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More on That Bladderwort "Junk DNA" Claim

Myth of Junk DNA.jpegHas a carnivorous plant proven that non-coding DNA is not essential, and thus that it is junk after all? Earlier we briefly explained why the claim is illogical, considering all the evidence to the contrary. Now let's take a closer look at the original paper and the ensuing media fallout.

The paper in Nature, with nearly 30 international authors, is called "Architecture and evolution of a minute plant genome." The carnivorous bladderwort, Utricularia gibba, has a smaller genome than many other plants (82 megabases). What inferences can be drawn from this evidence about the significance of non-coding DNA?

First of all, this is not the only case of extreme size reduction in a genome. We know that some pathogenic microbes have extremely sparse genomes, presumably because they rely on their hosts. As a carnivorous plant, could the bladderwort rely on its food source for some genomic processes that might otherwise be necessary? How do other carnivorous plants compare? These are some initial questions we might ask about this exceptional case.

The authors write in the abstract,

The compressed architecture of the U. gibba genome indicates that a small fraction of intergenic DNA, with few or no active retrotransposons, is sufficient to regulate and integrate all the processes required for the development and reproduction of a complex organism. (Emphasis added.)

In scientific practice, it can be reckless to extrapolate one finding to a universal principle, such as the claim that intergenic DNA is unnecessary in all organisms. Is there something unique about this plant?

The bladderwort, without question, is very unusual. Not differentiated into roots, leaves and stems, it relies for food on its sophisticated traps that capture small protozoa and metazoa in moist soil. The traps are faster than the blink of an eye. When the trigger hairs are touched, the prey is sucked from outside to inside in thousandths of a second. The intricate design of these traps has inspired some engineering attempts to imitate it -- but that's another story.
Because of floral similarities to snapdragons and monkeyflowers (Mimulus), the bladderwort is classified with them in the family Lentibulariaceae. Yet its genus is so unusual that the classification seems somewhat forced. The Nature paper forces affinities even further back, claiming its genome indicates that three whole-genome duplications (WGD) have occurred since its common ancestry with angiosperms. Yet the fine print in the Supplemental Information document shows that the evidence for these events is scrambled by polyploidy and mutation, requiring a bit of analytical license to support the claim. The authors say at one point, "at least two" WGD's occurred.

In discussing the bladderwort's homologies with tomatoes and other plants, the authors embed their Darwinian assumptions in their methods and conclusions. Those assumptions require them to believe that the sophisticated traps are products of unguided, directionless processes of natural selection. Notice how their statements are often theory-laden, and become more speculative the further back the presumed ancestor goes:

Our U. gibba genome assembly, produced using a hybrid (454/Illumina/Sanger) sequencing strategy, closely matches the genome size estimated by flow cytometry (77?megabases (Mb)) (Supplementary Information section 1). Remarkably, despite its tiny size, the (G+C)-rich U. gibba genome accommodates about 28,500 genes, slightly more than Arabidopsis, papaya, grape or Mimulus, but less than tomato (Supplementary Information section 2). Indeed, the U. gibba genome has experienced a small, approximately 1.5% net gain across a conserved set of single-copy genes (Supplementary Information section 2.6). Synteny analysis reveals that U. gibba has undergone three sequential WGD events since last common ancestry with tomato and grape, with one of these duplications possibly shared by the closely related species Mimulus (Fig. 1a and Supplementary Information section 7). Consequently, the U. gibba genome seems to be 8� with respect to the palaeohexaploid (3�) core eudicot ancestor (Fig. 1b), whereas Arabidopsis is 4� with a genome 1.5-times larger. Compared with independently polyploid tomato, the U. gibba genome shows extremely fractionated gene loss (Fig. 1c), with almost two-thirds of syntenic genes shared with tomato having returned to single copy (Supplementary Information section 7.4 and Supplementary Table 39).

The 91-page Supplemental Information document shows reliance for many of their evolutionary conclusions on software that assumes common ancestry. Here is one example:

In SynMap, when syntenic gene pairs are coloured by Ks values, syntenic regions derived from the same evolutionary event (e.g., polyploidy or divergence of lineages) tend to be coloured similarly. By using the syntenic path assembly, evolutionarily or structurally related contigs that would otherwise be scattered across a dotplot will cluster, permitting the visualisation of evolutionary patterns such as polyploidy.

Later, we see them stretching and shrinking the timing of events, mutation rates and evolution rates to maintain their assumption of common ancestry. On page 30, they have to overcome some evidence that appears to contradict the claim of three WGDs. The point is that their conclusions are highly dependent on the methods used to analyze the data. Watch them struggle at this spot on page 32:

Estimates of syntenic depth using structural syntenic comparisons are complicated by two major factors: evolutionary time and completeness of genomic sequence. Genomes change over time, which obfuscates identifying syntenic regions, specifically when polyploidy is involved since the diploidisation process fractionates duplicated genes. Since many genome sequences are generated by NextGen shotgun sequencing, the resulting assemblies have many small chromosome fragments. Such small contigs often lack enough genes to infer synteny through either a colinear arrangement of genes or through a local density of colinear genes, a problem that is exacerbated by genome evolution.

In addition, they had to propose a faster mutation rate and evolution rate in the bladderwort than for other plants. The charts show a lot of crisscrossing lines trying to line up genes of bladderwort with those of other plants. There appears to be ample opportunity for confirming one's biases when analyzing data sets this complicated.

But Is It Junk?

Returning to the question of whether non-coding DNA is essential, the paper indicates that it's a complicated question how or why the bladderwort reduced its genome so dramatically compared with other plants. The reductions are in the nuclear DNA only; no significant reductions were found in mitochondrial DNA or plastid DNA. "Therefore, the evolutionary forces acting to reduce U. gibba genome size seem to have affected only the nucleus," they say. Additionally, some of bladderwort's protein families are enriched compared to other plants, and some are diminished. It's not a simple story. The conclusions about this genome don't leap out of the data. You might say, "Some assembly required."

Since bladderwort does not possess true stem, root, and leaf distinctions, it is possible that many genes required for their development are not needed. "Interestingly, contractions and losses in all of these root-expressed MADS box gene clades/subfamilies account for much of the global reduction of the MADS box gene family in U. gibba," the authors say. Other gene families are expanded, however; "it is tempting to speculate that specific clade expansions may be related to the genus-wide diversity of branching patterns in Utricularia," they write. Why are they omitting the possibility that this genus, while having much in common with other plants, is designed differently?

Taken together, we infer from our analyses of U. gibba coding sequence that natural selection preserved a core set of gene functions, most of which have returned to single copy along with considerable genomic fractionation after three WGDs. Relaxed selection pressure for unnecessary functions probably led to gene losses, whereas in other cases, gene family expansions may have been promoted by selection. Evidence for localized selection on the U. gibba gene complement, however, does not provide support for the existence of genome-wide selective forces that might favour reduction of nonessential, non-coding DNA.

That last sentence precludes a simple evolutionary theory to account for the loss of non-coding DNA, or for considering it nonessential.

So other than stating their biases, did they find that non-coding DNA in other organisms is junk? They dispensed with a suggestion that U. gibba was under different selection pressure than Arabidopsis. In fact, it's not clear to them what exactly they did determine:

Collectively, our analyses highlighting total gene complement, sequential WGD and mutational diversity estimates for U. gibba raise quandaries regarding the evolution of its contracted genome. It is possible that inherent molecular mechanisms favouring deletion dominated nuclear genome size reduction in a population genomic background where selection was too weak to counteract such a burden.,,, Of course, a molecular-mechanistic deletion bias does not preclude that selection still enhances fixation of such deletions.

Indeed, since carrying and duplicating non-coding DNA is a burden, one would think that natural selection would hurry to fix such deletions if they were nonessential. Another quandary is why three whole-genome duplications did not buffer against gene loss.

With so many quandaries, the authors end by calling for more genomes from more species to figure out what causes these changes in certain plant families. After so much head scratching, you might hope they would be a little reticent to attack the results of the ENCODE project. But they do. That's what generated all the press.

In summary, U. gibba genome architecture demonstrates that angiosperms can evolve diverse gene landscapes while overall genome size contracts, not only during expansions. Furthermore, in contrast to recent publications that highlight a crucial functional role of non-coding DNA in complex organisms such as animals [here they cite the ENCODE paper], the necessary genomic context required to make a flowering plant may not require substantial hidden regulators in the non-coding 'dark matter' of the genome.

So there, after a theory-laden analysis of a very complicated data set, they made a mere suggestion that became the lead story in the press. Live Science reports, "'Junk' DNA Mystery Solved: It's Not Needed." PhysOrg writes, "Bladderwort genome contradicts notion that vast quantities of noncoding DNA crucial for complex life."

We hope this excursion into the sausage factory for evolutionary claims reinforces the need for discernment when evaluating simplistic headlines.