Cave animals at the dawn of speleogenomics

Cave animals at the dawn of speleogenomics

Markus Friedrich

Wayne State [email protected]

mailto:[email protected]

fix-Markus Friedrich


● Spelling correct?

○ I think so

○ Spelleochecked this morning...


● What does it mean?

○ Concepts and approaches


● Transcriptome or genome sequence based analyses of cave faunas:

■ Viruses■ Bacteria■ Algae■ Plants■ Obligate cave animals

● Extant● Extinct


● Transcriptome or genome sequence based analyses of cave faunas:

■ Specimen samples■ Environmental samples


● Transcriptome or genome sequence based analyses of:■ Regressive traits

Protas & William R. Jeffery WIREs Dev Biol 2012. doi: 10.1002/wdev.61


● Transcriptome or genome sequence based analyses of:■ Regressive traits■ Relaxed vs positive selection



● Transcriptome or genome sequence based analyses of:■ Regressive traits■ Relaxed vs positive selection■ Constructive (elaborated) traits



● Transcriptome or genome sequence based analyses of:■ Regressive traits■ Relaxed vs positive selection■ Constructive (elaborated) traits■ Convergent vs parallel evolution


Candidate gene studies harvesting molecular key signatures of the evolutionary process

○ Crandall, K. A., & Hillis, D. M. (1997). Rhodopsin evolution in the dark. Nature, 387(6634), 667–668.

○ Buhay, J. E., & Crandall, K. A. (2005). Subterranean phylogeography of freshwater crayfishes shows extensive gene flow and surprisingly large population sizes. Molecular Ecology, 14(14), 4259–4273.

○ Leys, R., Cooper, S. J. B., Strecker, U., & Wilkens, H. (2005). Regressive evolution of an eye pigment gene in independently evolved eyeless subterranean diving beetles. Biology Letters, 1(4), 496–499.

○ Aspiras, A. C., Prasad, R., Fong, D. W., Carlini, D. B., & Angelini, D. R. (2012). Parallel reduction in expression of the eye development gene hedgehog in separately derived cave populations of the amphipod Gammarus minus. Journal of Evolutionary Biology, 25(5), 995–1001.

○ Niemiller, M. L., Fitzpatrick, B. M., Shah, P., Schmitz, L., & Near, T. J. (2013). Evidence for repeated loss of selective constraint in rhodopsin of amblyopsid cavefishes (Teleostei: Amblyopsidae). Evolution; International Journal of Organic Evolution, 67(3), 732–748.

Speleogenomic studies harvest molecular signatures of evolutionary change

○ Cave-adaptive loss of function:■ Ortholog of surface genes not detectable

Gene x

○ Cave-adaptive loss of function:■ Ortholog of surface genes not detectable

Gene x


○ Cave-adaptive loss of function:■ Orthologs of surface genes not detectable■ Transcripts with deletions, nonsense or frameshift

mutations

Gene x

Gene x


○ Cave-adaptive loss of function:■ Orthologs of surface genes not detectable■ Transcripts with deletions, nonsense or frameshift

mutations■ Equal numbers of replacement (dN) vs silent

substitutions (dS) mutation ratio (dN/dS = 1)

Gene x

Gene x

dN/dS = 1

dN/dS < 0.2


○ Cave-adaptive gain of function:■ Orthologs of surface genes expressed at higher level

Gene x

Gene x


○ Cave-adaptive gain of function:■ Orthologs of surface genes expressed at higher level

Gene x

Gene x


○ Cave-adaptive gain of function:■ Orthologs of surface genes expressed at higher level■ Gene family expansions compared to surface forms

Gene x

Gene x 1

Gene x 2


○ Cave-adaptive gain of function:■ Orthologs of surface genes expressed at higher level■ Gene family expansions compared to surface forms■ High replacement vs silent substitution ratio

Gene x

Gene x

dN/dS > 2

dN/dS < 0.2


○ Cave-adaptive gain of function:■ Orthologs of surface genes expressed at higher level■ Gene family expansions compared to surface forms■ High replacement vs silent substitution ratio■ Selective sweep patterns


○ Purifying selection:■ Orthologs of surface genes intact■ Expressed at similar levels■ Low replacement vs silent substitution ratio

Speleogenomic studies harvest molecular signatures of evolutionary change or conservation

Gene x

Gene x

dN/dS < 0.2

dN/dS < 0.2

○ Cave-adaptive loss of function■ Orthologs of surface genes not detectable■ Transcripts with deletions, nonsense or frameshift

mutations■ Equal replacement vs silent substitution mutation ratio

○ Cave-adaptive gain of function■ Orthologs of surface genes expressed at higher level■ Gene family expansions compared to surface forms■ High replacement vs silent substitution ratio■ Selective sweep patterns

○ Purifying selection○ All of the above identify candidate trait changes

-> Predictive genomics across >5,000 genes

From sequence read to trait discovery:Predictive speleogenomics


● Spelling correct● Transcriptome or genome sequence based analyses of

cave adaptation● What have we learned so far?

Welcome to the age of Next Generation Sequencing!

http://www.slideshare.net/AlagarSuresh/ngs-introduction-51560394/2

2005

http://www.slideshare.net/AlagarSuresh/ngs-introduction-51560394/2

2005 2006 2007 2008 2009 2010

http://www.cnn.com/2016/03/28/health/cave-man-neanderthal-dna/index.html

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016

Homo n+s

http://www.cnn.com/2016/03/28/health/cave-man-neanderthal-dna/index.html

(Gross and Wilkens 2013; Bilandžija et al. 2013; Protas et al. 2006)

2005 2006 2007 2008 2009 2010 2011

OCA2 His615Arg

OCA2

OCA2

OCA2 1252G

Convergent evolution of pigment reduction in human and cavefish populations

Humans

Astyanax

2005 2006 2007 2008 2009 2010

Green et al. (2010)

Kim et al. (2011): Genome sequencing reveals insights into physiology and longevity of the naked mole rat. Nature, doi:10.1038/nature10533

● Strictly subterraneanoTolerate low oxygen and high carbon dioxide concentrations

● EusocialoReside in large colonies with a single breeding female (queen), who

suppresses the sexual maturity of her subordinate. ● Extraordinarily long-lived (>30 years)

oNegligible senescence▪No age-related increase in mortality▪High fecundity until death▪Resistant to both spontaneous cancer and experimentally induced

tumorigenesis● Unable to sustain thermogenesis● Insensitivity to certain types of pain● Microphthalmic

2005 2006 2007 2008 2009 2010 2011

● 2,4 Gb large genome● 22,500 genes● Apparent lack of age related expression level changes● 39 Proteins with unique amino acid changes

compared to 36 vertebrate genomes○ Thermoregulation○ DNA repair○ Oncogenesis○ Vision

2005 2006 2007 2008 2009 2010 2011


● Pain reception related neuropeptide gene ACT1 intact but ancestral promoter missing

● 244 pseudogenes:○ 183 frameshift events○ 119 premature termination events

Functional categories enriched for pseudogenes:SpermatogenesisOlfactory receptor activity Visual perception

2005 2006 2007 2008 2009 2010 2011



2005 2006 2007 2008 2009 2010 2011

Lack of function mutations in visual system genes:

2005 2006 2007 2008 2009 2010 2011

Friedrich et al. (2011): SB. 2011. Phototransduction and clock gene expression in the troglobiont beetle Ptomaphagus hirtus of Mammoth cave. J Exp Biol 214:3532–41.

2005 2006 2007 2008 2009 2010 2011

Friedrich et al. (2011): Phototransduction and clock gene expression in the troglobiont beetle Ptomaphagus hirtus of Mammoth cave. J Exp Biol 214:3532–41.

Bottom

Surface

Academy

1839-1905

Alpheus Spring Packard 1888. The cave fauna of North America, with remarks in the anatomy of brain and the origin of blind species. Memoirs of the National Academy of Sciences (USA) 4, 1-156

Academy


"Transverse and horizontal sections of the head of Adelops (=Ptomaphagus) hirtus, from Mammoth Cave, reveal no traces of the optic ganglia or the optic nerve."

Academy


"Transverse and horizontal sections of the head of Adelops (=Ptomaphagus) hirtus, from Mammoth Cave, reveal no traces of the optic ganglia or the optic nerve."

"It thus appears that Adelops must be blind though the eyes exist in a degenerate state."

Ommatidium

Schistocerca

Tribolium

Drosophila

A lens without photoreceptors?


Drosophila

Schistocerca

Tribolium

Schistocerca

Tribolium

Drosophila


RNA-seq: Deep sequencing transcriptome analysis

• NSF award NSF Award #0951886: Pax6 and the genetic regulation of eye development in Tribolium • Rui Chen, Bryce Daines (Baylor College)

25 dissected P. hirtus heads minus antennae

Deep sequencing of the P. hirtus head transcriptome

• NSF award NSF Award #0951886: Pax6 and the genetic regulation of eye development in Tribolium • Rui Chen, Bryce Daines (Baylor College)

● 27,428,409 short reads (75 bp) produced● 5,476,803 (19.97%) aligned by blastx to Tribolium refseq proteins● Orthologs of 8718 Tribolium transcripts observed with >10 reads● Concatemerization of blastx hit group reads

Drosophila (Adams et al 2000)

Ptomaphagus

Tribolium (TGSC 2008)

100200300 Mya

Deep sequencing of the P. hirtus head transcriptome

The insect phototransduction protein machinery

R opsin

Gqα

β

PLCβ PKC 53E

γINAD INAD

5

Arrestin 1

Arrestin 2

Gqα Gqα

R opsin

R opsin

PLCβ PKC 53E

2

TRP

4

3

1

5

2

4

3

1

Ca2+

The insect phototransduction protein machinery

R opsin

Gqα

β

PLCβ PKC 53E

γINAD INAD

5

Arrestin 1

Arrestin 2

Gqα Gqα

R opsin

R opsin

PLCβ PKC 53E

2

TRP

4

3

1

5

2

4

3

1

Ca2+

Conserved expression of the insect phototransduction protein machinery in P. hirtus

R opsin

Gqα

β

PLCβ PKC 53E

γINAD INAD

5

Arrestin 1

Arrestin 2

Gqα Gqα

R opsin

R opsin

PLCβ PKC 53E

2

4

3

1

5

2

4

3

1

TRP

Ca2+

Transcript conservation predicts a functional visual system in P. hirtus

•Structural evidence

•Behavioral evidence

Phototaxis

Circadian rhythm

Our first light-dark choice assay

Friedrich et al. (2011) The Journal of Experimental Biology 214: 3532-3541.

Light vs dark choice of P. hirtus in response to desktop lamp illumination

Friedrich et al. (2011) The Journal of Experimental Biology 214: 3532-3541.

Speleogenomic approach revealed:

Conservation of functional visionPresence of photoreceptor cellsNegative photoresponseDifferential wavelength specificity

Conservation of circadian gene expressionPreliminary evidence of circadian activity

rhythmsGenetic regression of the visual system:

Loss of UV opsinLoss of eye specific pigmentation genes:

cinnabarscarlet white

2005 2006 2007 2008 2009 2010 2011

Friedrich et al. (2011): Phototransduction and clock gene expression in the troglobiont beetle Ptomaphagus hirtus of Mammoth cave. J Exp Biol 214:3532–41.

2005 2006 2007 2008 2009 2010 2011 2012

● Genome size: 174 Mb● ~13,500 genes● “Dark-fly”: Drosophila melanogaster maintained in constant darkness > 60 years =

1,500 years○ Dark-fly produce more offspring in dark than in light○ Preserved strong phototactic behavior and circadian locomotor rhythm○ Nonsense mutation in the Rhodopsin7 (not expressed in eye) ○ Functional conservation of Rhodopsin 1-6 (all expressed in the eye)

Izutsu et al. (2012): Genome features of "Dark-fly", a Drosophila line reared long-term in a dark environment. PLoS One. 7(3):e33288.

2005 2006 2007 2008 2009 2010 2011 2012 2013

Hinaux et al (2011): De novo sequencing of Astyanax mexicanus surface fish and Pachón cavefish transcriptomes reveals enrichment of mutations in cavefish putative eye genes. PLoS One. 8(1):e53553.Gross et al. (2011): An integrated transcriptome-wide analysis of cave and surface dwelling Astyanax mexicanus. PLoS One. 8(2):e55659

2005 2006 2007 2008 2009 2010 2011 2012 2013

● 44,145 contigs assembled from expressed sequence tags generated by Sanger sequencing

● 8 eight cDNA libraries■ 4 from a surface stream population■ 4 from the Pachon cave population

● For both populations, libraries were generated from four developmental stages.

Hinaux et al (2011): De novo sequencing of Astyanax mexicanus surface fish and Pachón cavefish transcriptomes reveals enrichment of mutations in cavefish putative eye genes. PLoS One. 8(1):e53553.

2005 2006 2007 2008 2009 2010 2011 2012 2013

Hinaux et al (2011): De novo sequencing of Astyanax mexicanus surface fish and Pachón cavefish transcriptomes reveals enrichment of mutations in cavefish putative eye genes. PLoS One. 8(1):e53553.

● 31 non-conservative amino-acid changes specific to the lineage of the cave population.○ The significant majority function in carbohydrate

metabolism○ No enrichment of eye developmental genes

● 79 non-conservative amino-acid differences between the genes of surface and cave morphotypes tentative evidence of suggested accelerated evolution of vision genes

2005 2006 2007 2008 2009 2010 2011 2012 2013

Gross et al. (2011): An integrated transcriptome-wide analysis of cave and surface dwelling Astyanax mexicanus. PLoS One. 8(2):e55659

● 22,596 contigs from 454 sequencing data from Pachon and surface animals ○ > 600 transcripts were unique to cave or surface

populations■ 10% of which were related to genes known from

zebrafish○ Two classes of gene functions overrepresented in

the conserved population-specific subsamples:■ Metabolic genes were specific for, or

transcriptionally elevated in cave morphotype■ 16 homologs associated with visual functions

specifically missing or reduced in the cave morphotype

2005 2006 2007 2008 2009 2010 2011 2012 2013

Meng et al. (2013): Evolution of the eye transcriptome under constant darkness in Sinocyclocheilus cavefish. Molecular Biology and Evolution 30 :1527-1543

● Compared the mature retinal histology of surface and cave species○ Reduction in number and length of photoreceptor cells

● Generated transcriptomes for surface and cave species● Transcriptional downregulation of eye fate promoting

transcriptional factors

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016

Yang et al. (2016): The Sinocyclocheilus cavefish genome provides insights into cave adaptation. BMC Biol. 2016 Jan 4;14:1.

●Whole-genome sequencing and comparative analysis of:○ Macrophthalmous S. grahami, 1.75 Gb○ Micophthalmous S. rhinocerous 1.73 Gb ○ Anophthalmous S. anshuiensis.and 1.68 Gb

●~40,000 genes in each species●Several opsin genes missing in all three species:

○ Lws2, Rh2-1 and Rh2-2 (middle wavelength-sensitive)

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016


● Anophthalmous S. anshuiensis:○ Genetic regression related to pigmentation:

■ Reduced transcript levels of Oca2 and melanogenesis pathway genes

■ Loss of Mpv17○ Genetic regression related to vision:

■ Opsin gene Rh2-4 lost specifically in anophthalmic species (Sa)

■ Nine eye developmental regulators transcriptionally downregulated

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016


● Anophthalmous S. anshuiensis:○ Gain of function changes related to taste reception:

■ Duplication of taste receptor genes, such as Tas1r1 and Tas2r200-2

○ Gain of function changes related to taste reception:■ Hsp90α1

● Duplicated● Higher expression level

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014

Tierney et al. (2013): Opsin transcripts of predatory diving beetles: a comparison of surface and subterranean photic niches. R Soc Open Sci. 2015 Jan 28;2(1):140386.

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014

● Identified transcripts for UV, LW, and c-opsins from surface beetle transcriptomes

● 3 out of 3 subterranean beetles presented evidence of parallel loss of all opsin transcription

● Documents genetic regression of the visual system

Tierney et al. (2013): Opsin transcripts of predatory diving beetles: a comparison of surface and subterranean photic niches. R Soc Open Sci. 2015 Jan 28;2(1):140386.

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014

McGaugh et al. (2014): The cavefish genome reveals candidate genes for eye loss. Nat Communications 5:5307. doi: 10.1038/ncomms6307

● First de novo genome assembly for Astyanax mexicanus○ ~1 Gb large genome○ ~23,000 genes

■ ~16,000 1:1 orthologs to zebrafish● Identifies candidate genes underlying previously mapped quantitative trait

loci (QTL)

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014


● Identifies and tests developmental eye reduction candidate genes● Assays cave adaptive candidate genes for potential functional and

expression differences between surface and cave morphotype○ RT-PCR experiments define morphotype candidate gene losses as

false negatives

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014


● RT-PCR confirmation of species-generic candidate gene losses in several gene families:○ Retinol dehydrogenases○ Crystallins○ Sine oculis homeoboxes○ Opsins○ Fibroblast growth factors○ Gamma-aminobutyric acid A○ Dopamine receptors

Vision

Sleep and circadian clock

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015

Stahl et al. (2015): A Transcriptomic Analysis of Cave, Surface, and Hybrid Isopod Crustaceans of the Species Asellus aquaticus. PLoS One. 10:e0140484.

● Deep sequenced cDNA libraries○ Cave morphotype○ Surface morphotype○ Hybrid

● ~25,000 transcripts○ >4,000 with orthologs in “other” or non-model

species● Identifies candidate morphotype-specific alleles

Welcome to the tree of speleogenomic animal life!

Astyanax mexicanus SinocyclocheilusHeterocephalus

glaber

P. hirtus

Paroster macrosturtensis

Asellus aquaticus

Homo n+s

Speleogenomic coverage of cave animal species so far

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016

Speleogenomic coverage of cave animal species so far

Coverage of animal genomes so far

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016

http://www.ncbi.nlm.nih.gov/genome/browse/

http://www.ncbi.nlm.nih.gov/genome/browse/


● What have we learned so far?○ A lot

● What is the future holding?○ Genetic surveys of vanishing diversity○ High resolution genetic analysis of cave invasion○ High resolution analysis of troglomorphism○ Value of close relationship to model organisms○ Impact of genetic drift: population genomics○ Impact of time: Young vs old troglobites○ Quest for the oldest and the youngest troglobite...