G1 B3

Do branch lengths support the concept of “Living Fossils”? A phylogenomic analysis using Coelacanth as a model

Comentários dos revisores

A questão formulada para este trabalho é extremamente interessante, e ainda uma questão em aberto que merece ser explorada. A abordagem utilizada é bacana e promissora. O relatório está bem apresentado com boa escrita, boas figuras e clareza.

Os métodos estão incompletos, principalmente relativos aos resultados e configuração de parametros das reconstruções (seria necessário explicitar mais a fundo os modelos, o número de sítios, etc)

Nota: 9.5 (será aplicada igualmente aos membros a não ser que enviem pedido do contrário).

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Branch length, in a phylogenetic context, is a metric for the degree of evolutionary change¹. A slow rate of evolution theoretically yields a shorter branch. However, when considering living fossils (LF), it is interesting to examine the behaviour of branch length, given that this topic has not yet been fully explored. LF, in Darwin’s view, are conceptualised as extant organisms that closely resemble their extinct ancestors². Given minimal morphological changes, one would assume that LF have also undergone minimal genetic changes, resulting in short branch lengths. Nevertheless, whether the concept of morphological stasis can be applied at the genomic level is disputable³, as the apparent stasis in the phenotypes of genes that regulate morphology of LF may not be evident in genes that do not correspond to morphological features. Moreover, genomes are constantly subjected to neutral nucleotide changes, which are unaffected by natural selection⁴. Considering the long period of time during which LF are virtually unchanged, they should accumulate these genetic changes to a high level despite at a slow rate⁵. In light of the perplexity, this study aims to explore the behaviour of branch lengths in LF from a phylogenomic perspective, with coelacanth as a model. Three possible outcomes are expected (Fig. 1): (1) LF will exhibit longer branch lengths than other species, (2) LF will exhibit shorter branch lengths than other species, or (3) LF will exhibit branch lengths similar to other species.

Figure 1. The expected branch length from the phylogenomic analysis comparing the living fossil coelacanth with other extant species may result in one of three outcomes: (1) a longer branch length than other species, (2) a shorter branch length, or (3) a branch length similar to the other terminal taxa.

To model the behaviour of branch lengths of LF in relation to other taxa, protein sequences were compared in four species from the following groups: coelacanth (Latimeria chalumnae); Dipnoi (Protopterus annectens); anuran amphibians (Rana temporaria) and placental mammals (Mus musculus). All sequences were retrieved from the NCBI database. Comparing sequence datasets at the amino acid level confers advantages over nucleotide-level comparisons by reducing noise from synonymous mutations and allowing for higher levels of evolutionary signal preservation through codon-based analysis. The quality of the protein datasets was first assessed using BUSCO⁶, with a pre-defined metazoan gene set as the reference lineage. In total, 203 complete single-copy BUSCO genes were identified and extracted across taxa. These were subsequently aligned using the MAFFT algorithm⁷. The aligned sequences were then trimmed using TrimAl⁸ to retain only high-confidence alignment columns. Using the multiple sequence alignment as input, a ML-based phylogenetic tree was reconstructed in IQ-Tree⁹. The inferred, unrooted binary species tree was then visualised using FigTree.

Figure 2. Branch length comparison between coelacanth (Latimeria chalumnae), Dipnoi (Protopterus annectens), anuran amphibians (Rana temporaria) and placental mammals (Mus musculus). Bootstrap support = 100. A. Inferred unrooted tree from protein sequences of selected taxa. B. Comparison of individual branch length, measured from node to tip. Scale bar corresponds to 0.03 substitutions per nucleotide position.

In our experimental results, coelacanths exhibited a branch length approximately 32.02% shorter than the average branch length of all taxa analysed (Table 1; Fig. 2A-B). The approximate substitution rate per nucleotide position (Table 1) suggests that coelacanths have undergone genetic mutations at slower rates than the other species analysed, which is consistent with its apparent morphological stasis. In short, the branch length of coelacanths aligns with hypothesis 2 presented in Fig. 1. The three other taxa presented branch lengths greater than the average: the lungfish had the second shortest branch length, followed by the rat and the frog, respectively. The unrooted tree was supported by maximum bootstrap value (Fig. 2A).

Table 1. Approximate rate of substitutions per nucleotide position for each terminal taxa, and the proportion of branch length relative to the average.

Although the result presented herein corroborates the hypothesis 2 of Fig. 1, its interpretation should be approached with caution. The interpretation of branch lengths can be assessed through two different approaches: selection of terminal taxa, and the concept of LF. First, it is important to highlight the relativistic nature of branch lengths in relation to selected taxa. While the chosen Tetrapoda taxa are recently-evolved and rapidly-diverging compared to LF, the lungfish shares a more similar evolutionary trajectory with coelacanths. Both groups first appeared in the late Devonian period and experienced comparable patterns of diversification and stasis^{10, 11}. Hence, in a broader sense, lungfish could also be considered a LF. To enhance the validity of the observed branch length behaviour, a comparative analysis involving more distantly-evolved lineages could be conducted. Ultimately, our understanding of LF is based on fossil records, but missing links, e.g., morphologically distinct extinct forms, may not have been preserved nor discovered¹². This limitation, along with the absence of a reliable definition on LF, raises concerns about the biological significance of LF in such studies. It warrants further exploration in the literature.

References:

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1. Gregory, T.R. 2008. Understanding Evolutionary Trees. Evo Edu Outreach, 1:121–137. http://doi.org/10.1007/s12052-008-0035-x.

2. Darwin, C. 1859. On the Origin of Species by Means of Natural Selection. John Murray, London.

3. Lidgard, S. & Lova, A.C. 2021. The living fossil concept: reply to Turner. Biology & Philosophy, 36: 13. https://doi.org/10.1007/s10539-021-09789-z.

4. Kimura, Motoo (1968) Evolutionary Rate at the Molecular Level. Nature, 217:624.

5. Bennett, D.J., Sutton, M.D., and Turvey, S.T. 2018. Quantifying the Living Fossil concept. Palaeontologia Electronica 21.1.14A 1-25. https://doi.org/10.26879/750.

6. Manni, M., Berkeley, M. R., Seppey, M., & Zdobnov, E. M. (2021). BUSCO: Assessing genomic data quality and beyond. Current Protocols, 1, e323. https://doi.org/10.1002/cpz1.323.

7. Katoh, K., Misawa, K., Kuma, K. I., & Miyata, T. (2002). MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic acids research, 30(14), 3059-3066. https://doi.org/10.1093/nar/gkf436.

8. Capella-Gutiérrez, S., Silla-Martínez, J. M., & Gabaldón, T. (2009). trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics (Oxford, England), 25(15), 1972–1973. https://doi.org/10.1093/bioinformatics/btp348.

9. Nguyen, L. T., Schmidt, H. A., Von Haeseler, A., & Minh, B. Q. (2015). IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular biology and evolution, 32(1), 268-274. https://doi.org/10.1093/molbev/msu300.

10. Zhu, M., & Yu, X. (2002). A primitive fish close to the common ancestor of tetrapods and lungfish. Nature, 418(6899), 767-770. DOI: 10.1038/nature00871.

11. Simpson, G. G. (1984). Tempo and mode in evolution. Columbia University Press.

12. Casane, D. & Laurenti, P. 2013. Why coelacanths are not “Living Fossil”: a review of molecular and morphological data. BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology, 35(4): 332–8. https://doi.org/10.1002/bies.201200145.

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https://docs.google.com/presentation/d/1Z4fYedfnqsBFhKXPes8BSeJr2a2iBtdkAiF1RC4bvqI/edit?usp=sharing