, a 658 base-pair fragment of the mitochondrial gene, cytochrome oxidase subunit I (mtCOI) – consensus for iBOL consortium; 18S rDNA – Nematoda etc. •For plants, mitochondrial genes do not differ sufficiently to distinguish among closely related species. Promising markers are genes on cpDNA: matK and rbcL •For bacteria, a 16S-rDNA emerges as very useful marker (especially when using next-generation sequencing) • •For particular taxonomic groups, also other barcodes are widely used, e.g. cytochrome b for mammals Focus to date human chimp anopheles pip 26dec2004 Cytochrome c oxidase I (COI or CoxI) contains differences representative of those in other protein-coding genes Possible gains in accuracy or cost using a different protein-coding gene would likely be small. „barcoding gap“ What do barcode differences among and within animal species studied so far suggest? •barcodes identify most animal species unambiguously •approximately 2-5% of recognized species have shared barcodes with closely-related species - many of them hybridize regularly •in all groups studied so far, distinct barcode clusters with biological co-variation suggest cryptic species intra inter 28dec2004 Barcoding North American birds Tak co třeba znamená toto? Barcoding Hominidae2 Barcodes affirm the unity of the species Homo sapiens Comparisons show we differ from one another by only 1 or 2 nucleotides out of 648, while we differ from chimpanzees at 60 locations and gorillas at 70 locations. A barcoder? 1.Metagenomics -community of microorganisms -PCR of 16S (18S) rRNA -it is also possible to quantify (to some extent) 2. Diet composition -COI barcoding (carnivores) -chloroplast (cp)DNA (herbivores) Next generation sequencing of amplicons http://www.massgenomics.org/wp-content/uploads/2010/03/ion-torrent-sequencer-300x235.jpg 3. Analysis of contaminated samples A barcoder? ... COMING SOON What isn’t DNA Barcoding? •it is not intended to, in any way, supplant or invalidate existing taxonomic practice •it is not DNA taxonomy; it does not equate species identity, formally or informally, with a particular DNA sequence •it is not intended to duplicate or compete with efforts to resolve deep phylogeny (e.g., Assembling the Tree of Life, ATOL) Fly Didemnum Xmas_worm2 Fish2 What are the main limits to barcoding encountered so far? •horizontal gene transfer •gene tree vs. species tree •nuclear pseudogenes •hybrids – mtDNA introgression 1. Horizontal gene transfer File:Wolbachia.png Wolbachia within an insect cell (25-70% species of insects) http://upload.wikimedia.org/wikipedia/commons/f/fa/Protocalliphora.azurea.-.lindsey.jpg Results of nuclear and mitochondrial DNA do not match Horizontal transfer of mtDNA through Wolbachia (among closely related species, at the level of genera the barcoding is OK) AFLP mtDNA Ellipses = candidates for horizontal gene transfer Symbols correspond to the type of Wolbachia infection 2. Gene tree vs. species tree One gene (mitochondrial) is not (sometimes) sufficient for species delimitation https://www.math.duke.edu/mathbio/images/geneTreesInSpeciesTree.jpg http://2.bp.blogspot.com/-2Zby6vL-3SQ/TmpxkW6VECI/AAAAAAAABcs/GdsqmH7MTrQ/s400/Hemiplazie.gif Time to fix the polymorphisms = 2 Ne generations Incomplete lineage sorting Problems of species identification in young species Difficult to distinghuish from the gene flow (hybridization) Statistical species delimitation *BEAST (species trees) BEAST BPP and iBPP Lophuromys flavopunctatus complex in Ethiopia 4 nuclear markers (Komarova et al., submitted) (2 604 bp concatenated dataset) Sanger sequencing ddRADseq 15 623 informative loci (Mikula et al., in prep.) 100 100 100 100 100 100 99 100 100 100 100 100 100 100 100 100 97 44 88 100 100 94 95 100 98 92 83 91 94 77 86 77 brevicaudus flavopunctatus brunneus 2n = 68 melanonyx chrysopus menangeshae pseudosikapusi chercherensis 2n = 70 simensis 2n = 60 2n = 54 And what about mtDNA? ddRADseq 15 623 informative loci 100 100 100 100 100 100 99 100 100 100 100 100 100 100 100 100 brevicaudus flavopunctatus brunneus melanonyx chrysopus menangeshae pseudosikapusi chercherensis simensis 100 100 96 93 96 97 96 100 82 89 88 100 100 100 100 100 100 97 mtDNA cytochrome b (1140 bp) 2n = 68 2n = 70 2n = 60 2n = 54 ddRADseq 15 623 informative loci 100 100 100 100 100 100 99 100 100 100 100 100 100 100 100 100 brevicaudus flavopunctatus brunneus melanonyx chrysopus menangeshae pseudosikapusi chercherensis simensis 100 100 96 93 96 97 96 100 82 89 88 100 100 100 100 100 100 97 mtDNA cytochrome b (1140 bp) And what about mtDNA? „reticulate evolution“ 3. Pseudogenes Heterozygotes in mtDNA → be careful! NUMTS = „nuclear copy of mtDNA sequences Relatively often for cytochrome b How to recognize numt? - ultracentrifugation (fresh samples required) - the use of tissues with high proportion of mitochondria (e.g. muscles) - long-range PCR (or sequence complete mtDNA) - RT-PCR (pseudogenes are not transcribed) - indels, stop codons - cloning cryptic numts number of suggested barcoded taxa based on 3% divergence on COI with/without numts (identified by stop-codons and indels) http://www.mitochondrialncg.nhs.uk/images/3.jpg -well studied mitochondrial disorders in human -low Ne of mtDNA → usually fast fixation of new mutations – mitochondrial bottleneck - paternal leakage Heteroplasmy Paternal leakage •allele-specific real-time quantitative PCR (RT-qPCR) → heteroplasmie je asi častý jev •14 % jedinců, ale velmi nízká frekvence druhého haplotypu •paternal leakage 6 % 4. Introgression/replacement of mtDNA Berthier et al. 2006 Myotis myotis - Europe Myotis blythii - Asia http://www.naturfoto.cz/fotografie/andera/netopyr-velky-xxx4242.jpg http://zmmu.msu.ru/bats/rusbats/pictures/mblythi2.jpg Myotis blythii vs. Myotis myotis - mtDNA replacement M. myotis - Europe M. blythii - Asia male Myotis blythii vs. Myotis myotis - mtDNA replacement M. myotis - Europe M. blythii - Asia Tendency to back-crosses with males of M. blythii led to increase of proportion of M. blythii in Europe Colonizing (invasive) species often adopt mtDNA of original speices (Currat et al. 2008) male MLtree 98/99/1.00 70/-/- 94/100/1.00 100/100/1.00 96/96/1.00 -/97/- 66/77/0.95 89/93/1.00 A B C2 C1 D Příklad: Praomys cf. daltoni complex How many species? Four based on mtDNA genotype – cca 7% divergence (cyt b)? Two based on phenotype? C:\Documents and Settings\Pepa Bryja\Local Settings\Temporary Internet Files\Content.IE5\1VH7BA5N\MCAN04317_0000[1].wmf C:\Documents and Settings\Pepa Bryja\Local Settings\Temporary Internet Files\Content.IE5\1VH7BA5N\MCAN04317_0000[1].wmf C:\Documents and Settings\Pepa Bryja\Local Settings\Temporary Internet Files\Content.IE5\1VH7BA5N\MCAN04317_0000[1].wmf C:\Documents and Settings\Pepa Bryja\Local Settings\Temporary Internet Files\Content.IE5\1VH7BA5N\MCAN04317_0000[1].wmf C:\Documents and Settings\Pepa Bryja\Local Settings\Temporary Internet Files\Content.IE5\1VH7BA5N\MCAN04317_0000[1].wmf DSC_5869 C:\Documents and Settings\Pepa Bryja\Local Settings\Temporary Internet Files\Content.IE5\1VH7BA5N\MCAN04317_0000[1].wmf C:\Documents and Settings\Pepa Bryja\Local Settings\Temporary Internet Files\Content.IE5\1VH7BA5N\MCAN04317_0000[1].wmf daltoni - paraphyletic derooi - commensal Fig. 2: Phylogenetic relationships between haplotypes recovered by maximum likelihood (ML) analysis. Main clades are identified on the right border; broad lines separate presumed species.The numbers above branches represent ML and neighbour joining (NJ) bootstrap support, and Bayesian posterior probability, respectively. See text and Table 1 for more details on localities and haplotypes. NOT CORRECTED !!! Fig 1 zaklad.tif 48 51 41 7 37 12 17 13 20 27 19 21 31 8 35 26 16 24 11 30 33 38 29 34 1 49 44 50 46 40 3 2 6 47 43 45 39 42 67 71 61 68 55 54 57 56 53 32 10 23 15 60 63 58 18 70 66 62 65 64 82 83 86 72 75 88 84 73 77 85 78 74 80 91 4 Senegal river Niger river Volta river 79 89 90 Clade B Clade A Clade D Clade C1 Clade C2 SENEGAL GUINEA MALI BURKINA FASO GHANA BENIN TOGO IVORY COAST NIGER NIGERIA CAMEROON 69 36 14 25 9 5 59 81 76 87 52 22 28 C:\Documents and Settings\Pepa Bryja\Local Settings\Temporary Internet Files\Content.IE5\1VH7BA5N\MCAN04317_0000[1].wmf C:\Documents and Settings\Pepa Bryja\Local Settings\Temporary Internet Files\Content.IE5\1VH7BA5N\MCAN04317_0000[1].wmf C:\Documents and Settings\Pepa Bryja\Local Settings\Temporary Internet Files\Content.IE5\1VH7BA5N\MCAN04317_0000[1].wmf C:\Documents and Settings\Pepa Bryja\Local Settings\Temporary Internet Files\Content.IE5\1VH7BA5N\MCAN04317_0000[1].wmf C:\Documents and Settings\Pepa Bryja\Local Settings\Temporary Internet Files\Content.IE5\1VH7BA5N\MCAN04317_0000[1].wmf Phylogeographic structure at mtDNA Dahomey gap Fig. 1: Map of sampling points showing the distribution of principal phylogenetic clades identified on the basis of mtDNA analyses. Different symbols represent populations belonging to different clades: clade A (black triangles), clade B (dark purple circle), clade C1 (green triangles), clade C2 (yellow squares), and clade D (red circles). Numbers indicate collection sites. See Table 1 for description of haplotypes occurring at different localities. Rivers mentioned in the text are shown in blue. The presumed distribution of mtDNA clades is marked by dashed lines of particular colours. Partial mtDNA introgression in Dahomey gap Fig 1 zaklad.tif 48 51 41 7 37 12 17 13 20 27 19 21 31 8 35 26 16 24 11 30 33 38 29 34 1 49 44 50 46 40 3 2 6 47 43 45 39 42 67 71 61 68 55 54 57 56 53 32 10 23 15 60 63 58 18 70 66 62 65 64 82 83 86 72 75 88 84 73 77 85 78 74 80 91 4 Senegal river Niger river Volta river 79 89 90 Clade B Clade A Clade D Clade C1 SENEGAL GUINEA MALI BURKINA FASO GHANA BENIN TOGO IVORY COAST NIGER NIGERIA CAMEROON 69 36 14 25 9 5 59 81 76 87 52 22 28 What is species??? Fig. 1: Map of sampling points showing the distribution of principal phylogenetic clades identified on the basis of mtDNA analyses. Different symbols represent populations belonging to different clades: clade A (black triangles), clade B (dark purple circle), clade C1 (green triangles), clade C2 (yellow squares), and clade D (red circles). Numbers indicate collection sites. See Table 1 for description of haplotypes occurring at different localities. Rivers mentioned in the text are shown in blue. The presumed distribution of mtDNA clades is marked by dashed lines of particular colours. Fig 1 zaklad.tif 48 51 41 7 37 12 17 13 20 27 19 21 31 8 35 26 16 24 11 30 33 38 29 34 1 49 44 50 46 40 3 2 6 47 43 45 39 42 67 71 61 68 55 54 57 56 53 32 10 23 15 60 63 58 18 70 66 62 65 64 82 83 86 72 75 88 84 73 77 85 78 74 80 91 4 Senegal river Niger river Volta river 79 89 90 Clade B Clade A Clade D Clade C1 SENEGAL GUINEA MALI BURKINA FASO GHANA BENIN TOGO IVORY COAST NIGER NIGERIA CAMEROON 69 36 14 25 9 5 59 81 76 87 52 22 28 Morphology and ecology Fig. 1: Map of sampling points showing the distribution of principal phylogenetic clades identified on the basis of mtDNA analyses. Different symbols represent populations belonging to different clades: clade A (black triangles), clade B (dark purple circle), clade C1 (green triangles), clade C2 (yellow squares), and clade D (red circles). Numbers indicate collection sites. See Table 1 for description of haplotypes occurring at different localities. Rivers mentioned in the text are shown in blue. The presumed distribution of mtDNA clades is marked by dashed lines of particular colours. Fig 1 zaklad.tif 48 51 41 7 37 12 17 13 20 27 19 21 31 8 35 26 16 24 11 30 33 38 29 34 1 49 44 50 46 40 3 2 6 47 43 45 39 42 67 71 61 68 55 54 57 56 53 32 10 23 15 60 63 58 18 70 66 62 65 64 82 83 86 72 75 88 84 73 77 85 78 74 80 91 4 Senegal river Niger river Volta river 79 89 90 Clade B Clade A Clade D Clade C1 SENEGAL GUINEA MALI BURKINA FASO GHANA BENIN TOGO IVORY COAST NIGER NIGERIA CAMEROON 69 36 14 25 9 5 59 81 76 87 52 22 28 Karyotypes Fig. 1: Map of sampling points showing the distribution of principal phylogenetic clades identified on the basis of mtDNA analyses. Different symbols represent populations belonging to different clades: clade A (black triangles), clade B (dark purple circle), clade C1 (green triangles), clade C2 (yellow squares), and clade D (red circles). Numbers indicate collection sites. See Table 1 for description of haplotypes occurring at different localities. Rivers mentioned in the text are shown in blue. The presumed distribution of mtDNA clades is marked by dashed lines of particular colours. Fig 1 zaklad.tif 48 51 41 7 37 12 17 13 20 27 19 21 31 8 35 26 16 24 11 30 33 38 29 34 1 49 44 50 46 40 3 2 6 47 43 45 39 42 67 71 61 68 55 54 57 56 53 32 10 23 15 60 63 58 18 70 66 62 65 64 82 83 86 72 75 88 84 73 77 85 78 74 80 91 4 Senegal river Niger river Volta river 79 89 90 Clade B Clade A Clade D Clade C1 SENEGAL GUINEA MALI BURKINA FASO GHANA BENIN TOGO IVORY COAST NIGER NIGERIA CAMEROON 69 36 14 25 9 5 59 81 76 87 52 22 28 Mitochondrial DNA + microsatellites in Benin + karyotypes Fig. 1: Map of sampling points showing the distribution of principal phylogenetic clades identified on the basis of mtDNA analyses. Different symbols represent populations belonging to different clades: clade A (black triangles), clade B (dark purple circle), clade C1 (green triangles), clade C2 (yellow squares), and clade D (red circles). Numbers indicate collection sites. See Table 1 for description of haplotypes occurring at different localities. Rivers mentioned in the text are shown in blue. The presumed distribution of mtDNA clades is marked by dashed lines of particular colours. Fig 1 zaklad.tif 48 51 41 7 37 12 17 13 20 27 19 21 31 8 35 26 16 24 11 30 33 38 29 34 1 49 44 50 46 40 3 2 6 47 43 45 39 42 67 71 61 68 55 54 57 56 53 32 10 23 15 60 63 58 18 70 66 62 65 64 82 83 86 72 75 88 84 73 77 85 78 74 80 91 4 Senegal river Niger river Volta river 79 89 90 Clade B Clade A Clade D Clade C1 SENEGAL GUINEA MALI BURKINA FASO GHANA BENIN TOGO IVORY COAST NIGER NIGERIA CAMEROON 69 36 14 25 9 5 59 81 76 87 52 22 28 Splitting approach taking morphology and ecology into account – the reproductive barriers between clades A, B, C1, and D remains to be identified Fig. 1: Map of sampling points showing the distribution of principal phylogenetic clades identified on the basis of mtDNA analyses. Different symbols represent populations belonging to different clades: clade A (black triangles), clade B (dark purple circle), clade C1 (green triangles), clade C2 (yellow squares), and clade D (red circles). Numbers indicate collection sites. See Table 1 for description of haplotypes occurring at different localities. Rivers mentioned in the text are shown in blue. The presumed distribution of mtDNA clades is marked by dashed lines of particular colours. Identification of individuals DNA fingerprinting (DNA profiling) Identification of individuals – why? •if we do not see the individual •non-invasive genetics – elusive animals, samples from faeces, urines, hairs – can be joined with individual variation of their diet •forensic genetics – identification of DNA in animal products, poachers, etc. • •species conservation – e.g. in falconary (confirmation of parentage) Microsatellites •Tandem repetitions of short motifs • •DNA extraction • •PCR • •Detection of alleles → sequencer, fragment analysis CTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTT CTTTCTTTCTTTCTTTC CTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTT CTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTT tools_1a 3LOKCELE detector laser Individual human identification https://tools.lifetechnologies.com/content/sfs/prodImages/high/4473287_and_4473289_FINAL650x600.jpg http://www.cstl.nist.gov/strbase/kits/Identifiler.JPG http://www.bodetech.com/wp-content/uploads/2011/01/analyze-the-samples.png 16 loci = reliable individual identification (Euro-American population) Identification of individuals depends on level of polymorphism •multilocus microsatellite fingerprinting – power estimated as „probability of identity“ (P(ID)) (Waits et al. 2001) – e.g. GenAlex program • Number of loci (H=0.6) Random Sibs •pilot studies with tissue samples are required to identify P(ID) in a population studied by e.g. non-invasive methods Brown bears in Pyrenees Taberlet et al. 1997 •Faeces and hairs • •24 microsatellites • •4 males and 1 female with unique multilocus genotypes (more than according footprints and photos) • •Multiple-tube approach Spatial activity of otters •P. Hájková – PhD thesis • vydra_snezna Identified individuals - Hornád, NP Slovenský Raj mapa_komplet_orezana SR 1 SR 2 SR 3 SR 4 SR 5 SR 6 SR 7 SR 8 SR 9 SR 10 1 km vydra1 vysek02 kopie_orezana Ind. SR 3 otter_otocena_hneda 5. 4. 2003 4. 11. 2003 10. 11. 2003 11. 11. 2003 30. 12. 2003 500 m Hornad Spatial activity in mountain habitat Human forensic genetics •Pozůstatky vojáků z války Vietnam a Korea Identifikace na základě mtDNA příbuzných osob (lze jen někdy) V současnosti: vzorek DNA (krve) při odvodu, jiné markery Armed Forces Repository of Specimen Samples for the Identification of Remains • •Soudní pře Clinton-Lewinská Pozůstatky ruského cara Nikolaje II •Kriminalistika • •Oběti tragických událostí • • • 461921_Clinton_Lewinski News Czar-Nicholas-II- Klony Bambus Sasa senanensis •Suyama et al. 2000 • •Plocha 10 hektarů • •AFLP • •22 klonů • •Klon na ploše 300 m v průměru s_senanensis_M Slavní klonální bezobratlí •Rotifera – Bdelloidea • •Ostracoda (Darwinula) • •Partenogenetické klony vysokého stáří (milióny let) Darwinula stevensoni •organismy složené z buněk s různými genotypy • •Dictyostelium discoideum chimérismus je pravidelná součást života Genetické chiméry Genetické chiméry •Ficus srůst kořenů různých jedinců • •sumky Botryllus schlosseri chimérické kolonie příbuzní jedinci • •Diplosoma listerianum i nepříbuzní Celleporella hyalina (Bryozoa) Hughes et al. 2004 •Pravděpodobnost fůze koreluje s příbuzností • •Histokompatibilita • •Lepší rozpoznávání v pokročilejších fázích → dozrávání imunokompetence • •Speciální proteiny (spongikany...) Ceratioid anglerfish File:Representatives of ceratioid families.jpg •miniaturní samec po narození vyhledá samici, její kůže vyloučí hydrolytický enzym a samec přiroste • •vzniká hermafroditická chiméra Genetické chiméry – „microchimerism“ •kosman bělovousý Callithrix jacchus (asi i rod Saguinus) • •dizygotická dvojčata • •DNA fingerprinting krve - hematopoietické chiméry • •během embryonálního vývoje vzájemná výměna buněk kostní dřeně • •týká se to asi jen krve (neinvazivní metody – chlupy, trus → jeden genotyp) • •průnik embryonálních erytroblastů a volné DNA přes placentu i u člověka • •(pohlaví dítěte před narozením lze určit i pomocí PCR sekvencí typických pro Chr Y, jako templát je periferní krev matky) • title07-01 sagui_de_tufos_brancos Canine transmissible venereal tumor (CTVT) Devil facial tumour disease -parasitic cancer -„single cell parasitic wolf“ Známé „lidské chiméry“ - geneticky nepotvrzené mateřství - chiméra matky Foekje Dillema.jpg 46XX/46XY woman - holandská atletka, mistryně na 100 a 200 m - odmítla test na pohlaví - mozaika zjištěna až posmrtně (v r. 2007) Foekje Dillema Identification of sex DNA sexing Why? 1)species without sex dimorphism (birds, but also many mammals) 2)embryos, larvae 3)non-invasive methods 4) Genetic sex identification •genetic sex determination •birds (♂=ZZ, ♀=ZW) •mammals (♂=XY, ♀=XX) •DNA amplification of W/Y chromosome •W, Y – small chromosomes http://www.nature.com/scitable/content/18935/pierce_4_10_large_2.jpg Sex identification – birds Griffith et al. 1998 •CHD1W and CHD1Z, genes at sex chromosomes (chromobox-helicase-DNA-binding gene (CHD) – Griffiths & Tiwari 1995) • •Primers amplifying introns of both genes • •Introns differ by their length • •Up to three primer combinations • •Problematic species, e.g. Struthioniformes • ♂ ♂ ♂ ♂ ♀ ♀ ♀ ostrich Manorina melanocephala (Meliphagidae) Arnold et al. 2001 •Sons = „helpers“ • •In adults 2,31 males vs. 1 female • •Offspring in nests sex ratio 1:1 (57:57) • •Males are hatching first (in 17 out of 18 nests) they are bigger and heavier when leaving the nest • • OW_NoisyMinerThumbnail medosavka hlučná Sex identification - mammals •Amplification of a gene at Y-chromosome (Sry) (in duplex PCR with X-linked or autosomal fragment) • • •Microtus cabrerae Sry at Chr X Ellobius, Tokudaia Sry completely missing •Nannomys • Large variability • • Bryja and Konečný (2003) Ellobius Tokudaia osimensis M. cabrerae DSC_5565 Nannomys •Amplification of a gene at Y-chromosome (Sry) (in duplex PCR with X-linked or autosomal fragment) • • • • •Faecal analyses: species-specific primers are required to avoid a cross amplification with species in the diet bear3 odocoileus x Murphy et al. 2003 Bryja a Konečný 2003 Sex identification - mammals