Differentiation at the MHCIIα and Cath2 Loci in Sympatric Salvelinus alpinus Resource Morphs in Lake Thingvallavatn

Publisher's versionNorthern freshwater fish may be suitable for the genetic dissection of ecological traits because they invaded new habitats after the last ice age (∼10.000 years ago). Arctic charr (Salvelinus alpinus) colonizing streams and lakes in Iceland gave rise to multiple populations of small benthic morphotypes, often in sympatry with a pelagic morphotype. Earlier studies have revealed significant, but subtle, genetic differentiation between the three most common morphs in Lake Thingvallavatn. We conducted a population genetic screen on four immunological candidate genes Cathelicidin 2 (Cath2), Hepcidin (Hamp), Liver expressed antimicrobial peptide 2a (Leap-2a), and Major Histocompatibility Complex IIα (MHCIIα) and a mitochondrial marker (D-loop) among the three most common Lake Thingvallavatn charr morphs. Significant differences in allele frequencies were found between morphs at the Cath2 and MHCIIα loci. No such signal was detected in the D-loop nor in the other two immunological genes. In Cath2 the small benthic morph deviated from the other two (FST = 0.13), one of the substitutions detected constituting an amino acid replacement polymorphism in the antimicrobial peptide. A more striking difference was found in the MHCIIα. Two haplotypes were very common in the lake, and their frequency differed greatly between the morphotypes (from 22% to 93.5%, FST = 0.67). We then expanded our study by surveying the variation in Cath2 and MHCIIα in 9 Arctic charr populations from around Iceland. The populations varied greatly in terms of allele frequencies at Cath2, but the variation did not correlate with morphotype. At the MHCIIα locus, the variation was nearly identical to the variation in the two benthic morphs of Lake Thingvallavatn. The results are consistent with a scenario where parts of the immune systems have diverged substantially among Arctic charr populations in Iceland, after colonizing the island ∼10.000 years ago.The Palsson laboratory is supported by Icelandic Research foundation and the University of Iceland research fund. Icelandic research foundation (grant of excellence: nr 100204011) to S.S. Sigurdsson, A. Palsson, B.K. Kristjansson, Zophonias O. Jonsson and Ian A. Johnston paid for part of this work. Kalina H. Kapralova and Johannes Gudbrandsson were supported by the University of Iceland doctoral fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer ReviewedRitrýnt tímari

Differential gene expression during early development in recently evolved and sympatric Arctic charr morphs

Phenotypic differences between closely related taxa or populations can arise through genetic variation or be environmentally induced, leading to altered transcription of genes during development. Comparative developmental studies of closely related species or variable populations within species can help to elucidate the molecular mechanisms related to evolutionary divergence and speciation. Studies of Arctic charr (Salvelinus alpinus) and related salmonids have revealed considerable phenotypic variation among populations and in Arctic charr many cases of extensive variation within lakes (resource polymorphism) have been recorded. One example is the four Arctic charr morphs in the ∼10,000 year old Lake Thingvallavatn, which differ in numerous morphological and life history traits. We set out to investigate the molecular and developmental roots of this polymorphism by studying gene expression in embryos of three of the morphs reared in a common garden set-up. We performed RNA-sequencing, de-novo transcriptome assembly and compared gene expression among morphs during an important timeframe in early development, i.e., preceding the formation of key trophic structures. Expectedly, developmental time was the predominant explanatory variable. As the data were affected by some form of RNA-degradation even though all samples passed quality control testing, an estimate of 3′-bias was the second most common explanatory variable. Importantly, morph, both as an independent variable and as interaction with developmental time, affected the expression of numerous transcripts. Transcripts with morph effect, separated the three morphs at the expression level, with the two benthic morphs being more similar. However, Gene Ontology analyses did not reveal clear functional enrichment of transcripts between groups. Verification via qPCR confirmed differential expression of several genes between the morphs, including regulatory genes such as AT-Rich Interaction Domain 4A (arid4a) and translin (tsn). The data are consistent with a scenario where genetic divergence has contributed to differential expression of multiple genes and systems during early development of these sympatric Arctic charr morphs.he project was funded by The Icelandic Center for Research (grant number: 100204011) to Sigurður S. Snorrason, Arnar Pálsson, Zophonías O. Jónsson and Bjarni K. Kristjánsson. The University of Iceland Doctoral Fund to Jóhannes Guðbrandsson and University of Iceland research fund to Arnar Pálsson, Sigurður S. Snorrason and Zophonías O. Jónsson. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer Reviewe

The Underwater Photic Environment of Two Subarctic Icelandic Lakes

The underwater light fields of two Icelandic lakes of volcanic origin and differing trophic status, Thingvallavatn (oligotrophic) and Myvatn (eutrophic), were investigated. Gilvin and turbidity depth profiles were also measured. Diurnal variation in photosynthetically active radiation (PAR) reached almost 3 orders or magnitude. Downward irradiance spectra were variable near the surface, but with increases in depth transmission peaks at 510, 560 and 570 nanometres (nm) became apparent in Thingvallavatn, Myvatn-East Basin and Myvatn-South Basin respectively. Upward irradiance transmission maxima shifted from 480 to 500 nm with depth in Thingvallavatn, while in Myvatn they remained near 570 nm. An irradiance trough at 520 nm was noted in both the upward and downward spectra of Thingvallavatn. The importance of phytoplankton (chlorophyll) and gilvin in modifying the underwater light climate of Myvatn is clearly demonstrated. The mean downwelling and upwelling irradiance curves for Thingvallavatn coincide very well with the spectral sensitivities of resident adult arctic charr. This provides support for the sensitivity hypothesis.Key words: subarctic, limnology, imdiance, underwater light, arctic charr, spectral sensitivitjr, IcelandL'environnement photique sous-marin de deux lacs islandais, le Thingvallavatn (oligotrophe) et le Myvatn (eutrophe), d'origine volcanique et présentant un état trophique différent, ont été étudiés. La quantité de matière organique dissoute (gilvin) et la turbidité ont été mesurées en fonction de la profondeur dans chaque lac. La radiation disponible pour la photosynthèse (PAR) pouvait varier quotidiennement jusqu'à trois ordres de grandeur. Alors que les spectres de l'irradiance descendante variaient près de la surface, ils augmentaient avec la profondeur aux pics de transmission de 510, 560 et 570 nanomètres (nm) respectivement dans le Thingvallavatn et le bassin est du Myvatn et le bassin sud du Myvatn. Les maxima dans la transmission de l'irradiance ascendante ont été décalés avec la profondeur de 480 à 500 nm dans le Thingvallavatn tandis que dans le Myvatn ils sont demeurés près de 570 nm. Une zone d'absorption à 520 nm a également été observée dans les spectres descendants et ascendants du Thingvallavatn. L'importance du phytoplancton (chlorophylle) et de la matière organique dissoute (gilvin) dans la modification de l'environnement photique sous-marin du Myvatn a été nettement démontrée. Les spectres moyens de l'irradiance descendante et ascendante coïncident très étroitement avec les sensibilités spectrales chez les ombles-chevaliers adultes indigènes. Ceci appuie l'hypothèse de la sensibilité chez ces poissons.Mots clés: subarctique, limnologie, irradiance, lumière sous-marine, omble-chevalier, sensibilite spectrale, Island

Gene expression during early development and genetic variation among recently evolved sympatric Arctic charr (Salvelinus alpinus) morphs in Lake Þingvallavatn, Iceland

The four morphs of Arctic charr (Salvelinus alpinus) in Lake Þingvallavatn, Iceland, which differ in many phenotypic traits related to morphology, life history, and feeding ecology, are believed to have evolved locally within the lake after the retreat of the Ice-age glacier. The principal aims of the study were to get a handle on the molecular mechanisms underlying the phenotypic differences and assess genetic differentiation among the morphs. Gene expression during early development was surveyed to look for differential expression of genes thereby identifying developmental processes affecting divergent phenotypes. Genetic variation among the morphs was studied and patterns of biological function (gene ontology) and genomic position of highly differentiated variants were examined. Both candidate gene and transcriptome-wide profiling (RNA-sequencing) approaches were used. The results reveal both differentiation in expression and genetic composition of the sympatric morphs. Gene expression differences in multiple genes and biological pathways were discovered, pointing to substantial differentiation of morphs and that many genes may have been under selection. However, RNA degradation during sample handling created technical problems that complicated data analysis. The transcriptome-wide profiling was nonetheless useful as it revealed expression differences in genes relating to extra-cellular matrix formation and skeletogenesis and highlights differentiation in more biological pathways. The data show extensive genome-wide differentiation in allele frequencies, pointing to reproductive isolation of the morphs and/or natural selection operating on multiple parts of the genome. Strong genetic differentiation among morphs was for example discovered in immunological genes using a candidate gene approach and in genes involved in collagen metabolism and environmental sensing, by gene ontology enrichment tests of differentiated variants in the transcriptome. The expression differences in embryos and the clear genetic differentiation of the morphs suggest that they have advanced along "the speciation continuum" towards becoming reproductively isolated.Fjögur afbrigði bleikju (Salvelinus alpinus) finnast í Þingvallavatni. Afbrigðin eru talin hafa þróast innan vatnsins frá lokum síðustu ísaldar og eru ólík hvað varðar m.a. stærð, útlit, lífsferla og fæðuöflun. Meginmarkmið verkefnisins var að auka skilning á hvaða sameinda- og þroskunarferlar liggja að baki mismunandi svipgerð afbrigðanna og meta erfðafræðilega aðgreiningu þeirra á milli. Könnuð var genatjáning í snemmþroskun í leit að mismunandi tjáðum genum sem gætu þannig bent á hvaða ferlar móta þroskun ólíkra svipgerða. Erfðabreytileiki meðal afbrigðanna var metinn og athugað hvort að erfðaset með mikinn mun í tíðni samsæta, meðal afbrigða, fyndust í genum með svipuð líffræðileg hlutverk og/eða væru bundin við ákveðna staði innan erfðamengisins. Tvenns konar aðferðarfræði var beitt, í fyrsta lagi að kanna áhugaverð gen með sértækum aðferðum og í öðru lagi að rannsaka allt umritunarmengið (RNA-raðgreiningu). Niðurstöðurnar afhjúpa ólíka genatjáningu og erfðamun milli afbrigðanna. Munur í tjáningu finnst á genum í mörgum líffræðilegum ferlum sem bendir til töluverðar aðgreiningar afbrigðanna og að mögulega hafi mörg gen verið undir náttúrulegu vali. RNA-niðurbrot í sýnum, líklega vegna tæknilegra vandkvæða í meðhöndlun þeirra, hækkaði flækjustig greininga á RNA-raðgreiningargögnum. Greining umritunarmengisins var engu að síður gagnleg þar sem tjáningarmunur fannst í genum sem tengjast myndun utanfrumugrindar og beinmyndun. Gögnin sýna mikinn mun í tíðni samsæta meðal afbrigða og var mikinn mun að finna víða í erfðamenginu. Það getur bent til æxlunarlegrar einangrunar afbrigðanna og/eða náttúrulegs vals á mörgum litningasvæðum. Mikill erfðamunur fannst til dæmis í genum tengdum ónæmiskerfinu með sértækum aðferðum og aðgreinandi erfðabreytileiki í genum tengdum kollagen-efnaskiptum og umhverfisskynjum (sjón, heyrn) var áberandi í umritunarmenginu. Genatjáningarmunur í fóstrum og skýr munur í erfðasamsetningu afbrigðanna bendir til að þau séu komin áleiðis inn á "veg tegundamyndunar"

Fault and magmatic interaction within Iceland's western rift over the last 9kyr

We present high-resolution 'Chirp' sub-bottom profiler data from Thingvallavatn, a lake in Iceland's western rift zone. These data are combined with stratigraphic constraints from sediment cores to show that movement on normal faults since 9 ka are temporally correlated with magmatic events, indicating that movements were controlled by episodic dyke intrusion. Sediment depo-centres and the focus of subsidence migrated westwards over 3-4 kyr towards the locus of subsequent brittle failure. We interpret this subsidence as related to dyke intrusion a few km along strike, originating from the Hengill volcanic system, which occurred prior to major dyking, faulting and subsidence within the lake at 1.9 ka

Differential expression of the aryl hydrocarbon receptor pathway associates with craniofacial polymorphism in sympatric Arctic charr

Background The developmental basis of craniofacial morphology hinges on interactions of numerous signalling systems. Extensive craniofacial variation in the polymorphic Arctic charr, a member of the salmonid family, from Lake Thingvallavatn (Iceland), offers opportunities to find and study such signalling pathways and their key regulators, thereby shedding light on the developmental pathways, and the genetics of trophic divergence. Results To identify genes involved in the craniofacial differences between benthic and limnetic Arctic charr, we used transcriptome data from different morphs, spanning early development, together with data on craniofacial expression patterns and skeletogenesis in model vertebrate species. Out of 20 genes identified, 7 showed lower gene expression in benthic than in limnetic charr morphs. We had previously identified a conserved gene network involved in extracellular matrix (ECM) organization and skeletogenesis, showing higher expression in developing craniofacial elements of benthic than in limnetic Arctic charr morphs. The present study adds a second set of genes constituting an expanded gene network with strong, benthic–limnetic differential expression. To identify putative upstream regulators, we performed knowledge-based motif enrichment analyses on the regulatory sequences of the identified genes which yielded potential binding sites for a set of known transcription factors (TFs). Of the 8 TFs that we examined using qPCR, two (Ahr2b and Ap2) were found to be differentially expressed between benthic and limnetic charr. Expression analysis of several known AhR targets indicated higher activity of the AhR pathway during craniofacial development in benthic charr morphotypes. Conclusion These results suggest a key role of the aryl hydrocarbon receptor (AhR) pathway in the observed craniofacial differences between distinct charr morphotypes.This project was supported by The Icelandic Centre for Research (RANNIS/IRF, Grant 100204), The University of Iceland Research Fund and the Eimskip University Fund.Peer Reviewe

Adaptive radiation of Arctic charr (Salvelinus alpinus) in three Norwegian lakes - niche segregation, phenotypic and genetic variation

Å forstå hvordan økologiske og fysiske faktorer fører til opprinnelsen av nye arter, og hvilke faktorer som former ny intraspesifikk diversitet, er den “hellige gral” i evolusjonær biologi. Adaptiv radiasjon er den evolusjonære prosessen som kan føre til diversifisering av fenotyper og genotyper i ulike miljøer, og som kan splitte en forfaders form eller linje i nye former og arter. I den økologiske artsdannelsesprosessen vil lokal tilpasning gjennom naturlig seleksjon drive oppsplittingen av populasjoner, noe som vil føre til evolusjon av reproduktiv isolasjon og dermed dannelsen av nye økotyper, morfer, populasjoner og til slutt arter. Et godt eksempel på en polymorf art er røya (Salvelinus alpinus) som kan ha ulike nisjer (det vil si tilpasning til spesifikke abiotiske og biotiske forhold) i en innsjø. Spesialiseringen til en bestemt nisje kan favorisere divergensen mellom morfene, noe som kan lede til forskjeller i utseende, vekst, kjønnsmodning, gytetid og sted, og over tid utvikle reproduktive barrierer mellom morfene. Denne doktorgradsavhandlingen fokuserer på fenotypisk og genetisk divergens av røyemorfer. Hovedmålet er å undersøke nisjesegregering på trofisk nivå (det vil si diettvalg og habitatbruk), ved å studere morfologiske og genetiske forskjeller mellom røyemorfer fra tre forskjellige ferskvannsystemer i Norge (Tinnsjøen, Tårnvatn og Skøvatn). To røyemorfer ble funnet eksisterende sammen i Skøvatn, tre morfer i Tårnvatn, og fire i Tinnsjøen. To nye morfer ble funnet i Tinnsjøen og Skøvatn. Alle morfene viste forskjeller i livshistorie, genetikk, fenotype, diettvalg og habitatbruk. De fiskespisende morfene ernærte seg hovedsakelig av fisk, og ble funnet i den profundale delen av Tinnsjøen og Tårnvatn. Planktivore morfer spiste hovedsakelig dyreplankton, og ble funnet i flere ulike habitater i disse tre innsjøene. Livshistoriekarakterer og habitatbruk var lignende hos de småvokste dypvannsmorfene, men morfen i Skøvatn viste forskjeller i diettvalg sammenlignet med morfen fra Tårnvatn. Den nye dypvannsmorfen som ble funnet i den dypere delen av Tinnsjøen har likhetstrekk med hulefisk som hvitt skinn, underutviklede øyne og små hjerneregioner. Parallell evolusjon kan være en forklaring for likhetene som er funnet blant noen av morfene i disse tre ferskvannssystemene. Funnene viser hvordan seleksjonspress kan føre til lignende resultater i samme miljøer, der fenotypisk plastisitet også kan være en viktig mekanisme i tidlige stadier av nisjespesialisering. Disse morfene er sannsynligvis i en økologisk artsdannelsesprosess, der naturlig seleksjon spiller en viktig rolle i den adaptive divergensen av morfer, og bidrar til reproduktiv isolasjon. Adaptiv radiasjon kan forklare tilpasninger og diversitet hos den polymorfe røya i ulike vann.Abstract Understanding the ecological and physical factors driving the origin of species, and which ones are shaping new intraspecific diversity, are the “holy grail” of evolutionary biology. Adaptive radiation is the evolutionary process that can generate diversification of phenotypes and genotypes across different environments, differentiating a single ancestor into different forms and species. Under ecological speciation, local adaptation through natural selection drives the divergence of populations, evolving reproductive isolation and leading to the formation of new eco morphs, populations, and ultimately, species. A good example of polymorphic species is Arctic charr (Salvelinus alpinus), which has the flexibility to occupy different niches (i.e. a specific range of abiotic and biotic factors that a species has specialised) in a lake. For example, in fish, the specialisation into a specific niche can favour divergence among morphs, showing differences in morphology, growth, maturity, spawning time and site, developing reproductive barriers among the morphs. This thesis focuses on phenotypic and genetic divergence of Arctic charr morphs. The main objectives are to investigate trophic niche segregation (i.e. diet choice and habitat use), morphological and genetic differences among sympatric Arctic charr morphs from three different lakes in Norway (Tinnsjøen, Tårnvatn and Skøvatn). Two Arctic charr morphs were found coexisting in Lake Skøvatn, three morphs in Lake Tårnvatn and four morphs in Lake Tinnsjøen. Two novel morphs were found in Lake Tinnsjøen and Skøvatn. All morphs showed divergence in life history, genetics, phenotype, diet and habitat use. The piscivore morphs fed mainly on fish and were found in the profundal habitat of Lake Tinnsjøen and Tårnvatn. The planktivore morphs were feeding mainly on zooplankton, and were found across different habitats from these three lakes. Life-history traits and habitat use was similar among the small-sized profundal morphs, but the morph in Skøvatn presented differences in diet choice compared to the morph from Lake Tårnvatn. Finally, the Abyssal morph was found in the deepprofundal habitat in Lake Tinnsjøen, presenting similarities with cave fish such as white coloration, reduced eyes and small brain regions. Parallel evolution could be responsible for the similarities found among some of the Arctic charr morphs across these three lakes. These findings show how selection pressures can sometimes lead to similar outcomes in similar environments. However, phenotypic plasticity may also be an important component during the early stages of niche specialization. These morphs are likely under ecological speciation, where natural selection could play an important role in the adaptive divergence of morphs, contributing to reproductive isolation. Arctic charr polymorphism could be a case of adaptive radiation, explaining their diversity across different freshwater systems