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Just as a phylogeny encodes the evolutionary relationships among a group of organisms, a cophylogeny represents the coevolutionary relationships among symbiotic partners. Both are primarily reconstructed using computational analysis of biomolecular sequence data. The most widely used cophylogenetic reconstruction methods utilize an important simplifying assumption: species phylogenies for each set of coevolved taxa are required as input and assumed to be correct. Many studies have shown that this assumption is rarely – if ever – satisfied, and the consequences for cophylogenetic studies are poorly understood. To address this gap, we conduct a comprehensive performance study that quantifies the relationship between species tree estimation error and downstream cophylogenetic estimation accuracy. We study the performance of state-of-the-art methods for cophylogenetic reconstruction using in silico model-based simulations. Our investigation also assessed cophylogenetic reproducibility using genomic sequence data from two important models of symbiosis: soil-associated fungi and their endosymbiotic bacteria, and bobtail squid and their bioluminescent bacterial symbionts. Our findings conclusively demonstrate the major impact that upstream phylogenetic estimation error has on downstream cophylogenetic reconstruction. Relative to other experimental factors such as cophylogenetic estimation method choice and coevolutionary event costs, phylogenetic estimation error ranked highest in importance based on a random forest-based variable importance assessment. We conclude with practical guidance and future research directions. Among the many considerations needed for accurate cophylogenetic reconstruction – choice of computational method, method settings, sampling design, and others – just as much attention must be paid to careful species phylogeny estimation using modern best practices. 

In our continued investigations of microbial globins, we solved the structure of a truncated hemoglobin from Shewanella benthica, an obligate psychropiezophilic bacterium. The distal side of the heme active site is lined mostly with hydrophobic residues, with the exception of a tyrosine, Tyr34 (CD1) and a histidine, His24 (B13). We found that purified SbHbN, when crystallized in the ferric form with polyethylene glycol as precipitant, turned into a green color over weeks. The electron density obtained from the green crystals accommodated a trans heme d, a chlorin-type derivative featuring a γ-spirolactone and a vicinal hydroxyl group on a pyrroline ring. In solution, exposure of the protein to one equivalent of hydrogen peroxide resulted in a similar green color change, but caused by the formation of multiple products. These were oxidation species released on protein denaturation, likely including heme d, and a species with heme covalently attached to the polypeptide. The Tyr34Phe replacement prevented the formation of both heme d and the covalent linkage. The ready modification of heme b by SbHbN expands the range of chemistries supported by the globin fold and offers a route to a novel heme cofactor.