Evolution of a Behavioral Character in a Sparrow Species

            Although the mutational study of behavioral characters was initiated by Seymour Benzer in the 1960’s, his study was concerned primarily with laboratory mutations of fruitflies and did not give much insight into the evolutionary mechanisms of behavioral characters in the wild. For this reason, many authors are now investigating this problem at the molecular level. Horton et al. (2014) recently published an interesting result with respect to the social behavior in the white-throated sparrow Zonotrichia albicollis.

In the white-throated sparrow there are two polymorphic phenotypes with respect to the color pattern of the head crown: (1) tan-striped (TS) and (2) white-striped (WS) (Fig. 1). The male WS phenotype is known to be more aggressive than the male TS with respect to territoriality and mate-finding. In song-birds, these behaviors are typically dependent on sex steroid during the breeding season. WS birds have higher plasma testosterone than the same sex TS birds. However, morph differences in behavior cannot be entirely explained by these hormones, because the differences persist even when plasma levels are experimentally equalized. Therefore, individual variation in steroid-dependent behavior may be better explained by neural sensitivity to the hormones, for example by variation in the distribution and abundance of steroid receptors (Horton et al., 2014).

  From Horton et al. (2014).

By the way, the WS and TS are associated with two inversion haplotypes of chromosome 2 (Thomas et al. 2008). That is, the genes controlling WS and TS are apparently located in the inverted segment of haplotypes ZAL2^m and ZAL2. However, because WS is dominant over TS and the frequency of ZAL2^m is relatively low, individuals can roughly be divided into two groups, WS (ZAL2^m/ZAL2,) and TS (ZAL2/ZAL2), genotype ZAL2^m/ZAL2^m, being practically absent. It has also been inferred that ZAL2^m was derived from ZAL2 about 2 million years ago by chromosomal inversion and therefore the polymorphism has existed for a long time. Note also that there is practically no recombination between two inverted chromosomes.

Horton et al. (2014) looked for steroid receptor genes in the inverted segment and found that the gene (ESR1) encoding estrogen receptor α (ERα) is located in the inverted segment and that the two proteins encoded by the ESR1 genes from the WS and TS phenotypes showed one amino acid difference but this difference did not affect the gene expression pattern appreciably. They then hypothesized that the phenotypic difference between WS and TS is caused by the difference in the gene regulatory region of the gene. In fact, when the binding sites of transcription factors in the cis-regulatory region upstream of the ESR1 gene were examined by a computer program, there was considerable difference between the WS and TS haplotypes (Fig. 2A).

 From Horton et al. (2014).

However, to prove that this difference is indeed responsible for the behavioral difference, it was necessary to show that the expression level of the ESR1 gene is higher in haplotype ZAL2^m than in ZAL2. For this purpose, Horton et al. used several molecular techniques such as the luciferase reporter method with HeLa cells and radioimmunoassay. Their results showed that the expression level of the ESR1 gene is about 1.5 times higher in haplotype ZAL2^m than in ZAL2 (Fig. 2B,C).

This finding indicates that the difference in expression level of a single major gene generates a clear phenotypic difference, which in turn affects an important behavioral character. At the present time, this type of data is rare, but it is possible that many behavioral characters are controlled by similar molecular mechanisms, and it is desirable that more studies will be conducted in the future. In practice, behavioral characters are generally controlled by many genes, and eventually we may be able to understand the molecular basis of the characters. Yet, the basic principle of gene expression could be simpler than our intuition suggests as in the case of the above example. In their “significance” statement, Horton et al. write:

In this series of studies, we provide a rare illustration of how a chromosomal polymorphism has affected overt social behavior in a vertebrate. White-throated sparrows occur in two alternative phenotypes, or morphs, distinguished by a chromosomal rearrangement. That the morphs differ in territorial and parental behavior has been known for decades, but how the rearrangement affects behavior is not understood. Here we show that genetic differentiation between the morphs affects the transcription of a gene well known to be involved in social behavior. We then show that in a free-living population, the neural expression of this gene predicts both territorial and parental behavior. We hypothesize that this mechanism has played a causal role in the evolution of alternative life-history strategies.

Symbiosis and Genome Degeneration: Micro-niche Evolution

            Symbiotic association of bacteria with animals and plants are ubiquitous, and about 15% of insect species have been estimated to harbor bacterial species as symbionts (Oakeson et al. 2014). These bacterial symbionts generally have reduced genome size compared with ancestral free-living bacteria (McCutcheon and Moran 2011). However, these symbionts produce nutrients that are essential for survival of the host, and therefore a mutualistic symbiosis is generated. In many of these bacteria, the initiation of symbiosis occurred a long time ago, so that it is difficult to know how the genome size reduction occurred. In these bacteria, the rate of amino acid substitution is generally higher than that of ancestral free-living bacteria (Moran 1996; Lynch 1996), and this high rate has been attributed to advantageous mutation (Fares et al. 2002) or to Muller’s ratchet effect (Lynch 1996). However, Itoh et al. (2002) and Dale et al. (2003) suggested that the loss of DNA repair enzymes in these bacteria is responsible for the high rate of amino acid substitution. To resolve this controversy, however, it is important to know how gene loss occurs in the early stage of evolution of endosymbiosis.

             In recent years a number of investigators (e.g., Dale et al 2002; Burke and Moran 2011) have identified bacterial species which started a symbiotic life very recently so that they could study their early stage of genome reduction. In particular, the group of Clayton et al. (2012) and Oakeson et al. (2014) discovered a novel human-infective bacterium designated “strain HS.” This strain was isolated from a patient who had a hand wound following impalement with a tree branch. Phylogenetic analysis showed that the strain HS is a member of the Sodalis-allied clade of insect endosymbionts and that close relatives of strain HS gave rise to symbiotic association in a range of insect species. Using 165 rRNA genes, Clayton et al. (2012) showed that strain HS is closely related (by 98% sequence identity for synonymous sites) to the bacteria Solidas glossinidius and Sitophilus oryzae primary endosymbiont (SOPE) that are endosymbionts of grain weevils. The genome size (5.16 Mb) of HS was only slightly greater than those of S. glossinidius and SOPE (see Table 1). This result suggests that the latter two species have become endosymbionts only recently and HS is a free-living bacterium. (The symbiont bacterium (Buchnera aphidicola) of aphids has only 20% of the genome of the ancestral free bacteria.)

Table 1. General features of the strain HS, SOPE, and S. glossinidius genome sequences

            Clayton et al. (2012) and Oakeson et al. (2014) sequenced the genomes of HS and SOPE and compared the genomic sequences with the sequence of S. glossinidius, which was available from the literature. The results indicated that the number of pseudogenes has increased substantially in the symbiont bacteria whereas the number of intact genes (supposedly functional genes) has been reduced (see Table 1 and Fig. 1). Furthermore, a large number of mobile insertion sequences (IS) and a substantial number of duplicate genes have accumulated in the symbiotic bacteria.

Figure 1. Alignments of three regions of the S. glossinidius, strain HS, and SOPE chromosomes. Alignments of three regions of the S. glossinidius, strain HS, and SOPE chromosomes, corresponding to SG0948–SG0977 (A), ps_SGL0466–SG0918 (B) and ps_SGL0318–ps_SGL0330 (C) in the most recent S. glossinidius annotation [25]. Putative ORFs and intergenic regions are drawn according to scale, oriented according to their inferred direction of transcription and color-coded according to COG functional categories. While all of the depicted strain HS genes have intact reading frames, the status of their orthologs in S. glossinidius and SOPE are shown in the outer bars (green = intact, purple = inactivated). Nonsense mutations (premature stop codons) are depicted by purple diamonds, and frameshifting indels are depicted by purple triangles. Light grey connecting bars are syntenic nucleotide alignments, while brown bars illustrate IS-element acquisitions that occur more frequently in SOPE.

            These results suggest that when free-living bacteria entered into a host insect many genes of the free-living bacteria was nonfunctionalized because they were not necessary in the insect host. At the same time normally harmful IS elements have accumulated because the destruction of many functional genes by IS elements appear to be harmless under the condition of symbiosis. It is interesting to note that these evolutionary changes have occurred very rapidly because in the initial stage of symbiosis many useless genes can be pseudogenized but the pseudogenes may be retained in the genome for some time. (In the present case symbiosis was estimated to have occurred only about 28,000 years ago, though this estimate seems to be too low; Concord et al. 2008.) This rapid regressive evolution occurred apparently because a small number of free-living bacteria colonized in the bacteriocytes of the host and the number of vertically inherited bacteria has remained to be small. I would like to call this type of evolution small-niche or micro-niche evolution. In micro-niche evolution, the bacterial population was effectively homozygous, and many mutational changes are expected to be fixed at the rate of mutation unless they are deleterious under the symbiotic condition.

             Of course, if the evolutionary time becomes long, many pseudogenes and other useless DNA elements would be lost by deletion and the genome size is expected to become small as is observed in ancient endosymbionts. Furthermore, in the long run some mutations that would enhance the mutual dependency of the symbiont and the host are expected to occur, and eventually the symbiont bacteria and the host organism become inseparable.

             Note also that because many mutations are not harmful under the symbiotic condition, even DNA repair genes such as recA and recF may be lost (Shigenobu et al. 2000; Itoh et al. 2002). In fact, the loss of recA has been confirmed even in the symbiont bacteria S. glossinidius and SOPE (Dale et al. 2003). This suggests that a relatively high rate of amino acid substitution in endosymbionts has evolved in the early stage of symbiotic life.

             Microniche evolution is different from the evolution by population bottlenecks proposed by Mayr (1963), because in the latter theory population size shrinks temporarily but increases later to the original level whereas in the former theory the population size remains small throughout the evolutionary process. Micro-niche evolution occurs in many different organisms, and it is often associated with regressive evolution of various characters. For example, many organisms living in ancient caves have lost pigmentation because in the dark cave condition pigments are not necessary. In the case of Mexican cavefish Astyanax mexicanus even the eyes are degenerated. In this case a number of authors (e.g., Jeffrey 2009; Yoshizawa et al. 2012) proposed that the degeneration of eyes in A. mexicanus has occurred by positive Darwinian selection. Their argument is that in the dark cave condition there is no need of having eyes and therefore natural selection operates to reduce the eye size so that the nutrition saved by elimination of eye formation can be used for other purposes.

             I have opposed this view by noting that the first step of degenerative evolution is likely to be the reduction of eye size by destructive mutations in the small cavefish population and therefore the degeneration of cavefish eyes can easily be explained by micro-niche evolution (Nei 2013). Of course, positive selection after degeneration of the eyes may have occurred in the cave condition. For example, cavefish are known to have thin skin to cover the degenerated eyes. It is quite possible that any mutations that cause the development of this skin have been subjected to positive selection. However, the most important event of eye degeneration must be caused by degenerative mutations. In fact, this view is supported by the micro-niche evolution of symbiotic bacteria mentioned above.

References

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2. McCutcheon, J. P. & Moran, N. A. (2011). Extreme genome reduction in symbiotic bacteria. Nat Rev Microbiol 10(1):13-26.

3. Moran NA. 1996. Accelerated evolution and Muller's ratchet in endosymbiotic bacteria. Proc. Natl. Acad. Sci. USA 93:2873-2878.

4. Lynch M. 1996. Mutation accumulation in transfer RNAs: molecular evidence for Muller's ratchet in mitochondrial genomes. Mol Biol Evol 13:209-220.

5. Fares, M. A., Barrio, E., Sabater-Munoz, B., & Moya, A. (2002). The evolution of the heat-shock protein GroEL from Buchnera, the primary endosymbiont of aphids, is governed by positive selection. Mol Biol Evol 19(7):1162-70.

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9. Clayton et al. (2012). A novel human-infection-derived bacterium provides insights into the evolutionary origins of mutualistic insect–bacterial symbioses. PLoS Genetics 8(11):e1002990.

10. Concord, C., Despres, L., Vallier, A., Balmand, S, Miquel C., et al. (2008). Long-term evolutionary stability of bacterial endosymbiosis in curculionoidea: additional evidence of symbiont replacement in the dryophthoridae family. Mol Biol Evol 25:859-868.

11. Shigenobu, S., Watanabe, H., Hattori, M., Sakaki, Y., & Ishikawa, H. (2000). Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407(6800):81-6.

12. Mayr E. 1963. Animal species and evolution. Harvard University Press, Cambridge.

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14. Yoshizawa, M., Yamamoto, Y., O’Quin, K. E., & Jeffery, W. R. (2012). Evolution of an adaptive behavior and its sensory receptors promotes eye regression in blind cavefish. BMC Biology 10:108.

15. Nei, M. (2013). Mutation-driven evolution. Oxford University Press, Oxford.