Multivariate analysis of morphology, behaviour, growth and developmental timing in hybrids brings new insights into the divergence of sympatric Arctic charr morphs

This work was fully funded by the Icelandic Centre of Research, RANNÍS (Icelandic Research Fund grant no.173802-051).Background: Studying the development of fitness related traits in hybrids from populations diverging in sympatry is a fundamental approach to understand the processes of speciation. However, such traits are often affected by covariance structures that complicate the comprehension of these processes, especially because the interactive relationships between traits of different nature (e.g. morphology, behaviour, life-history) remain largely unknown in this context. In a common garden setup, we conducted an extensive examination of a large suit of traits putatively involved in the divergence of two morphs of Arctic charr (Salvelinus alpinus), and investigated the consequences of potential patterns of trait covariance on the phenotype of their hybrids. These traits were measured along ontogeny and involved growth, yolk sac resorption, developmental timing (hatching and the onset of exogeneous feeding), head morphology and feeding behaviour. Results: Growth trajectories provided the strongest signal of phenotypic divergence between the two charr. Strikingly, the first-generation hybrids did not show intermediate nor delayed growth but were similar to the smallest morph, suggesting parental biases in the inheritance of growth patterns. However, we did not observe extensive multivariate trait differences  between the two morphs and their hybrids. Growth was linked to head morphology (suggesting that morphological variations in early juveniles relate to simple allometric effects) but this was the only strong signal of covariance observed between all the measured traits. Furthermore, we did not report evidence for differences in overall phenotypic variance between morphs, nor for enhanced phenotypic variability in their hybrids. Conclusion: Our study shed light on the multivariate aspect of development in a context of adaptive divergence. The lack of evidence for the integration of most traits into a single covariance structure suggested that phenotypic constraints may not always favour nor impede divergence toward ecological niches differing in numerous physical and ecological variables, as observed in the respective habitats of the two charr. Likewise, the role of hybridization as a disruptive agent of trait covariance may not necessarily be significant in the evolution of populations undergoing resource polymorphism.Publisher PDFPeer reviewe

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

Extensive genetic differentiation between recently evolved sympatric Arctic charr morphs

Publisher's version (útgefin grein)The availability of diverse ecological niches can promote adaptation of trophic specializations and related traits, as has been repeatedly observed in evolutionary radiations of freshwater fish. The role of genetics, environment, and history in ecologically driven divergence and adaptation, can be studied on adaptive radiations or populations showing ecological polymorphism. Salmonids, especially the Salvelinus genus, are renowned for both phenotypic diversity and polymorphism. Arctic charr (Salvelinus alpinus) invaded Icelandic streams during the glacial retreat (about 10,000 years ago) and exhibits many instances of sympatric polymorphism. Particularly, well studied are the four morphs in Lake Þingvallavatn in Iceland. The small benthic (SB), large benthic (LB), planktivorous (PL), and piscivorous (PI) charr differ in many regards, including size, form, and life history traits. To investigate relatedness and genomic differentiation between morphs, we identified variable sites from RNA‐sequencing data from three of those morphs and verified 22 variants in population samples. The data reveal genetic differences between the morphs, with the two benthic morphs being more similar and the PL‐charr more genetically different. The markers with high differentiation map to all linkage groups, suggesting ancient and pervasive genetic separation of these three morphs. Furthermore, GO analyses suggest differences in collagen metabolism, odontogenesis, and sensory systems between PL‐charr and the benthic morphs. Genotyping in population samples from all four morphs confirms the genetic separation and indicates that the PI‐charr are less genetically distinct than the other three morphs. The genetic separation of the other three morphs indicates certain degree of reproductive isolation. The extent of gene flow between the morphs and the nature of reproductive barriers between them remain to be elucidated.This project was supported by The Icelandic Center for Research (RANNIS #100204011) to SSS and coworkers, The University of Iceland Doctoral Fund to JG and University of Iceland research fund to AP, SSS and ZOJ.Peer reviewe

Transcriptional dynamics of a conserved gene expression network associated with craniofacial divergence in Arctic charr

Background Understanding the molecular basis of craniofacial variation can provide insights into key developmental mechanisms of adaptive changes and their role in trophic divergence and speciation. Arctic charr (Salvelinus alpinus) is a polymorphic fish species, and, in Lake Thingvallavatn in Iceland, four sympatric morphs have evolved distinct craniofacial structures. We conducted a gene expression study on candidates from a conserved gene coexpression network, focusing on the development of craniofacial elements in embryos of two contrasting Arctic charr morphotypes (benthic and limnetic). Results Four Arctic charr morphs were studied: one limnetic and two benthic morphs from Lake Thingvallavatn and a limnetic reference aquaculture morph. The presence of morphological differences at developmental stages before the onset of feeding was verified by morphometric analysis. Following up on our previous findings that Mmp2 and Sparc were differentially expressed between morphotypes, we identified a network of genes with conserved coexpression across diverse vertebrate species. A comparative expression study of candidates from this network in developing heads of the four Arctic charr morphs verified the coexpression relationship of these genes and revealed distinct transcriptional dynamics strongly correlated with contrasting craniofacial morphologies (benthic versus limnetic). A literature review and Gene Ontology analysis indicated that a significant proportion of the network genes play a role in extracellular matrix organization and skeletogenesis, and motif enrichment analysis of conserved noncoding regions of network candidates predicted a handful of transcription factors, including Ap1 and Ets2, as potential regulators of the gene network. The expression of Ets2 itself was also found to associate with network gene expression. Genes linked to glucocorticoid signalling were also studied, as both Mmp2 and Sparc are responsive to this pathway. Among those, several transcriptional targets and upstream regulators showed differential expression between the contrasting morphotypes. Interestingly, although selected network genes showed overlapping expression patterns in situ and no morph differences, Timp2 expression patterns differed between morphs. Conclusion Our comparative study of transcriptional dynamics in divergent craniofacial morphologies of Arctic charr revealed a conserved network of coexpressed genes sharing functional roles in structural morphogenesis. We also implicate transcriptional regulators of the network as targets for future functional studies.This project was supported by The Icelandic Centre for Research (RANNIS/IRF, grant 100204) and The University of Iceland Research Fund.Peer Reviewe

Study of morphogenesis and miRNA expression associated with craniofacial diversity in Arctic charr (Salvelinus alpinus) morphs

Trophic diversification of Arctic charr (Salvelinus alpinus, Linn. 1758) into four genetically distinct morphs, varying in life history characteristics, behavior, and trophic morphology, has occurred in lake Thingvallavatn following the last glaciation. The aim of this study was to investigate the genetic and developmental aspects of this diversification thereby gaining insights into the evolution and the maintenance of the Thingvallavatn morphs. In chapter I a population genetic screen of immunological candidate genes revealed differences among morphs for Cath2 and MHCII alpha that far exceeded differentiation at neutral loci. This is consistent with a scenario were selection has led to divergence in parts of the immune system. In chapter II embryonic and early post-hatching craniofacial cartilage development is described. The ontogenetic trajectories of shape and size indicated developmental heterochrony as a possible mechanism of morph divergence. Chapter III describes subtle but significant differences in early post-hatching craniofacial morphology between the progeny of three morphs of Arctic charr. Moreover hybrid progeny of two contrasting morphs showed extreme (or transgressive) phenotypes well outside of the parental range, indicating that the ecological divergence within the lake might be enhanced by lowered fitness of hybrids. In chapter IV the level of integration and modularity in craniofacial traits in morphs and hybrids is analysed. Chapter V describes the annotation of Arctic charr miRNAs expressed during development and analyses of candidate miRNAs involved in Arctic charr morphogenesis and diversification.Frá lokum síðustu ísaldar hafa þróast fjögur afbrigði bleikju (Salvelinus alpinus, Linn. 1758) innan Þingvallavatns. Afbrigðin eru erfðafræðilega aðgreind og eru ólík hvað snertir lífsferla, atferli og útlit, og á það sérstaklega við um líkamshluta er tengjast fæðuöflun. Markmið þessarar rannsóknar var að kanna erfðafræðilegar og þroskunarfræðilegar orsakir þessa fjölbreytileika og öðlast þannig innsýn í þróun og varðveislu bleikjuafbrigðanna í Þingvallavatni. Stofnerfðafræðilegri leit að genum tengdum ónæmiskerfinu sem sýna mismun milli afbrigða er lýst í fyrsta kafla ritgerðarinnar. Þar á meðal eru Cath2 og MHCII alpha sem sýna breytileika sem getur ekki talist hlutlaus og líklegast er að áhrif náttúrulegs vals á ónæmiskerfið hafi leitt til aðgreiningar á þessum erfðasetum. Annar kafli lýsir þroskun brjósks og beina í höfði fóstra og seiða stuttu eftir klak. Sá munur sem fram kemur milli afbrigða í þroskunarfræðilegum brautum útlits og stærðar þessara stoðeininga bendir til þess að orsakanna sé að leita í breytingum á tímasetningu þroskunaratburða. Í þriðja kafla segir frá litlum en marktækum mun í útliti höfuðbeina á fyrstu stigum eftir klak seiða þriggja afbrigða bleikju. Auk þess sýna blendingar tveggja ólíkra afbrigða svipgerð sem fellur að verulegu leyti fyrir utan útlitsmengi beggja foreldra-afbrigðanna. Það bendir til þess að aðskilnað afbrigðanna í vatninu megi rekja til minni hæfni blendinga. Fjórði kafli fjallar um þroskunarfræðileg tengsl valinna stoðeininga í höfði, þ.e. hversu sjálfstæðar eða samþættar þær eru, og hvernig þeim er háttað hjá kynblendingum ólíkra afbrigða Í fimmta kafla er miRNA sameindum bleikjunnar og tjáningu þeirrra í þroskun lýst í mismunandi afbrigðum. Sérstaklega athygli fengu miRNA-gen sem sýndu mismunandi tjáningarmynstur á afbrigðunum en slík gen kunna að leika mikilvægt hlutverk í formþroskun höfuðbeina og þannig verið undirstaða útlitsmunar milli afbrigða

Remote sensing of salmonid spawning sites in freshwater ecosystems: The potential of low-cost UAV data.

Salmonids are especially vulnerable during their embryonic development, but monitoring of their spawning grounds is rare and often relies on manual counting of their nests (redds). This method, however, is prone to sampling errors resulting in over- or underestimations of redd counts. Salmonid spawning habitat in shallow water areas can be distinguished by their visible reflection which makes the use of standard unmanned aerial vehicles (UAV) a viable option for their mapping. Here, we aimed to develop a standardised approach to detect salmonid spawning habitat that is easy and low-cost. We used a semi-automated approach by applying supervised classification techniques to UAV derived RGB imagery from two contrasting lakes in Iceland. For both lakes six endmember classes were obtained with high accuracies. Most importantly, producer's and user's accuracy for classifying spawning redds was >90% after applying post-classification improvements for both study areas. What we are proposing here is an entirely new approach for monitoring spawning habitats which will address some the major shortcomings of the widely used redd count method e.g. collecting and analysing large amounts of data cost and time efficiently, limiting observer bias, and allowing for precise quantification over different temporal and spatial scales