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 Identification of species DNA barcoding WHAT THE SPECIES IS AND DO WE NEED THEM? It is really laughable to see what different ideas are prominent in various naturalists minds, when they speak of „species“; ... It all comes, I believe, from trying to define the indefinable. C. Darwin, 24 Dec 1856 (Letter to J.D. Hooker) Is it possible to define a species? Species concepts in biology •Agamospecies •Biological •Biosimilarity •Cladistic •Cohesion •Compilospecies •Differential Fitness •Ecological •Evolutionarily Significant Unit •Evolutionary •Genealogical •Genealogical Concordance •General Lineage •Genetic •Genic •Genoypic cluster •Hennigian •Internodal •Least Inclusive Taxonomic Unit •Morphological •Non-dimensional •Nothospecies •Phenetic •Phylogenetic (Diagnosability Version) •Phylogenetic (Monophyly Version) •Phylo-Phenetic •Pragmatic •Recognition •Reproductive Competition •Successional •Taxonomic •Unified Morphological Species Concept •= the smallest groups that are consistently and persistently distinct, and distinguishable by ordinary means •Aristoteles → Linnaeus → rules of zoological nomenclature •does not take evolution into account Linnaeus, Carolus (1707 - 1778) Biological Species Concept •= interbreeding natural populations reproductively isolated from other such group •reproductive isolation mechanisms (RIM) = post- or prezygotic barriers of reproduction •most popular – it is intuitive and it was promoted most successfully (e.g. by influential evolutionary biologists of the 20th century as main concept of Modern Synthesis) • •problems: allopatric and allochronic populations/species, ... • Mayr, 1942 Complications: Parapatric contact zones •Erinaceus – distinct species (minimal hybridization) • • • • • • •house mice – distinct subspecies (substantial hybridization) Bolfiková and Hulva 2012 Macholán et al. 2008 E. europaeus E. roumanicus Complications: Ring species seaguls 495px-Rings_species_example Larus 404px-Ring_species_diagram greatdane Complications: Physical constraints Phylogenetic species concept (Diagnosability Version) •= the smallest population or group of populations, within which there is a parental pattern of ancestry and descent •two populations are considered species if they are 100% diagnostic (e.g. discriminant analysis of morphometric data or allele frequency data) •recent paradigmatic shift from the Biological Species Concept to Phylogenetic Species Concept •extreme cases: descendants of a single mother with a mutation at mtDNA can be 100% diagnosed (i.e. should be considered species) • Cracraft, 1983, Cracraft 1997 Example: Taxonomy of ungulates •increase from 143 (Grubb 2005) to 279 (Groves and Leslie 2011 -Handbook of the Mammals of the World) species of bovid ungulates klipspringers Oreotragus – from one to 11 species Consequences intensively debated •„pros“papers •„cons“ papers Why does it matter? The power of names! •description of Nessiteras rhombopteryx in Nature •„the Loch Ness monster“ – if indeed it does exist, it exists in small numbers and deserves protection •to be protected it must have a taxonomic name Rinnes and Scott 1975, Nature Why does it matter? Taxonomic inflation. •inflation leads to devaluation •tigers (Panthera tigris) have been split into two species based on 3 diagnostic bp in the mitochondrial cytochrome b (P. tigris and P. sumatrae) (Cracraft et al. 1998) •genetic drift of some other populations in India has already led to the fixation of unique haplotypes •„The fact that tigers are dwindling towards extinction will thus cause a multitude of new tiger „species“ – before they all vanish“ (Zachos 2016) •PSC → increase of threatened species – many species will have low population sizes and distribution ranges (IUCN RED List criteria) – e.g. US Endangered Species Act – increase from US$4.6 billion to US$7.6 billion for full recovery of all species • Why does it matter? Biodiversity research. •species richness is a function of the underlying species concept •often comparing „apples and oranges“ •36 biodiversity hotspots (at least 1500 endemic vascular plants species and 70% of primary vegetation has been destroyed) •more than US$1 billion for conservation in biodiversity hotspots Mittermeier et al. 2011 Eastern Afromontane Biodiversity Hotspot (EABH) •Albertine Rift – considered as the most diverse part of EABH •Ethiopian Highlands – the most neglected part of EABH despite the large area and geomorphological diversity •examples from rodents NUBIAN PLATE SOMALIAN PLATE ARABIAN PLATE „Ethiopian craddle“ NUBIAN PLATE SOMALIAN PLATE ARABIAN PLATE e.g. Bryja et al. 2019, Folia Zoologica Mus (Nannomys) Otomys typus group Lophuromys flavopunctatus group 600_3187 Tachyoryctes „out of Ethiopia“ Šumbera, ..., Bryja 2018, Mol Phyl Evol •the highest evolutionary diversity in Ethiopia (5 species using PSC) •a single colonization of Kenyan Highlands and Albertine Rift Mts. root rats (Tachyoryctes) T. macrocephalus (ETH) T. „splendens“ (1 sp. in Ethiopia, 12 spp. in Kenya and Albertine rift ) HOW CAN GENETICS BE HELPFUL IN SPECIES IDENTIFICATION? first idea in 2003 CBOL CBOL in 2005 iBol iBOL 2010-2015 500 000 species barcoded in 2015 „DNA barcode“ – short fragment of mitochondrial DNA 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? •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. 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. • •3. 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. • •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. barcoding_gap •Barcoding principle barcoding_gap Barcoding principle 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. For animals, a 658 base-pair fragment of the mitochondrial gene, cytochrome oxidase subunit I (mtCOI) – consensus for iBOL consortium •for particular taxonomic groups, also other barcodes are widely used - e.g. cytochrome b for mammals 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. •For animals, a 658 base-pair fragment of the mitochondrial gene, cytochrome oxidase subunit I (mtCOI) – consensus for iBOL consortium •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) • Focus to date 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 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 Metabarcoding/eDNA • Bold black line = Total number of studies per year; thin straight lines: high‐throughput next‐generation sequencing techniques; thin dashed lines: Sanger sequencing method or other traditional molecular technique, e.g., RFLP) Diet analysis •identification of MOTUs („molecular operational taxonomic units“) 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? What are the main limits to barcoding encountered so far? 1)horizontal gene transfer 2)nuclear pseudogenes 3)heteroplasmy (paternal leakage) 4)gene tree vs. species tree 5)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. 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 3. 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 % - mtDNA transmission from both parents occurs regularly in certain bivalves (Bivalvia) 4. GENE TREES VS. SPECIES TREES STATISTICAL MULTI-LOCUS SPECIES DELIMITATION (see lecture of O. Mikula) Allopatric speciation model Mayr 1942 Dobzhansky-Muller incompatibility Dobzhansky 1936, Muller 1942 A A B B a A B B A A b B A A b b a a B B a A B b genetic incompatibility (negative epistatic interaction) Phylogeny at the level of populations and species D:\Fleliaer\My Documents\Congressen\2011_5thEPC_Rodos\allelic genealogies.jpg >> A B C Species are metapopulation lineages > new methods for DNA-based species delimitation Gene trees •Vital to understanding the process of speciation •Span intraspecific and interspecific evolution Obsah obrázku řada/pruh, diagram, design Popis byl vytvořen automaticky gene tree (e.g. mtDNA) species tree The idea that species are metapopulation lineages has led to the development of new DNA-base species delimitation methods. Genes that show variation within species will be phylogenetically informative below and above the species level. Gene trees are vital to understanding the process of speciation because they span intraspecific and interspecific evolution. When we think about a phylogeny, we typically think about species relationships in a tree. But within the branches of this tree there is population genetics going on, which can be modeled as a coalescence process. D:\Fleliaer\My Documents\Congressen\2011_5thEPC_Rodos\allelic genealogies_mutations_3.jpg Multilocus data, multi-species coalescence A B C D:\Fleliaer\My Documents\Congressen\2011_5thEPC_Rodos\allelic genealogies_mutations_2.jpg A B C locus 1 locus 2 Now what this figure shows is that although gene genealogies below and above the species level are different in nature, young lineages reside within a zone where both processes meet. Therefore species delimitation should incorporate aspects of both population genetics and phylogenetics. 5. MITOCHONDRIAL INTROGRESSION AND A UNIFYING SPECIES CONCEPT (?) Dobzhansky-Muller incompatibility A A B B A A b b a a B B a A B b genetic incompatibility (negative epistatic interaction) „MAGIC TRAITS“ A A A A a a a A mitonuclear incompatibility M m M a A M m Hill, 2019 X X Oxidative phosphorylation (OXPHOS) by electron transport system (ETS) Rand et al. 2004 red = mt genes blue = N-mt genes (mtDNA) (nDNA) Co-adaptation of mt and N-mt genes Hill 2019 mitonuclear incompatibility Mitonuclear coevolution as the genesis of speciation Hill 2016, 2017 Mitonuclear compatibility species complex •= a species is a population that is genetically isolated from other populations by incompatibilities in uniquely coadapted mt and N-mt genes •the need of mitonuclear coadaptation is universal among eukaryotes •species boundaries become objective and defensible •determining species boundaries would no longer be an esoteric intellectual exercise •„species“ is exclusively a eukaryotic concept Hill 2016, 2017, 2018, 2019 first idea in 2003 CBOL CBOL in 2005 iBol iBOL 2010-2015 500 000 species barcoded in 2015 „DNA barcode“ – short fragment of mitochondrial DNA Introgression/replacement of mtDNA in Myotis 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 •three species of Lepus in Iberia have often mtDNA of L. timidus •but L. timidus dissappeared from Iberia at the end of last glacial period •neutral process – consequence of spatial expansion Hares in Spain and Portugal Interspecific mtDNA introgressions in Lophuromys 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 Komarova, Kostin, Bryja et al., Mol. Ecol., 2020 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) Interspecific mtDNA introgressions in Lophuromys Photo: D. Mizerovská Interspecific mtDNA introgressions can be surprisingly frequent Borena Saynt National Park (central Ethiopia) prezentacia_uvodnyobrazok2.jpg Mizerovská et al. 2020, J. Vert. Biol. Stenocephalemys (Ethiopian endemics) (B) complete mitogenomes (A) anchored phylogenomics (388 nuclear loci) Borena Saynt NP Distribution of S. sokolovi sp. nov. Borena Saynt NP Lake Tana Stenocephalemys Borena Saynt NP Distribution of S. sokolovi sp. nov. Borena Saynt NP Lake Tana Distribution of S. albipes albipes sokolovi 2800 m a.s.l. 3500 m a.s.l. Stenocephalemys Borena Saynt NP albipes sokolovi Stenocephalemys menageshae simensis yaldeni sp. B Lophuromys ddRADseq ddRADseq anchored phylogenomic Crocidura (Mizerovská et al. 2020) (Komarova et al. 2021) (Konečný et al. 2020) Possible evolutionary explanations •Non-adaptive explanation •rodents have male-biased dispersal – expanding males captures mtDNA of local species (see examples of hares or bats) •expansion due to climate change → low-elevation species should move up and capture high-elevation mtDNA ♂ Possible evolutionary explanations •Adaptive explanation no. 1 •advantageous OXPHOS genes of low-elevation taxon •→ studies of energetic metabolism and co-introgression are required •climate change → increase of temperature co-introgression of N-mt genes (?) „speciation in reverse“ Possible evolutionary explanations •Adaptive explanation no. 2 •„mutational erosion“ - replacement of non-functional high-elevation mtDNA (accumulation of mutations in small populations in fragmented Afroalpine habitats) – „best of bad options“ •higher mutation rate (UV?) •lower Ne – higher fixation rate •lower mutation rate •higher Ne CONCLUSIONS Conclusions •species concepts and species delimitations are crucial parts of biodiversity studies •enormous value in applied conservation biology •genomics can be useful tool for species delimitation (analytical approaches still in development) – once they will be able to discriminate between population- and species-level structuring •mitonuclear compatitibility species concept is a good candidate for a unifying species concept