Retention, erosion, and loss of the carotenoid biosynthetic pathway in the nonphotosynthetic green algal genus Polytomella

The evolutionary loss of carotenoid biosynthesis is often tied to the loss of photosynthesis, which is not surprising. In plants and algae, carotenoids are primarily associated with photosynthetic processes, from light absorption to photosystem assembly to protection from photodamage (Lohr, 2009; Cazzonelli, 2011; Santabarbara et al., 2013), and they are also key constituents of algal eyespots – specialized, typically plastid-located optical devices that detect light and direct phototaxis (Kreimer, 2009; Ladygin & Semenova, 2014). In fact, a nonphotosyntheticmutant of themodel green alga Chlamydomonas reinhardtii lacking phytoene synthase – one of the first enzymes in carotenoid biosynthesis (Fig. 1a) – bears a remarkable resemblance to naturally occurring colourless algae, exhibiting starch accumulation, a disorganized eyespot, and no pyrenoid (Inwood et al., 2008). This observation has led some to suggest that mutations to the carotenoid pathway could be responsible for the evolution of nonphotosynthetic algae (Inwood et al., 2008), many of which are missing the genes for carotenoid production (Borza et al., 2005; Pombert et al., 2014; FigueroaMartinez et al., 2015), with some notable exceptions (Tonhosolo et al., 2009). One algal system that could prove particularly useful for investigating the relationship between carotenoid biosynthesis and a heterotrophic existence is Polytomella: a monophyletic genus of nonphotosynthetic, free-living unicells closely related to C. reinhardtii (Pringsheim, 1955; Smith et al., 2013), and not to be confused with the polyphyletic genus Polytoma, which lost photosynthesis independently of Polytomella (Figueroa-Martinez et al., 2015). There are currently four well-described Polytomella lineages (Fig. 1b), represented by P. magna, P. capuana, P. piriformis, and P. parva (G€ottingen Culture Collection of Algae, SAG, strains 63-9, 63-5, 63-10 and 63-3, respectively) (Smith et al., 2013). The three latter species have no discernable eyespot and are white in colour, whereas P. magna, the deepestbranching of the four species, has an eyespot and is pinkish (Pringsheim, 1955; Smith et al., 2013; MacDonald & Lee, 2015). Recently, our laboratory group in collaboration with Robert W. Lee ofDalhousie University (Halifax, NS) generated large amounts of Illumina RNA and DNA sequencing data from all four Polytomella lineages, which can be found in GenBank’s Sequence Read Archive (accession numbers SRX363995, SRX377397, SRX377560, SRX551283, and SRX710730–SRX710732). These data have already been used to explore certain plastid-located pathways, revealing thatPolytomella spp. likely do not have a plastid genome (Smith & Lee, 2014), and to validate the species name P. piriformis (MacDonald & Lee, 2015). In our ongoing work towards developing a complete and polished Polytomella genome and transcriptome, we discovered an interesting feature regarding the carotenoid biosynthetic pathway, which to the best of our knowledge has heretofore not been observed in any other group of nonphotosynthetic or photosynthetic algae.