Genetic and Developmental Basis of Evolutionary Pelvic Reduction in Threespine Sticklebacks Michael D. Shapiro, Melissa E. Marks, Catherine L. Peichel, Benjamin K Blackman, et al. Presentation By Phillip Calender and Adam Gray

Introduction  Threespine Sticklebacks are small fish native to northern rivers, seas, oceans and lakes worldwide  Marine threespine sticklebacks have pelvic structures to protect themselves against gape- limited, soft-mouthed predators by presenting a lacerating structure.  Freshwater Sticklebacks have lost this pelvic structure as a probable response to local absence of predatory fish, low calcium ion availability, to to avoid predation by macroinvertebrates that grasp the fish by their dorsal and pelvic spines.

Introduction (cont.)  The goals of the study were to determine the number and type of genetic changes underlying this pelvic reduction.  Aspects studied were: –Genetic architecture of pelvic reduction through examination of the number and location of chromosome regions controlling pelvic reduction –Isolation and mapping of candidate genes –Comparison of left-right asymmetry in pelvic reduction –Sequence comparison of candidate gene –Testing for possible regulatory change in the candidate gene –Comparison to other Threespine Stickleback populations

Genetic architecture of pelvic reduction  First, the researchers crossed a marine female with a Paxton Lake male.  375 F2 progeny were then studied and measured for extent of development of pelvic features  Scoring pelvic reduction as a qualitative trait involved normal pelvic structures versus any form of size reduction, loss, or asymmetry

Genetic architecture of pelvic reduction  The scoring revealed a near 3:1 Mendelian ratio of unaffected to affected fish, meaning that pelvic reduction is dominant.  Mapping of the qualitative trait connected linkage group 7, which showed strong control over all pelvic traits  Several chromosome regions with smaller effects (QTL’s) were also detected  Remaining variance was deemed to be from additional minor loci with phenotypic effects too small to detect, or to environmental or epistatic factors

Genetic architecture of pelvic reduction  Even fish with one Paxton and one marine allele near a given QTL showed significant change in the size of pelvic structures, indicating incomplete dominance of pelvic reduction, and the effects of the Paxton QTL’s was additive  “Rare, favourable, new alleles with semi-additive effects are expected to spread more repidly in an evolving population than purely recessive alleles.”  Traits controlled by a relatively small number of genes should evolve more quickly than traits that require mutation and fixation across many loci.  Results suggest that major morphological mutations in pelvic reduction here occurred through relatively few chromosome regions (in a natural population)

Isolation and mapping of candidate genes  Several genes are known to be expressed specifically in hindlimbs.  Tbx4, Pitx1, Pitx2  BAC clones were prepared for stickleback orthologues of each gene  Sequenced portions of the clones were used to identify polymorphic genetic markers  These markers were then used to determine the segregation pattern of these genes in the F2 population  Pitx1 mapped to linkage group 7 while Tbx4 and Pitx2 mapped to linkage groups 1 and four respectively.

Left-right asymmetry in stickleback pelvic reduction  Pitx1 pelvic limb reduction in mice is directionally asymmetric, with greater reduction on the right than left side.  Does this also occur in the Threespine stickleback F2 population?

Left-right asymmetry in stickleback pelvic reduction YES Directional asymmetry of pelvic reduction in sticklebacks also occurs. Longer spines were seen on the left than the right in 78% of the animals with asymmetric development (as opposed to complete loss. Granted, this is a weak test for Pitx1

Pitx1 Sequence comparison in marine and benthic fish  Determined the exon/intron structure of the gene  Sequenced the entire coding region and exon/intron junctions of the gene in both parents  Pitx1 in threespine sticklebacks is very identical to that of other known Pitx1 sequences  But, no coding region mutations were found that would alter this gene between the two parents.  Both parents form the exact same protein product according to reverse transcriptase mediated polymerase chain reaction and sequencing studies. So why the difference?

Altered Pitx1 gene expression in pelvic reduced fish  To test for possible regulatory changes in the gene, the researchers examined the spatial pattern of gene expression during normal development of the F2 progeny  Pitx1 is expressed in many different regions, including the developing thymus, olfactory pits, sensory neuromasts on the head, trunk, and tail, and the ventral portion of the developing caudal fin in addition to the site of the pelvic fin bud.

Altered Pitx1 gene expression in pelvic reduced fish

 For the freshwater fish, no Pitx1 expression was seen in the prospective pelvic region, but the gene was expressed in all other normal regions.  But the caudal fins are normal, so the altered expression cannot be due solely to an absence of some structures  The expression was also not due to a developmental delay or a difference in timing. No expression was ever detected in the freshwater larvae

Altered Pitx1 gene expression in pelvic reduced fish

Parallel Evolution of Pelvic Reduction  Complementation cross was created between Paxton fish and Lake Vifilsstadavatn (Iceland) fish to see if the genetic basis of pelvic loss is similar in both locations.  The pelvic reduction alleles in the two populations failed o complement and restore normal pelvic morphology.

Parallel evolution of pelvic reduction  Cross between Icelandic fish (same lake, this is just easier to pronounce) produced F1 fish that had strong pelvic features, implying that the failure of pelvic development in the complementary cross (Paxton X Iceland) was not due to dominant genetic changes in the Hard-to- pronounce lake fish.  Pelvic Reduction in these two distinct Pacific and Atlantic basin populations likely results from defects in similar genes

Discussion  Cis-acting regulatory mutations in Pitx1 are a major cause of pelvic reduction in this rapidly evolving system (10,000 yrs old, many conserved non-gene regions around the Pitx1 locus)

Discussion  Paxton Benthic fish show no alterations in Pitx1 coding sequences and changes in gene regulation disrupt expression only at specific sites in developing larvae.  Regulatory mutations in key developmental control genes may provide a general mechanism to selectively alter expression in specific structures yet preserve expression at all other sites

Discussion  This variation has been seen in many other species, but DNA sequences pertaining to tissue specific expression are still unknown  Pitx1 gene is flanked by a 300+kb region that is highly enriched in conserved non- coding sequences  Future research will be to identify these regions and compare them between stickleback populations

Discussion  Complementation cross suggests that this mode of pelvic reduction is the mechanism used in populations 5,700 km apart.  May be similar in other species, and the methods of this paper can be used in other species.

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