Jumping genes…

Jumping genes capture
deep relationships between
parrots and songbirds

A new study adds support to two earlier reports that songbirds and parrots are each other’s closest relatives (Psittacopasserae), indicating that vocal learning abilities appeared in this group of birds 30 million years earlier than originally assumed

GrrlScientist Tuesday 23 August 2011, guardian.co.uk

Passerines and parrots share a common ancestor as well as the ability to learn vocalization. Vocal learning may have evolved 30 million years earlier than previously thought.
Image: Corn bunting, Miliaria calandra. Kriegs/LWL (with permission)

Birds share many characteristics with humans. Unlike our fellow mammals, which learn about the world primarily by sniffing crotches, birds and humans have excellent colour vision, are highly auditory and some groups possess superior vocal learning abilities. Thus birds — especially songbirds and parrots — are the most important teachers that we have, particularly for scientists who study higher cognitive processes such as vocal learning and memory. Yet even though birds are the most studied group of animals in the world, the evolutionary origins of several major avian groups, particularly passerines and psittacines, remain shrouded in the mists of time.

Roughly 50 percent of all birds are passerines (songbirds), yet their relationships remain enigmatic. For example, morphological studies suggested that songbirds are a relatively young group within Neoaves (Neoaves includes all birds except the ratites — ostriches and similar — and the Galloanserae — "waterfowl", "wildfowl" and "landfowl"), and that the sister group to the songbirds may be woodpeckers [doi:10.1111/j.1096-3642.2006.00293.x] and rollers [doi:10.1007/BF02101113] or cuckoos [doi:10.1046/j.1439-0469.2003.00230.x]. But mitochondrial studies indicate a number of conflicting relationships between passerines and the other Neoaves, and nuclear DNA analyses provide yet more conflicting data. Basically, none of our methodologies were providing clear information, so our understanding of songbird relationships were confused and confusing.

Then three years ago, a fascinating paper was published describing a DNA study that shook the foundations of our understanding of the avian tree of life. This study examined almost 32 kilobases of nuclear DNA sequences from 19 independent loci for 169 species representing all major extant avian groups — a tremendous number at the time. The most stunning revelation was that parrots and songbirds are sister taxa — each other’s closest living relatives. Was that proposed psittacine-passerine phylogenetic relationship real or was it artifact? Even though this finding agreed with an earlier study that proposed the same relationship [doi:10.1098/rsbl.2006.0523], this finding had the scientific and bird-watching communities abuzz. Everyone, it seemed, had an opinion about this.

The scarlet macaw, Ara macao, and other parrots learn vocalizations. Their closest living relatives, passerines, are also adept vocal learners, indicating that vocal learning may have evolved in their common ancestor.
Image: Kriegs/LWL (with permission) [velociraptorise] doi:10.1038/ncomms1448

Now there’s another study that adds yet more support to those original findings. Published by a team of German investigators led by Münster University graduate student Alexander Suh, this hot-off-the-presses research takes an even closer and more detailed look at the proposed relationship between parrots and songbirds by using yet another method: retroposons.

Retroposons are "jumping genes". They are repetitive DNA fragments that insert randomly into the genome after being copied ("reverse transcribed") from an RNA intermediary. Whilst the original repetitive retroposon sequence identities and locations are inherited like any other genetic loci, the new insertions (along with any changes in sequence) are unique and are reliably inherited from the time they are inserted. The resulting patterns of change are straightforward and can be traced through ensuing generations for more than 100 million years — major evolutionary timescales. In short, retroposons are molecular fossils.

Based on what we know of retroposons, it is reasonable to predict that they could be used as phylogenetic markers that may provide some intriguing insights into previously unresolved deep evolutionary relationships found early avian evolution, but no one was quite sure what Mr Suh and his team would actually discover.

There are more than 200,000 retroposed elements (REs) present in the chicken and zebra finch genomes, so Mr Suh and his team chose the two most numerous, comprising more than 97 percent of all avian REs: the chicken repeat 1 (CR1) family of long interspersed elements (LINEs) and the long terminal repeat elements (LTRs) of endogenous retroviruses.

Despite this huge number of loci, when the team analysed their chosen REs for bird-specific insertions in key avian groups, they identified several consistent patterns: (1) some retropsons (black balls) had a small deletion that was present in some avian CR1s, but was absent in all other avian and reptilian CR1s; (2) the presence or absence of specific RE markers (dark grey balls) are consistent with one another; and (3) some REs were inserted at the beginning of neoaves (light grey balls on grey gradient [label F]) and the RE dimorphisms were then subjected to incomplete lineage sorting. And the data were used to reconstruct an unambiguous family tree (figure 1):

Figure 1 | Retroposon evidence for the early branching events in the avian tree of life. [velociraptorise]

As you can see in the above phylogenetic tree, the retroposon evidence within Neoaves shows unmistakable presence/absence patterns. For example, the previously reported "landbird" assemblage (figure 1, branch G; two REs; also see doi:10.1098/rsbl.2006.0523) was recovered, as was a novel clade consisting of all "landbirds", excluding mousebirds (figure 1, branch H; two REs; also see doi:10.1126/science.1157704) and these data also indicated a close affinity among seriemas, falcons, parrots and passerines (figure 1, branch I; two REs). Also note that these letters correspond to the labeled branches: A, Aves; B, Neognathae; D, Neoaves; F, incongruent markers; G, ‘landbirds’; H, ‘landbirds’ without mousebirds; I, Eufalconimorphae + seriemas; J, Eufalconimorphae; K, Psittacopasserae; L, Passeriformes.

In short, the most important take-away message from the above phylogram is that songbirds and parrots are sister taxa, and their closest relative is falcons (Falco) — and falcons are not closely related to the other raptors (hawks and eagles; Buteo). For the specialist, the parrot-passerine (Psittacopasserae) relationship is an important finding, because until very recently, parrots and songbirds were considered to be only distantly related.

The consistency between these markers surprised the team.

"I was very surprised that (after all these controversies between and within mitochrondrial, nuclear and DNA-hybridization datasets regarding the neoavian radiation) we actually recovered something that appears to be non-conflicting retroposon markers", wrote Mr Suh in an email message.

But when did these retroposons do most of their "jumping"? The team looked for nested retroposons (sequence data not shown here); younger REs inserted inside older (inactive) REs that were already in place (figure 3):

Figure 3 | Chronology of Mesozoic retroposon activity in the zebra finch genome. [velociraptorise]

The team examined 995 nested retroposons in the zebra finch genome (blue balls are CR1 or red balls are LTRs; numbers indicate specific RE subtypes and the horizonal blue or red lines indicate "activity periods"). They identified which retroposon markers were inserted during the neoavian radiation (dashed bracket) at specific times during avian evolution up until the estimated end of the Mesozoic Era at the Cretaceous/Tertiary boundary (grey dashed vertical line).

These divergences are denoted in the above figure, starting from the left side, as follows: the first pale grey vertical bar indicates when the Paleognaths (ratites; ostriches and similar birds) split away from the Neognaths (all the other birds); the second pale grey vertical bar indicates when the Galloanserae ("fowls") split away from the Neoaves; the broader pale grey vertical bar corresponds to the rapid Neoavian radiation; and the pale grey vertical bar on the right side denotes when the Acanthisittidae split away from the oscines (Acanthisittidae are similar to suboscines because they do not learn their songs).

The most important thing to note in the chromogram above is that some RE subtypes were active during relatively short periods whilst others were not, yet these REs provide a surprisingly consistent estimate for retroposon nesting events during the Mesozoic evolution of birds.

Based on the topology of the phylogenetic tree (figure 1), we can see that vocal learning first appeared in the shared ancestor for parrots and songbirds — after the falcons diverged, as summarised below (figure 4):

Figure 4 | Evolution of vocal learning in birds. [velociraptorise]

This study underscores the striking neuroanatomical and gene expression similarities between parrots and songbirds, indicating that their vocal learning ability originally appeared in their shared ancestor 30 million years earlier than first predicted.

"With the strong retroposon support for Psittacopasserae (parrots + passerines), it seems plausible (and quite parsimonious) to assume that their similarities are not due to convergent evolution, but to shared ancestry", wrote Mr Suh.

"Passerines and parrots are among those few avian groups that are capable of vocal learning and the new study suggests that this capability already originated in the last common ancestor of the group," wrote Gerald Mayr, in email. Dr Mayr, a paleo-ornithologist and curator of ornithology at the Senckenberg Museum in Frankfurt, Germany, was not involved in the study.

"Many studies on vocal learning used either zebra finches [passerines] or parrots as model organisms, and recognition of close affinities between parrots and passerines may now make it much easier to directly compare and understand the data obtained from these different bird groups."

Another interesting thing to note is that even though hummingbirds are also vocal learners, this study shows that they are only distantly related to Psittacopasserae, so their vocal learning capability evolved independently after they diverged from the swifts, their sister group, which lacks this ability.

The lesser kestrel, Falco naumanni, and all other falcons are not related to other raptors. Instead, they are the closest living relatives of Psittacopasserae (passerines and parrots). Together, these are the Eufalconimorphae.
Image: Kriegs/LWL (with permission) [velociraptorise] doi:10.1038/ncomms1448

Apart from the fact that everyone is familiar with both parrots and songbirds, and is interested to learn more about them, this study also shows that analyses of "jumping genes" are a powerful new tool that can help to clarify evolutionary relationships among birds.

Even though the retroposon data is fully independent of nucleotide sequence analyses it still verified previous findings and provided robust support for some relationships, such as the Psittacopasserae, noted Mr Suh.

"If an understanding of the basic neural mechanisms of vocal learning is eased by recognition of close affinities between parrots and songbirds, this will perhaps also help to better understand vocal learning in humans," Dr Mayr concluded.

Sources:

Suh, A., Paus, M., Kiefmann, M., Churakov, G., Franke, F., Brosius, J., Kriegs, J., & Schmitz, J. (2011). Mesozoic retroposons reveal parrots as the closest living relatives of passerine birds. Nature Communications, 2 DOI: 10.1038/ncomms1448

Alexander Suh [emails; 18 & 23 August 2011]

Gerald Mayr, curator of ornithology, Senckenberg Museum [email: 23 August 2011]

Background:

Here’s what I wrote about a Science paper that might be of interest regarding the deep phylogenetic relationships between birds.

Hackett, S., Kimball, R., Reddy, S., Bowie, R., Braun, E., Braun, M., Chojnowski, J., Cox, W., Han, K., Harshman, J., Huddleston, C., Marks, B., Miglia, K., Moore, W., Sheldon, F., Steadman, D., Witt, C., & Yuri, T. (2008). A Phylogenomic Study of Birds Reveals Their Evolutionary History. Science, 320 (5884), 1763-1768 DOI: 10.1126/science.1157704

Ericson, P., Anderson, C., Britton, T., Elzanowski, A., Johansson, U., Kallersjo, M., Ohlson, J., Parsons, T., Zuccon, D., & Mayr, G. (2006). Diversification of Neoaves: integration of molecular sequence data and fossils. Biology Letters, 2 (4), 543-547 DOI: 10.1098/rsbl.2006.0523

Universität Münster News aus dem Bereich Presse (in Deutsch)

Funding for this research was provided by Deutsche Forschungsgemeinschaft (the German Research Foundation) and the Medizinische Fakultät der Westfälischen Wilhelms-Universität Münster (Medical Faculty of the Westphalian Wilhelms-University of Muenster).

Jumping genes reveal birds and their sex chromosomes evolved together

Avian retroposons — "jumping genes" — reveal that birds and their sex chromosomes evolved together, and provide us with important clues into the evolution of sex chromosomes and sex in general

by GrrlScientist Monday 24 October 2011, guardian.co.uk

Like mammals, the sex of individual birds is determined by the combination of sex chromosomes they get from their parents at fertilization. But unlike mammals, where females are the homogametic sex possessing two copies of the same sex chromosome, males are the homogametic sex. This difference to the mammalian sex chromosome system is indicated by the name: instead of X and Y, avian sex chromosomes are known as Z and W. Similar to mammalian sex chromosomes, avian sex chromosomes consist of one large chromosome (Z) and one very small, degenerate chromosome (W), which evolved from a pair of autosomes (non-sex chromosomes). However as one might predict, Z and W arose independently and their evolution followed an independent trajectory from that of mammalian X and Y chromosomes.

But within Aves, the sex chromosomes of different groups, or clades, of birds, are at different stages of evolution. For example, the neognaths, which include most of the birds that commonly visit your bird feeders, have ZW chromosomes that resemble mammalian XY chromosomes: one very large chromosome paired with one tiny, degenerate chromosome. On the other hand, the paleognaths, which include the ostriches and similar mostly terrestrial, flightless birds, have sex chromosomes that look like autosomes. In fact, their sex chromosomes behave a lot like autosomes, too; recombining along much of their length to swap genetic information (doi:10.1023/A:1009278914829). But that said, there is one paleognath lineage, the neotropical tinamous, that shows an intermediate level of Z–W differentiation (doi:10.1007/s00412-006-0088-y).

But have the genes located on the sex chromosomes evolved similarly to each other? And what does the pattern of sex chromosome evolution tell us about the evolution of birds?

You may recall that I recently wrote about retroposons, often referred to in the mainstream media as "jumping genes".

Retroposons are repetitive DNA fragments that are sprinkled throughout the genome. They are copied ("reverse transcribed") from an RNA intermediary that randomly "jumps" to another locus within the genome where they insert themselves. The original repetitive retroposon sequence identities and locations are inherited like other genetic loci, but the new insertion locations (along with any changes in sequence) are unique and are reliably inherited from the time they are inserted. The resulting patterns of change are easy to see and can be followed through ensuing generations for more than 100 million years — major evolutionary timescales. In short, retroposons are molecular fossils.

Like other chromosomes, sex chromosomes carry retroposons. Since similar genes (gametologues) on the avian Z and W chromosomes are not swapping genetic information, their evolution can be mapped by the presence or absence of retroposons (figure 1):

Figure 1.

The above diagramme shows how a pair of gametologues change as retroposed elements (REs) insert into each member of the pair at different times. The large gray boxes are exons (genes), small gray boxes are untranslated regions (not encoding any proteins), and the white boxes are retroposons [figure 1, larger view].

The gene on the left represents the pair whilst they were crossing over frequently. At that point, they look the same. However, after recombination stops, these regions on the sister chromosomes are free to diverge independently along their own evolutionary pathways, giving rise to two very similar, but no longer identical, gametologues (right side). REs that inserted prior to the cessation of crossing over (asterix) are inherited by both gametologues, whilst those REs that "jumped" after crossing over ceased (circle) are unique to just one of the gametologues. Of course, the corresponding regions of the two gametologues are different lengths, too.

Alexander Suh, a graduate student who studies phylogenomics at Münster University in Germany, sees retroposons as an unique way to study the evolution of avian sex chromosomes. Mr Suh spearheaded the effort to screen all 12 gametologous genes known from the chicken genome. His team was specifically looking to identify Z-/W-presence or Z-presence/W-absence regions amongst the 126 sequenced intron pairs for all birds (doi:10.1534/genetics.108.090324). They identified four such regions in three gametologues: introns 9 and 16 of the chromodomain helicase DNA–binding protein 1 (CHD1) gene, intron 16 of the avian homologue to the Drosophila Nipped-B (NIPBL) gene, and intron 3 of the ATP synthase α-subunit isoform 1 (ATP5A1) gene. Analyses of RE insertion patterns for each locus yielded three phylogenetic trees (data for CHD1 introns 9 & 16 are combined into one tree) (figure 2):

Figure 2.

The overall shape of the CHD1 gene tree (figure 2a; larger view) corroborates previous research showing an independent differentiation of this gene pair in tinamous and in neognaths (doi:10.1007/s00412-006-0088-y). These RE insertion patterns were also confirmed with sequence analyses.

Not surprisingly, a different tree topology was obtained for NIPBL, because recombination ceased independently in this gene pair in the neoavian and in the galloanseran lineage (figure 2b; larger view). Unfortunately, it’s not clear from the DNA data whether recombination ceased in the ancestor of Galloanserae or shortly after they diverged.

The gene tree of ATP5A1 is quite complex. It looks as though recombination ceased independently in all six neoavian representatives that were included in this study (figure 2c; larger view), but more study and more avian representatives are necessary to better understand these patterns and when they occurred.

"We have looked at sex chromosome evolution in birds from a perspective based on rare genomic changes," writes Mr Suh in email.

As I already mentioned, retroposon insertions are very useful for uncovering evolutionary relationships. But this new study shows yet another use of retroposons: helping us to understand the temporal sequence of sex chromosome evolution and gametolog differentiation. By tracing the pattern of RE insertions in gametologues of living bird species, it is possible to follow this back to the common ancestor of their sex chromosomes.

"To our knowledge, this has not been done in any other sex chromosomal system yet — so it should be promising to have a look at the many other sex chromosomal systems (e.g., in mammals, snakes, some turtles, some fish, some plants) from this perspective."

Retroposons provide another source of molecular information that is independent of autosomal or mitochondrial DNA sequences. But taken together, the RE insertions and other rare genomic changes suggest that the CHD1Z/CHD1W genes were already differentiated in the common ancestor for neognaths (119–105 mya; doi:10.1534/genetics.108.090324). Additionally, the NIPBLZ/NIPBLW genes also diverged early, in the neoavian ancestor (105–97.3 mya), whereas the ATP5A1Z/ATP5A1W genes appear to have diverged fairly recently (53 mya) (doi:10.1534/genetics.108.090324).

"The process of sex chromosome evolution and the dynamics of their regional differentiation is a fascinating topic," says Mr Suh. "Understanding the similarities and differences in the many independent evolutions of sex chromosomal systems promises to yield insights into general processes involved in the evolution of sex determination (and sex in general)."

Besides being fascinating research, the ornithologists, conservation biologists and aviculturists in the crowd have probably already noticed a practical application for this research: using the visible size difference of these REs to identify the sex of birds.

As we already saw, retroposon insertions increase the size of a particular region within a gametologue by several hundred basepairs. Female birds, having a Z and W chromosome, will carry two distinct copies of these gametologues, whilst males, being ZZ, have just one. Thus, when any of these four regions are copied a million-fold using PCR, then separated by size using gel electrophoresis, the resulting PCR amplicons provide an unambiguous and fast method for identifying sex of birds (eggs, feathers, etc.) from nearly all Neoavian or Neognathan species, depending upon the region amplified. Further, amplifying more than one region can provide an internal check against false results (figure 3):

Figure 3.
DOI: 10.1093/molbev/msr147

"[I]t’s nice to see that ‘by chance’ it’s possible to find something that is
of relevance to be patented and might be of use to ornithologists for
convenient bird sex identification," said Mr Suh in email.

"The nice thing about these three sexing tests is that because we know when the retroposon inserted into each test locus, we know for which species the test should be (virtually) applicable. All three retroposon insertions are quite ancient, so now there are three independent sexing tests for almost all birds and with a distinct size difference between the female-specific W band and the Z band."

Sources:

Suh, A., Kriegs, J., Brosius, J., & Schmitz, J. (2011). Retroposon Insertions and the Chronology of Avian Sex Chromosome Evolution. Molecular Biology and Evolution DOI: 10.1093/molbev/msr147

Alex Suh [emails]

Some background reading on avian retroposons.

Further reading:

Kiwoong Nam and Hans Ellegren. (2008). The Chicken (Gallus gallus) Z Chromosome Contains at Least Three Nonlinear Evolutionary Strata. Genetics 180:1131-1136. doi:10.1534/genetics.108.090324

Akira Ogawa, Koichi Murata, and Shigeki Mizuno. (1998). The location of Z- and W-linked marker genes and sequence on the homomorphic sex chromosomes of the ostrich and the emu. PNAS 95(8):4415-4418. [abstract with link to free PDF]

Swathi Shetty, Darren K. Griffin and Jennifer A. Marshall Graves. (1999). Comparative Painting Reveals Strong Chromosome Homology Over 80 Million Years of Bird Evolution. Chromosome Research 7(4):289-295. doi:10.1023/A:1009278914829