Organellar Genome Evolution in Red Algal Parasites: Differences in Adelpho- and Alloparasites

Parasitism is a common life strategy throughout the eukaryotic tree of life. Many devastating human pathogens, including the causative agents of malaria and toxoplasmosis, have evolved from a photosynthetic ancestor. However, how an organism transitions from a photosynthetic to a parasitic life history strategy remains mostly unknown. Parasites have independently evolved dozens of times throughout the Florideophyceae (Rhodophyta), and often infect close relatives. This framework enables direct comparisons between autotrophs and parasites to investigate the early stages of parasite evolution. Parasitic red algae have traditionally been defined as either ‘adelphoparasites’, which infect hosts within their same family or tribe, or ‘alloparasites’ which infect hosts in other families. Prior to this research, investigations have primarily focused on understanding the development and evolution of ‘adelphoparasites’. All adelphoparasites studied to date have been shown to have lost their native plastid and instead incorporate a host plastid when packaging their spores. Additionally, previously published ‘adelphoparasite’ mitochondrion genomes have reduced coding capacity. The goal of this research was to investigate 1) the evolutionary impact on the plastid of the ‘alloparasite’ Choreocolax polysiphoniae, 2) the coding capacity of the C. polysiphoniae mitochondrion, and 3) the evolutionary origins of Rhodomelaceae ‘alloparasites’. A combination of Sanger-sequencing of targeted PCR products and nextgeneration sequencing of genomic DNA and total RNA was used to investigate the organelles of the ‘alloparasites’ Choreocolax polysiphoniae, Harveyella mirabilis, Leachiella pacifica, and a previously undescribed species of Leachiella, the ‘adelphoparasites’ Gracilariophila oryzoides and Gonimophyllum skottsbergii, and the host of C. polysiphoniae, Vertebrata lanosa. Organellar genomes were assembled using CLC genomics workbench and Geneious Pro and subsequently manually annotated using BLAST and Pfam. Comparative analyses of organellar genomes were completed using MAUVE genome alignment software. Total RNA was assembled using the Trinity based pipeline, Agalma and annotated using InterProScan. Analyses of transcriptomic data were completed using Silix and HiFix. This research generated plastid genomes for C. polysiphoniae and its host V. lanosa. The C. polysiphoniae plastid represents the first plastid genome sequenced for a red algal parasite. Interestingly, this plastid has reduced coding capacity and has lost genes involved with photosynthetic processes and its presence challenges the previously proposed paradigm of red algal parasite evolution. Investigations of red algal parasite mitochondria demonstrated that parasites retain fully functional and typical Florideophyceae mitochondria. Finally, an investigation of parasites typically considered as alloparasites supports a monophyletic clade of parasites, which all retain their native plastid genomes, arose and radiated to infect different hosts within the Rhodomelaceae. Data generated here supports previous findings that ‘alloparasites’ rarely infect hosts in different families. Therefore the terms ‘adelphoparasite’ and ‘alloparasite’, which are based on evolutionary relationships to their hosts and do not accurately distinguish types of red algal parasites. Based upon this research, we propose to redefine red algal parasites by their plastid origins as either Archaeplastic parasites (parasites that retain a native plastid) or Neoplastic parasites (those which incorporate a host plastid).


LIST OF TABLES
and Yang et al. (2015) or otherwise unannotated.

Introduction
Parasitism has evolved innumerable times throughout the eukaryotic tree of life . Some of the more virulent parasites have transitioned from a once photosynthetic ancestor, including the causative agents of malaria and related mammalian diseases (Wilson et al. 1996. Therefore, understanding the evolutionary trajectory between photosynthesis and abandoning autotrophy for a parasitic strategy, is of particular importance. Red algal parasites are uniquely valuable to study this path because they have independently evolved many times, providing literally dozens of discrete events to compare . This system may provide novel insights into the evolution of parasitism, especially with regard to the early stages of transitioning from a photosynthetic past. Red algal parasites exclusively infect other red algae, typically ones with which they share a recent common ancestor ). The relationship between host and parasite was first recognized using morphological similarities in the life-cycles of parasites and their hosts (Setchell 1918). More recently, molecular data have confirmed this hypothesis ).
Traditionally red algal parasites have been placed into two different groups, based on their phylogenetic relationships with their hosts . Adelphoparasites (adelpho is Greek for "kin") are closely related to their host and often infect only one host, whereas alloparasites are more divergent from their host(s) . Currently, adelphoparasites are believed to make up roughly 90% of all red algal parasites ).
Among the Florideophyceae, parasites belonging to at least 66 different red algal genera have evolved independently over 100 times (Table 1) (Verbruggen et al. 2010). The accepted evolutionary paradigm proposes that adelphoparasitism is the initial state, followed by parasite diversification, which leads to the development of alloparasites (Fig. 1). These "older" parasites can infect more distantly related taxa, and make up roughly 10% of red algal parasites ).
Due to their rarity, alloparasites are relatively unstudied, with the exception of Choreocolax polysiphoniae.

The Importance Of Red Algal Pit Connections
One of the defining characteristics of the florideophycean red algae is the ability of cells to form connections with their adjacent cells (Pueschel and Cole 1982).
There are two distinct forms of these "pit-connections" formed by red algae. Primary pit-connections arise between a mother and daughter cell during apical growth ). These connections result from a seemingly incomplete cell division where the septum begins to develop from the cell walls growing inward to separate the daughter nuclei . However, cytokinesis is incomplete and the septum does not fuse, leaving an opening that connects the two cells . A pit-plug composed of a polysaccharide-protein complex then forms sealing the pit connection and separating the two cells . Though pit connections result in an aperture that is not entirely sealed by a septum, the pit plug prevents the transfer of cellular contents and photosynthate between adjacent cells (Turner and Evans 1978).
In addition to the primary pit connections, florideophytes also form secondary pit connections between adjacent non-daughter cells. These secondary pit connections are known to occur in a wide range of Florideophyceae and form between two genetically similar red algal cells (Hawkins 1972, Goff and. Red algal parasite spores utilize secondary pit connections as a way to enter the cells of the host (Wetherbee and Quirk 1982a. As evidence for the importance of secondary pit connections in parasitic infections, parasites are not known from red algal orders where secondary pit connections do not occur ). An advantage of this strategy is that the similarity between host and parasite at the genetic level allows parasite spores to simply dump their organelles into the host cell and take over, spreading through primary or secondary connections Quirk 1982b, Goff and. The widespread existence of secondary pit connections among red algae is undoubtedly a primary factor in the promiscuous nature of parasitism as a life history strategy in the lineage.

Spore Germination and Host Infection
Rhodophytes lack flagella in all stages of their life cycle, making the initial stages of locating a host a passive process. Once a parasite spore lands upon a susceptible host, the parasite carpospore (2N) or tetraspore (1N) will germinate and undergo an initial cell division . Adelphoparasite cells will divide between 1 and 3 more times before one of the cells forms a rhizoid that penetrates the surface of the host, growing into the wall of a host epidermal cell . The tip of the rhizoid swells isolating a single parasite nucleus along with its organelles into a conjunctor cell which divides from the infection rhizoid . This conjunctor cell then fuses, via a secondary pit connection, with the adjacent host cell (Fig. 2a). The contents of the conjunctor cell, which include the parasite nucleus and organelles, are deposited into the host cell, thus forming a heterokaryotic cell (containing both parasite and host nuclei) (Wetherbee and Quirk 1982a. This connection between the parasite infection rhizoid and the transformed host cell is sealed by a pit-plug that was formed previously during the initial fusion between the conjunctor cell and the parasite infection rhizoid . The differences between adelpho-and alloparasites become evident at the initial infection of the host cell. In alloparasites, rather than going through a few cell divisions before penetrating the surface, the parasite spore attaches to a suitable host, penetrating and forming a hyphae-like network of multicellular filaments between the host cells . These filaments enable the parasite to spread numerous cells deep into the host away from the initial site of infection ( Fig.   2b). Each alloparasite cell in these filaments contains a single nuclei and can form a conjunctor cell and secondary pit connection through which the parasite deposits its cellular contents into the host, creating a heterokaryotic cell .

Inside the Heterokaryon
After the host cell becomes heterokaryotic, both adelpho-and alloparasites take control of the host cellular machinery . Almost immediately upon infection, the hosts' central vacuole tonoplast is lysed, allowing cytoplasm to spread throughout the space previously occupied by the vacuole . Subsequently, the number of organelles including plastids, mitochondria, and ribosomes increases throughout the cytoplasm causing it to appear more dense . Along with the increase in cytoplasmic organelles comes an increase in cell size (hypertrophy), a process in which the cell can grow to 40 times its original size . In addition to the increased organelles, the host nuclei also increase in size and/or number Coleman 1984a, 1985).
Their study showed that infected V. lanosa central cells will become enlarged and the nuclei will undergo DNA synthesis but not nuclear division resulting in polyploid host nuclei . Alternatively, in infected V. lanosa pericentral cells, the host nuclei will either increase in size, increase in number, or some combination of both . An increase in the number of host nuclei is the most common response . Host cells that are adjacent to infected cells and connected by pit connections will not show any cytological transformation Coleman 1984a, 1985).
Conversely, no DNA synthesis or nuclear division has been observed from the host nucleus after infection by the adelphoparasite Janczewskia gardneri . Instead, the adelphoparasite nucleus rapidly undergoes DNA synthesis, generating numerous parasite nuclei inside a single host cell ( Fig. 2) . The adelphoparasite subsequently spreads to additional host cells through the formation of conjunctor cells that can infect adjacent host cells . Adelphoparasites can also form rhizoidal infection cells, which are multinucleate and contain large numbers of mitochondria, ribosomes, and dedifferentiated host-derived proplastids and can fuse with more distant host cells .
Alloparasites are capable of mitotic divisions to create the multicellular filaments that spread between host cells Coleman 1984a, 1987). However, the alloparasite nucleus does not undergo DNA synthesis inside the host cell . Therefore, parasite nuclei remain at a 1:1 ratio with the number of secondary pit connections between parasite and host cells . Once inside a heterokaryon the adelphoparasite utilizes the host to progress through its lifecycle and reproduce rather than continue to spread to additional host cells.

Formation of Reproductive Structures
As the adelphoparasite spreads throughout the host, a gall or "erumpent pustule" begins to form as host cells continually expand upon infection by the parasite.
Eventually the adelphoparasite will start to form reproductive structures . If the original infecting spore was haploid the parasite will form caprosporangia that can be fertilized by a spermatia from another parasite forming a diploid carposporophyte that will eventually release carpospores . If the original infection was from a diploid carpospores, the parasite will undergo meiosis forming haploid tetraspores that will be released from the erumpent pustule ).
An alloparasite does not spread through the host like the adelphoparasite.
Rather than forming a gall from invaded cells, a host pericentral cell containing a parasite nuclei and many host nuclei will form a protuberance . This protuberance will become isolated from the original host cell and undergo mitotic divisions, which produces the mature parasite pustule containing reproductive cells similar to that of adelphoparasites . After the reproductive cells are released, the former host tissue that made the erumpent pustule becomes necrotic . While the fate of future adelphoparasites lies in the released spores, alloparasites are able to continue their infection of the same host as parasite filaments continue to grow into uninfected areas .

Organelles
Studies of cellular organelles have yielded particularly interesting findings during investigations of red algal parasite biology. Early studies established the role of secondary pit connections between the parasite and host cells and demonstrated their role in transferring the parasite nucleus to the host cell (Wetherbee and Quirk 1982a. However, it was unclear whether the parasite maintained its own mitochondrion and plastid or it if utilized the host organelles once the parasite nucleus was transferred info the host cell. Cytoplasmic organelles support many major metabolic pathways as well as play major roles in cellular energy and carbohydrate production. It has been well established that purifying selection is relaxed on parasite organellar genes that become unnecessary, leading to genome reduction in parasites as they increasingly rely on a host for energy and carbohydrates , Cai et al. 2003, Borza et al. 2005, de Koning and Keeling 2006, Vaidya and Mather 2009). Therefore, it seems likely that some red algal parasite mitochondrion and plastid genes would be truncated or even lost over time. The origin and roles of mitochondria and plastids in the parasite-host interaction may reveal key information regarding red algal parasite biology and their ability to infect and control the host cellular machinery.
First using the alloparasite Choreocolax polysiphoniae, and later the adelphoparasites Gracilariophila oryzoides and Gardneriella tuberifera, researchers noticed that in addition to the parasite nuclei, organelles are also transferred to the host cell via the conjunctor cell upon infection . Once molecular tools became more widely available, Goff and Coleman utilized restriction fragment length polymorphisms (RFLPs) to investigate the origin of mitochondria in the adelphoparasites Plocamiocolax pulvinata, Gracilariophila oryzoides, and Gardneriella tuberifera and their respective hosts, Plocamium cartilagineum, Gracilariopsis lemaneiformis, and Sarcodiotheca gaudichaudii . This work revealed that P. pulvinata and G.
oryzoides maintain a genetically unique mitochondrion and that both the parasite and host mitochondria are present within the heterokaryotic cells . The study was unable to conclusively demonstrate that the mitochondrion from Gardneriella tuberifera is unique from that of Sarcodiotheca gaudichaudii, due to the extremely close relationship between the two species .
The mitochondrion genomes of the adelphoparasites Plocamiocolax pulvinata and Gracilariophila oryzoides, as well as its host Gracilariopsis andersonii, were recently sequenced. These data paved the way for a new level of fine-scaled investigations of red algal parasites, elucidating details of the organellar genome architecture that was previously unattainable . When comparing the mitochondrion genome sequences of the parasites with the free living host, the atp8 and sdhC genes from G. oryzoides were determined to be pseudogenes . Furthermore, atp8 was determined to be absent from P. pulvinata.
However, the authors noted that according to sequenced cDNA libraries, the genes were still transcribed . Recent second generation sequencing of additional samples of these species has revealed that the "missing" genes are all present and lack frameshift mutations (Salomaki & Lane, unpublished). These data suggest that purifying selection is maintained on red algal parasite mitochondria and that even though much of the parasite life cycle exists inside a host cell, red algal parasites still require their own mitochondrion for their survival.
While evident that parasites maintain their native mitochondrion, microscopy and molecular studies have demonstrated that red algal parasites do not maintain their own plastid . Microscopy shows that the spores of adelphoparasites Gracilariophila oryzoides, and Gardneriella tuberifera contain proplastids lacking photosynthetic pigments, phycobilisomes, and thylakoids . Once the parasite injects its nuclei and organelles into a host cell, the host plastids transform and their light harvesting pycobilisomes disappear from the thylakoids . After rapid dedifferentiation, simple proplastids that are similar to the infecting parasite plastid bud off the host plastid . As the parasite nuclei, mitochondrion, and host derived proplastids spread to adjacent cells through pit connections, plastids from the newly infected host cells also rapidly dedifferentiate into proplastids . Eventually cells emerge from the heterokaryotic host cell containing only parasite nuclei, parasite mitochondrion and a host-derived proplastid . RFLP analysis was utilized to investigate whether this plastid was a genetically unique parasite plastid, or instead, the parasite was incorporating a host-derived proplastid .
This study revealed that adelphoparasites Plocamiocolax pulvinata, Gracilariophila oryzoides, and Gardneriella tuberifera and their hosts had identical banding patterns . Subsequent DNA sequencing of the variable plastid rbcL-rbcS spacer region revealed that the plastid from both hosts and parasites were genetically identical, confirming that the parasite plastid is a dedifferentiated host plastid   (Callow et al. 1979). The authors note that many of the parasite pustules had a pinkish hue and incorporated radioactively labeled C 14 into their thallus (Callow et al. 1979). Furthermore, the carbon fixation rate increased over time (up to 66 hours) leading the authors to conclude that C.
polysiphoniae is capable of photosynthesis on its own. However, the source of photosynthetic activity in dissected C. polysiphoniae pustules may be from host cells that have been incorporated into the pustule as observed in that study, and independently, by Kugrens and West, and Goff examining Janczewskia gardneri , Callow et al. 1979. Additionally, the status of J. gardneri as a parasite or obligate epiphyte has been debated due to its pigmentation , Court 1980. Most recently, it was noted that during the early stages of the interaction between J. gardneri and its host, Laurencia spectabilis, J. gardneri exists as colorless cells and 'infects' host cells in the same manner as other adelphoparasites. As J. gardneri cells erupt from the host they remain colorless but the cells become pigmented once the adelphoparasite becomes reproductively mature . Whether this pigmentation originates from host cells in the pustule matrix, or if the proplastid differentiates back into a photosynthetic plastid remains unknown.

Nutrient Transfer
With the exception of a few adelphoparasites that gain pigmentation upon reproductive maturity, red algal parasites are not capable of photosynthesis on their own and must obtain carbohydrates and other nutrients from a host. After parasite infection, the host (now heterokaryotic) cell loses the ability to photosynthesize as a result of plastid dedifferentiation . This leads to a differential gradient of carbon between the heterokaryotic cell and the adjacent normally functioning host cells . To account for the loss of carbon fixation, uninfected host cells direct photosynthate to heterokaryon and parasite cells that they are connected to via pit connections .
The first studies investigating carbon transfer between a red algal host and its parasite found three products of photosynthesis (floridoside, isofloridoside, and manitol) were transferred from the host to its parasite via a concentration gradient (Evans et al. 1973, Callow et al. 1979. Later five different sugar species were identified to be assimilated by the host Rhodomela confervoides and translocated to its parasite Harveyella mirabilis . Investigations into carbon translocation in H. mirabilis demonstrated the localization of carbon, from being fixed by the photosynthetic host through its movement into the parasite cells and revealed that heterokaryon cells incorporated more C 14 than neighboring uninfected host cells . Furthermore, it was determined that starch was not distributed evenly throughout the parasite cells as might be expected, but instead was being directed preferentially to parasite reproductive cells .
Given the capabilities of parasites for obtaining carbon from the host, the role of the maintained proplastid in parasite cells remains in question.

Host Specificity and Parasite Resistance
Red algal parasites are known to be extremely host specific, usually infecting one to a few, closely related host species . A study using the adelphoparasite Janczewskia morimotoi tested its ability to infect 15 other species including close relatives of its natural host, Laurencia nipponica, as well as members of different genera . While J. morimotoi was capable of infecting two close relatives of its natural host, the more distantly related potential hosts prevented parasite infections . Additionally, the host specificity of Leachiella pacifica was assessed through culture studies attempting to use parasites isolated from Polysiphonia paniculata to infect Pterocladia bipinnata and vice-versa . Parasites isolated from P. paniculata could infect other populations of the same species as well as some other Polysiphonia species, however they could not infect Pt. bipinnata populations that were susceptible to parasites isolated from other Pt. bipinnata specimens . These L. pacifica isolates showed strong genus-level host specificity. However, due to the greatly reduced morphology of red algal parasites, it cannot be ruled out that parasites isolated from different genera are, in fact, different host-specific species. Revisiting this study with molecular data would strengthen our understanding of host specificity and potentially reveal cryptic parasite species.
Dawsoniocolax bostrychiae and Bostrychiocolax australis are parasites that infect Bostrychia radicans . A study on host range and specificity of these parasites on a variety of potential hosts, yielded similar results to the J. morimotoi study: the genetic distance between parasite and host has a strong negative correlation with susceptibility to parasite penetration and infection . The authors note that they encountered hosts that are resistant to parasite infection, including some host populations that contained resistant and susceptible specimens . In several cases the parasite was capable of forming an initial infection in a resistant host, however the host cell or cells adjacent to the infected cell died off, preventing the parasite from spreading further into the host . However, subsequent molecular studies revealed phenotypic plasticity and cryptic diversity in B. radicans . Therefore, the possibility remains that resistant and susceptible hosts from the host resistance study were actually different species. These findings emphasize the need for ongoing taxonomic evaluation of red algal parasites and their hosts. Without the taxonomic framework questions about whether or not the host is actually resisting parasite infection cannot be answered conclusively.

Many Questions Remain
Why does the parasite maintain a copy of the host plastid as it is forming its own reproductive cells and spores? Other parasites that have evolved from a plastid bearing ancestor, including the apicomplexans Eimeria tenella and Plasmodium falciparum, the parasitic plant Epifagus virginiana and many others, maintain a reduced plastid for cellular functions other than photosynthesis, such as fatty acid biosynthesis , Wilson et al. 1996, Cai et al. 2003. However, none of these plastid-bearing parasites steal a plastid from their host like the red algal adelphoparasites. Are adelphoparasites genetically similar enough to their hosts that they can target nuclear-encoded proteins to the host-derived proplastid and utilize those products for fatty acid biosynthesis? Genomic analyses of signaling and targeting peptides for plastid targeted nuclear genes in red algal parasites, combined with transcriptomic and proteomic approaches, will provide valuable insight into the role of the plastids in the infection mechanism and parasite life cycle. The use of additional molecular tools including in-situ hybridization would enable researchers to localize parasite nuclear-encoded proteins in the heterokaryotic cell.
Furthermore, the taxonomic range and multiple independent origins of red algal parasites makes it difficult to make generalizations based on a few observations.
Thus far, the origin of red algal parasite plastids has only been investigated in adelphoparasites and the origin of the alloparasite plastid remains unknown. The assumption is that alloparasites first progress through an adelphoparasite stage and also maintain a co-opted host plastid. However, there are distinct developmental differences between adelpho-and alloparasites, including the initial steps of infection and alloparasites inability to synthesize DNA in heterokaryon cells (Fig. 2). Therefore, it seems plausible to propose an alternative hypothesis that, rather than passing through an adelphoparasite stage, alloparasites are capable of directly evolving infection mechanisms to parasitize distantly related hosts. In the proposed scenario, alloparasites would presumably maintain their own plastid, as they are likely incapable of utilizing such a genetically distant host plastid. Preliminary genomic data from C.
polysiphoniae indicates this may, in fact, be the case (Salomaki & Lane, unpublished) Future research investigating red algal parasite evolution will provide unique insight into the effects of transitioning from a free-living to a parasitic life strategy. Molecular data has supported morphological observations that red algal parasites share a recent common ancestor with their hosts ). However, further use of molecular tools is essential to provide a robust taxonomic framework of red algal parasites and their hosts. Only then can meaningful observations be made about host specificity and parasite resistance. With the technological advances of the past few decades and continually decreasing costs of DNA sequencing, information about the relationships between parasites and their hosts, unraveling the roles of parasite and host interactions, and the origins and function of organelles is within our grasp.

Red Algal Parasite Evolution
With over 100 extant species of parasitic red algae Lane 2012, Salomaki and, most of which evolved independently, red algae are a spectacular group to investigate the evolutionary mechanism by which a species transitions from free-living to parasitic. Early after the discovery of red algal parasites, phycologists postulated that parasites arise sympatrically and infect the species with which they share their most recent common ancestry (Setchell 1918). Subsequent molecular studies have provided additional support for this hypothesis ). These red algal parasites take advantage of the close relationship with their host in their unique infection mechanism.
Elegant studies by Goff and colleagues provided the fundamental understanding of red algal parasite biology and how they interact with their hosts.
Upon infection, red algal parasites fuse with a host cell and deposit their cellular contents . The resulting heterokaryotic host cell rapidly increases carbohydrate production and starch formation, becoming enlarged . The parasite eventually directs the host to form spores, which will be released to start the cycle again . Interestingly, all red algal parasites studied, including specimens from three different orders, have lost their own plastid.
Instead of maintaining their native plastid, they incorporate a dedifferentiated host plastid into their spores .
Until now, only the plastids of red algal adelphoparasites (adelpho is Greek for "kin") that share a recent common ancestor with their host have been examined. Here we describe the plastid of Choreocolax polysiphoniae Reinsch, a parasite that is evolutionarily distant from its host (termed an alloparasite), and that of its host Vertebrata lanosa (Linnaeus) T.A. Christensen. These data reveal the parasite-host plastid interactions may be quite different in alloparasites. The presence of a native non-photosynthetic plastid in C. polysiphoniae, indicates that multiple evolutionary pathways to parasitism exist among red algae.

Descriptions of the Plastids
Vertebrata lanosa is a multicellular polysiphonious red alga that belongs to the Rhodomelaceae (Supplemental Figure  Choreocolax polysiphoniae is also a member of the Rhodomelaceae, however it is an obligate parasite of V. lanosa, which appears as a multicellular unpigmented erumpent pustule growing from the V. lanosa thallus (Supplementary Figure 1). In the course of gathering genome-scale data from C. polysiphoniae, a 90 kb contig with high coverage (815x) emerged from the data. Closer examination revealed that the contig represents a highly reduced plastid genome sequence (GenBank Accession KP308096) in C. polysiphoniae ( Figure 1). The plastid encodes 71 protein coding genes, 3 rRNAs, and 24 tRNAs. All 71 protein-coding genes are shared with the V.
lanosa plastid. However petF is the only photosynthesis-related protein encoded by the C. polysiphoniae plastid. In C. polysiphoniae, as in Chlamydomonas reinhardtii, it is likely to serve as an electron carrier in additional metabolic pathways Naber 1993, Jacobs et al. 2009). Although the C. polysiphoniae plastid is no longer capable of light harvesting and photosynthesis, genes involved in amino acid, fatty acid, isoprene, and protein biosynthesis, transcription and translation as well as other cellular maintenance, are conserved.
The C. polysiphoniae ftsH gene, which is involved in photosystem II repair, is missing the first ~150 residues, however the conservation of the remaining 452 amino acid residues does not indicate a loss of selection pressure on the gene. Conversely, only the remnants of gltB are detectable by BLAST. Unlike the conservation observed in ftsH, the gltB region of the plastid genome contains no substantial ORFs and is laden with stop codons, indicative of a vanishing pseudogene. BLAST searches against the preliminary assembly of C. polysiphoniae genomic data was used to locate plastid genes found in the V. lanosa plastid genome, but absent from C. polysiphoniae.
These searches were performed to account for the possibility of transfer from the plastid to the nucleus. None of the 'missing' plastid genes could be found in the assembly of nuclear data, however low coverage of these data (<10x) does not rule out transfer as a possibility.

Comparisons to Other Florideophyceae Plastids
Typical plastid genes used for Rhodophyte phylogenies, including the large subunit of RuBisCo (rbcL) and the photosystem I & II genes psaA and psbA  Table 1). The C. polysiphoniae plastid maintains blocks of synteny with other Florideophytes, but it is clear that the C. polysiphoniae plastid has undergone many losses and genome rearrangements ( Figure 2). This is likely a result of relaxed selection due to the parasite relying on the host for light harvesting and carbohydrate production.
Despite the annular representation of the C. polysiphoniae plastid in Figure 1, we have thus far been unable to unequivocally demonstrate that the genome is a circular molecule like most other plastid genomes. Reports of linear plastids have been made from some flowering plants (Bendich andSmith 1990, Bendich 2004). While the read mapping data indicates there are reads spanning from one end of the assembly to the other, multiple attempts to PCR across the 'gap' have been unsuccessful.
Additionally, two of the three independent C. polysiphoniae data sets (and combined datasets) assembled the plastid with the same beginning and end. The third dataset assembled the plastid in two separate contigs though this was a result of lack of overlapping coverage between the tufA and rps12 genes and the ends of those contigs corresponded to the same start and end of the previous assemblies (see Supplemental Materials for additional information). If the molecule were circular, a random "breakpoint" in the assembly would be expected. This similarity between the beginning and end to the sequence suggests either there is a distinct region resistant to DNA amplification and sequencing, or that it may actually be a linear molecule.
Future work using restriction digests and pulse-gel electrophoresis will help to confirm the annular or linear nature of the C. polysiphoniae plastid.

Comparisons to Other Parasite Plastids
Most "crown" Apicomplexa are parasites that contain a relict plastid (apicoplast) obtained through an ancient secondary endosymbiosis (Fast et al. 2001, Zhu et al. 2002, illustrating plastid genome reduction in a lineage that lost its photosynthetic abilities long ago (Douzery et al. 2004). The apicoplast genomes from Plasmodium falciparum, Theileria parva, and Eimeria are more reduced than C. polysiphoniae (all three are ~35 kb), but maintain function in fatty acid biosynthesis, heme biosynthesis, iron-sulfur cluster synthesis, and isoprenoid biosynthesis ( Figure 1) (Wilson et al. 1996, Cai et al. 2003, Gardner et al. 2005.
Non-photosynthetic plastids from parasites in the Viridiplantae lineage have also been sequenced , Borza et al. 2005 Koning and

Implications for Red Algal Parasite Evolution
The description of a plastid in C. polysiphoniae suggests there are multiple mechanisms for parasite evolution in Rhodophytes. Current dogma suggests that red algal parasites first evolve sympatrically with their host . However, there are several differences in the cellular interactions between adelpho-and alloparasites and their respective hosts . Furthermore, the non-photosynthetic plastid of the alloparasite, C. polysiphoniae, indicates this is not the sole evolutionary pathway. If C. polysiphoniae had passed through an adelphoparasite stage, previous research suggests it would no longer maintain a plastid . Conversely, if parasites can directly infect distantly related hosts, the remaining non-photosynthetic parasite plastid implicates an alternative to recognized organelle evolution in these parasites.

Adelphoparasites and alloparasites rely on different infection mechanisms, based on
the relationship with their host . The lack of a native plastid in all adelphoparasites examined to date suggests that the genetic similarity between adelphoparasites and their hosts allows the parasite to utilize the host plastid rather than maintaining its own. Clearly it is necessary for some red algal parasites to maintain their own plastid for certain functions while relying on the host for carbohydrate production. Perhaps future studies targeted at alloparasites will determine a link between phylogenetic relatedness and ability to utilize the host plastid.

Methods and Materials
Specimens of both Vertebrata lanosa and Choreocolax polysiphoniae were collected at Beavertail State Park, Jamestown, RI. The V. lanosa was inspected for parasites and epiphytes under a dissecting microscope and clean material was ground under liquid nitrogen. Individual C. polysiphoniae were excised from the thallus of V.
lanosa and collected in a 1.5 mL microcentrifuge tube. Specimens were hand-ground using a Corning Axygen® PES-15-B-SI disposable tissue grinder pestle in a 1.5 mL microcentrifuge tube, submerged in 100µL of DNA extraction buffer .
DNA was extracted from two batches containing ~50 individual C. polysiphoniae using a standard phenol/chloroform extraction   All C. polysiphoniae assemblies were aligned and found to be similar with few indels and single nucleotide polymorphisms between them. The assembly from the amplified library was chosen as the representative plastid genome sequence for C.
polysiphoniae due to the quality and depth of coverage. Open reading frame (ORF) prediction on the V. lanosa and C. polysiphoniae plastid sequences was done using Geneious Pro v6.1 ) and the resulting ORFs were manually annotated using GenBank and UniProt databases. Genes from other red algal plastid genomes that were absent from the V. lanosa and C. polysiphoniae plastids were BLAST searched against the plastid sequences and the genomic assemblies to verify their absence and check for evidence of transfer to another genetic compartment. The assembled plastid genome sequences were submitted to the tRNAscan-SE online server v1.21 for identification of tRNA sequences . Attempts to 'close the gap' by using PCR to sequence from one end of the C. polysiphoniae plastid genome was attempted unsuccessfully using 12 different primer pairs.    Mitochondrial genomes have experienced widespread gene loss and genome reduction within eukaryotes and DNA sequencing has revealed that most of these gene losses occurred early in eukaryotic lineage diversification. On a broad scale, more recent modifications to organelle genomes appear to be conserved and phylogenetically informative. The first red algal mitochondrial genome was sequenced more than 20 years ago, and an additional 29 Florideophyceae mitochondria have been added over the past decade. A total of 32 genes have been described to have been missing or considered non-functional pseudogenes from these Florideophyceae mitochondria.
These losses have been attributed to endosymbiotic gene transfer or the evolution of a parasitic life strategy. Here we sequenced the mitochondrial genomes from the red algal parasite Choreocolax polysiphoniae and its host Vertebrata lanosa and found them to be complete and conserved in structure with other Florideophyceae mitochondria. This result led us to resequence the previously published parasite Gracilariophila oryzoides and its host Gracilariopsis andersonii, as well as reevaluate reported gene losses from published Florideophyceae mitochondria. Multiple independent losses of rpl20 and a single loss of rps11 can be verified. However by reannotating published data and resequencing specimens when possible, we were able to identify the majority of genes that have been reported as lost or pseudogenes from Florideophyceae mitochondria.

Introduction
Endosymbiotic events have had a profound impact on eukaryotic evolution (Lane & Archibald 2008;Keeling 2010;Koonin 2010;Zimorski et al. 2014;Martin et al. 2015). All eukaryotes (with one recent exception (Karnkowska et al. 2016)) possess a mitochondrion or mitochondrion-related organelle (MRO) that was initially acquired from an alpha-proteobacteria endosymbiont Lang et al. 1999;Koonin 2010;Gray 2012). Additionally, photosynthetic lineages maintain a plastid that originated as a cyanobacterial endosymbiont in the shared ancestor of Glaucophytes, Rhodophytes, and Viridiplantae (Chlorophytes and Streptophytes), and was subsequently spread through the eukaryotic tree of life via secondary and tertiary endosymbiotic events Keeling 2004;Stiller 2007;Gould et al. 2008;Lane & Archibald 2008;Keeling 2010;Stiller et al. 2014). There is evidence of massive gene transfer from the endosymbiont to the host nucleus upon the initial acquisition of these organelles, resulting in host control and regulation of the organelle's function (Martin et al. 1998;Timmis et al. 2004;Qiu et al. 2013;Ku, Nelson-Sathi, Roettger, Garg, et al. 2015). Further organellar genome modifications appear to be mostly lineage specific, with gene losses and transfers being restricted within lineages (Tucker 2013;Janouškovec et al. 2013;Ku, Nelson-Sathi, Roettger, Sousa, et al. 2015;Qiu et al. 2015;Tanifuji et al. 2016). The conservation among organellar genomes, in addition to their being inherited predominately uniparentally, has made organelles prime targets for understanding evolutionary relationships across and within the eukaryotic tree of life.
Red algae (phylum Rhodophyta) diversified from their last common ancestor, shared with green algae, more than 1 billion years ago ). There are ~7,100 currently described species of rhodophytes that are divided into 7 classes; Bangiophyceae, Compsopogonophyceae, Cyanidiophyceae, Florideophyceae, Porpyridiophyceae, Rhodellophyceae, and Stylonematophyceae (Guiry & Guiry 2016). The Florideophyceae exhibit a wide range of morphological complexity and are by far the most species rich class, containing ~6,750 species spread across 30 orders (Guiry & Guiry 2016). Understanding the evolutionary relationships within the Florideophyceae has traditionally been complicated by phenotypic plasticity (Cianciola et al. 2010). More recently, molecular data have been analyzed and great progress has been made in describing new genera and species (Cianciola et al. 2010;Saunders & McDonald 2010;. However, teasing apart the evolutionary histories of red algal orders has proven quite difficult even with the abundance of sequence data currently available (Verbruggen et al. 2010;Lam et al. 2016). Resolving the evolutionary relationships among florideophytes will provide a robust framework for asking a wide range of evolutionary questions including, but not limited to, transitions from marine to freshwater habitats, the evolution of the complex triphasic life-cycle found in many Florideophyceae orders, and the evolution of parasitism, a life strategy that has arisen many times among the Florideophyceae The use of the maternally inherited mitochondrial genome to resolve evolutionary relationships among the Florideophyceae shows promise .
The number of sequenced red algal organellar genomes has been increasing exponentially over the past decade. In part, this is a result decreasing sequencing costs allow for increasing use of next-generation sequencing technologies. Currently there are 30 published Florideophyceae mitochondrial genomes species available on GenBank (Table 1). However, only 16 of the 30 florideophycean orders are represented in these data, and 10 of those orders are represented by a single mitochondrion genome sequence.
Analyses of mitochondrion and MRO genomes across the tree of life have shown they are highly variable in gene content, arrangement, and structure (Smith & Keeling 2015). More recently, the oxymonad Monocercomonoides was shown to have entirely lost its MRO and all genes of mitochondrial origin that had been transferred to the nucleus (Karnkowska et al. 2016). Wide variability of mitochondrial genome content and structure has been implicated in the Florideophyceae as well. A study investigating the impact of adopting a parasitic life strategy on mitochondrial genomes of red algae described the atp8 and sdhC genes of red algal parasite Gracilariophila oryzoides as pseudogenes, and that the atp8 gene in the parasite Plocamiocolax pulvinata has been lost entirely . The authors concluded that the products of these genes may be provided either from the parasite nucleus as a result of endosymbiotic gene transfer, or perhaps the proteins are being obtained from their hosts. More recently, Yang et al. (2015) sequenced 11 Florideophyceae mitochondrial genomes. Analysis of their data, in combination with all previously sequenced red algal mitochondria led them to describe widespread variation in gene arrangement and multiple independent losses of atp8, nad4L, rpl20, rps11, sdhC, sdhD, secY, and ymf39 across the Florideophyceae .
Prior to this study, 30 Florideophyceae mitochondrial genomes have been sequenced. Of those, 19 are reported to be missing a functional copy of at least one gene. A total of 8 different genes have been reported as pseudogenes or missing entirely from a Florideophyceae mitochondrial genome. Previous speculation on what is driving gene loss from Florideophyceae mitochondria include endosymbiotic gene transfer (EGT) from the mitochondrion to the nucleus Yang et al. 2015), and decreasing selective pressures in parasite mitochondria as a parasite may be obtaining products of those genes from the host ). Both explanations seem plausible, with the later hypothesis being directly responsible for the sequencing of the mitochondrial genome from the parasitic red alga, Choreocolax polysiphoniae and its host Vertebrata lanosa (this study).
The mitochondrial genomes of the parasitic red alga, Choreocolax polysiphoniae and its host Vertebrata lanosa represent the first mitochondrial genomes available from the family Rhodomelaceae, which comprises ~1/7 th of species diversity within the phylum Rhodophyta (Guiry & Guiry 2016). In 2010 our lab reported that two mitochondrial respiratory protein-coding genes were degraded in the red algal parasites, Gracilariophila oryzoides and Plocamiocolax pulvinata . Unexpectedly, the C. polysiphoniae mitochondrion has no degradation of respiratory mitochondrial genes. To reconcile these datasets we resequenced the mitochondrial genomes of the parasite Gracilariophila oryzoides and its host Gracilariopsis andersonii. Furthermore, we systematically reevaluate the described gene losses from the other 30 previously published Florideophyceae mitochondrial genomes, revealing that more than two-thirds of the described losses are the result of errors in sequencing or downstream analyses. We find Florideophyceae mitochondrial genomes to be highly conserved and that gene losses are rare and predominately, if not entirely, observed in genes encoding ribosomal proteins.

Mitochondrial Genome Sequencing
Specimens of Vertebrata lanosa and Choreocolax polysiphoniae were collected from Beavertail State Park, Jamestown, RI, USA (voucher RI 0423).  . DNA was extracted from all specimens using a standard phenol/chloroform extraction . oryzoides mitochondria were mapped back to the previously published mitochondrion to compare the two assemblies and confirm support for differences.

Gracilariopsis andersonii and
Open reading frame (ORF) prediction on the V. lanosa and C. polysiphoniae mitochondrion sequences was done using translation table 4 (Protazoa Mitochondrion) using ATG as a start-codon in Geneious Pro v6.1 ). The resulting ORFs were manually annotated using blastN against GenBank. If blastN was insufficient for annotating an ORF, the ORF was translated to an amino acid sequence and then searched against the non-redundant protein sequence database (nr) in GenBank using blastP and the Pfam database (Finn et al. 2010(Finn et al. , 2015. If no conserved domain or sequence similarity could be found after searches using blastP or Pfam, the ORF remained without further annotation. Mitochondrion genome sequences were submitted to the tRNAscan-SE online server v1.21 for identification of tRNA sequences . Ribosomal RNA predictions were based on annotations produced by MFannot (http://megasun.bch.umontreal.ca/cgibin/mfannot/mfannotInterface.pl).

Red Algal Mitochondrial Genome Conservation
All 30 currently available Florideophyceae mitochondrial genomes (Table 1) were downloaded from GenBank and imported into GeneiousPro v9.1. These mitochondrial genomes were combined with those from V. lanosa and C.
polysiphoniae to create a database of Florideophyceae mitochondrial genomes.
Sequences that have previously been found to have missing genes or pseudogenes were reanalyzed for ORFs in GeneiousPro v9.1. In cases where an ORF was found in a conserved location that had not previously been annotated as a gene, the ORF was translated and searched against the non-redundant protein sequence database (nr) in GenBank using blastP and Pfam. If this was insufficient to annotate an ORF in a conserved location, representatives of the missing genes were mapped back to the genome of interest for further evaluation and the region was manually curated.
Translations of ORFs from locations of missing genes were aligned with annotated copies of those genes to manually assess annotation. When an apparent premature stop codon was found in a conserved location for a missing gene or pseudogene, the region was resequenced using PCR amplification for confirmation when material or DNA from that species could be obtained.
To determine the AT content (%) and nonsynonymous to synonymous substitution ratio (dN/dS ratio), all protein-coding genes widely shared throughout the 29 Florideophyceae mitochondrial genomes were aligned using GeneiousPro v9.1.
The average AT content (% )was calculated for each gene across the Florideophyceae in GeneiousPro v9.1. The CODEML program in PAML v. 4.8 (Yang 2007) was utilized to estimate the pairwise dN/dS ratio across all published Florideophyceae mitochondrion genes. For each gene analyzed, a nucleotide alignment was created using the translation align function in GeneiousPro v9.1 utilizing the Blosum62 cost matrix. Additionally, a Neighbor-Joining tree was constructed from the alignment in GeneiousPro v9.1 using the Tamura-Nei substitution model and the gene from Hildenbrandia rubra was used to root the tree. For the rpl20 gene, which has been lost in H. rubra, Palmaria palmata was used to root the tree. The alignment and Neighbor-Joining tree were used as input files and the following parameters were specified in CODEML: runmode = 0; seqtype = 1; codonfreq = 0; model = 0; icode=4; and omega (measures dN/dS ratio) and kappa (measures transitions/transverstions) were estimated.

Description of a Red Algal Alloparasite and Host Mitochondrion
The mitochondrial genomes of the red algal alloparasite, Choreocolax polysiphoniae (KX687877) and its host, Vertebrata lanosa (KX687880) represent the first mitochondrial genomes sequenced from the Rhodomelaceae (Florideophyceae, Rhodophyta), further expanding the diversity of available red algal mitochondrial genomes. The C. polysiphoniae mitochondrial genome is a 25,357 basepair (bp) long circular molecule with an AT content of 79.4%. The mitochondrial genome of C. polysiphoniae encodes 23 protein coding genes, 2 rRNAs and 20 tRNAs and contains only 9.9% intergenic, non-coding DNA. The V. lanosa mitochondrial genome is also a circular molecule that is 25,337 bp long, has an AT content of 76.4%, and encodes 23 protein coding genes, 2 rRNAs and 19 tRNAs with 10.4% of the mitochondrial DNA being non-coding. Both the C. polysiphoniae and V. lanosa mitochondrial genomes maintain a similar genome architecture to other published red algal mitochondria.

Parasitic Red Algal Mitochondrial Genomes are Conserved
The atp8 gene has previously been reported missing from five Florideophyceae mitochondria Yang et al. 2015) including the parasites Gracilariophila oryzoides and Plocamiocolax pulvinata. A re-annotation of the mitochondrion of P. pulvinata, identified the atp8 as an ORF that was annotated only as a hypothetical protein CDS in the sequence downloaded from GenBank (Tables 1 &   2). Subsequently, resequencing the Gracilariophila oryzoides mitochondrial genome (KX687879) revealed a complete copy of the atp8 gene, rather than the pseudogene that was previously reported using both Illumina and Sanger sequencing ). The sdhC gene was also previously reported to be a pseudogene in the adelphoparasite Gracilariophila oryzoides . As with atp8, resequencing of the G. oryzoides mitochondrion (KX687879) demonstrated that there was no frameshift mutation, as originally published, and that sdhC remains complete in red algal parasite mitochondria. These findings indicate that red algal parasites have not found alternative mechanisms for acquiring mitochondrion proteins and rely on their own mitochondrion for generating cellular energy as was previously hypothesized .

Gene Loss in Other Red Algal Mitochondria
As a result of identifying conserved copies of genes originally reported to have been lost, we reevaluated all reported gene losses from red algal mitochondria.
Investigation of the other reported atp8 losses revealed that the gene was an ORF annotated as a hypothetical protein CDS in Plocamium cartilagineum, and that an ORF corresponding to atp8 could be found in the published Hildenbrandia rubra sequence data that was not previously annotated (Tables 1 & 2). The Ahnfeltia plicata mitochondrion had a premature stop codon resulting in a pseudogene where atp8 is normally found, however targeted PCR and sequencing showed the gene (KX687876) is complete. Analysis of the ratio of non-synonymous to synonymous substitutions (dN/dS ratio) in Florideophyceae copies of the atp8 gene show a higher rate on nonsynonymous mutations in atp8 than atp6 and atp9, which combine with atp8 to make up the F 0 domain of the F 1 F 0 -ATP synthase complex involved in ATP synthesis (Table   3). However the dN/dS ratio of all three proteins remains <1 indicating that purifying selection is acting on the mitochondrial F 1 F 0 -ATP synthase complex in red algae, as is expected from genes essential for mitochondrion function.
Although the biological implications of losing the nad4L gene was not discussed in previous literature, the gene was noted as being absent in the mitochondrial genomes of both Plocamiocolax pulvinata and Plocamium cartilagineum Yang et al. 2015). In Plocamiocolax pulvinata an ORF was identified in the same location as other red algal copies of nad4L, between the 16S ribosomal RNA and the 26S ribosomal RNA, and both Pfam sequence search and blastP search of the translation strongly supports it coding for a functional nad4L.
The published sequence for the mitochondrial genome of Plocamium cartilagineum splits an ORF here identified as the nad4L gene in two pieces, with the 5' portion of the sequence found from bases 26,172 -26,431 and the 3' portion of the sequence is located from bases 1-43. With a dN/dS ratio of 0.14619, the nad4L gene remains under strong purifying selection in red algal mitochondria. Therefore, the loss of nad4L in any red algal mitochondria would represent a strong departure from this heavy selective pressure.
The sdhD gene encodes an essential protein that serves to anchor the succinate dehydrogenase complex II to the inner-membrane of the mitochondrion (Elorza et al. 2004;Bayley et al. 2005Bayley et al. , 2006. The sdhD gene was reported missing from the mitochondria of Ceramium japonicum and Asparagopsis taxiformis  and the gene is also not annotated in the more recently published mitochondrial genome of Ceramium sungminbooi (Hughey & Boo 2016). Upon reanalysis, an ORF was identified between nad4 and nad2, in the conserved Florideophyceae location of sdhD in the published mitochondrial genomes for all three species (see Tables 1 & 2). Furthermore, a translated alignment of these ORFs with other Florideophyceae copies of sdhD show they are conserved in frame, retaining several critical conserved residues (Figure 1), and therefore should be annotated as sdhD.
Four mitochondria are reportedly missing copies of sdhC. Similarly to our findings with the sdhD genes, unannotated ORFs that are conserved with other Florideophyceae sdhC genes were identified from the mitochondrial genomes of Ceramium sungminbooi and Dasya bingamiae ( Table 2). Based on the published Sebdenia flabellata mitochondrial genome, using an ATG as the only start-codon, there is no ORF that can be attributed to sdhC. However, using all start-codons in translation table 4 (Protozoa Mitochondrion) an ORF that is highly conserved in comparison with other Florideophyceae copies of sdhC is found with a TTA startcodon (Tables 1 & 2). Alternative start codons have previously been invoked for annotating red algal mitochondrion genes with variable support, which is discussed in more detail below (and see Table 4). The Sebdenia flabellata sdhC appears to be a well-justified case for using an alternative initiation codon.
The Ceramium japonicum mitochondrion is the other reported case of an sdhC gene loss . Although it appears to be highly conserved throughout the 5' region in comparison to other species, the C. japonicum sdhC is truncated by ~81 nucleotides (27 amino acids) at the 3' end when aligned with copies of the sdhC gene from other Florideophyceae. The Coeloseira compressa sdhC is similarly conserved at the 5' region and truncated at the 3' end. A Pfam search of the C. japonicum and Co. compressa sequences, translated to amino acids, confirms their identity as Succinate dehydrogenase/Fumarate reductase transmembrane subunit proteins though suggests they may be truncated as well. Although material was not available for experimental validation, we speculate that this observed truncation has little effect on the functionality of sdhC as an anchor protein in succinate dehydrogenase complex II. The length of Florideophyceae sdhC genes (excluding C. japonicum and Co. compressa) is quite variable, ranging from 339 bp in Plocamiocolax pulvinata to 411 bp in Asparagopsis taxiformis. Furthermore, the dN/dS ratio remains at 0.47084 indicating that purifying selection is acting fairly strongly on deleterious mutations in sdhC. The alternative would seem that the sdhC gene in C. japonicum and Co. compressa is losing its functional capacity, which would hinder the ability of these free-living species to generate cellular energy.
Although not reported as a loss, the published Gracilariopsis andersonii rps11 gene is inverted in comparison with all other Florideophyceae copies of the gene Yang et al. 2015). Resequencing this genome revealed an ORF in the conserved location between nad3 and atp9 that was not inverted and maintained strong homology with red algal rps11 genes. Analysis of this ORF in comparison to the previously published G. andersonii mitochondrion identified a string of seven 'A's stretching from bases 9,158-9,164 correspond to only six 'A's in the newly sequenced mitochondrion. This apparent frameshift mutation resulted in a premature stop codon in the conserved direction that led to identifying an ORF in the same location but inverted as rps11 in the earlier publication. The rps11 gene in the resequenced G.
andersonii mitochondrion, extending from bases 14,568 to 14,209, maintains strong homology with, and is encoded in the same direction as other Florideophyceae copies of rps11.
Although no genes were explicitly described as being lost in the recently sequenced Dasya binghamiae mitochondrial genome (Tamayo and Hughey 2016), annotations for rpl20 and sdhC are absent from the published sequence. Additionally, alignments demonstrate that the cox3, rps3 and TatC genes are truncated in comparison with other Florideophyceae. Perhaps even more interesting is the report of two inverted multi-gene rearrangements that are unprecedented in light of the highly conserved synteny in florideophycean mitochondria. Unfortunately a thorough evaluation of the losses, truncations and rearrangements in this mitochondrial genome is difficult as the publication is extremely brief (<500 words) and lacks essential details such as the sequencing platform from the materials and methods.

Frameshift Mutations are Overstated
In addition to the annotation of an inverted rps11 in Gracilariopsis andersonii, frameshift mutations have been described as the cause for genes being lost or becoming pseudogenes in Florideophyceae mitochondria including atp8 in Ahnfeltia plicata and Gracilariophila oryzoides and sdhC in Gr. oryzoides. The Gracilaropsis andersonii rps12 gene is another case of an apparent frameshift mutation causing a gene to be truncated. In G. andersonii, the rps12 gene is annotated at 240 nucleotides in length while other red algal copies of the gene range from 366-390 basepairs long.
As a part of this study we resequenced the G. andersonii mitochondrion (KX687878) and identified that the 'CT' found at bases 25,864-25,865 in the previously published G. andersonii mitochondrion appears to be the result of sequencing or assembly error.
Without these additional bases, the rps12 gene remains conserved and is 369 basepairs long.
The Ceramium japonicum nad3 gene appears to be an instance of an unreported frameshift mutation causing a gene to be truncated. Although the C. The Ceramium japonicum TatC (secY) initially appears to be another case of a Florideophyceae mitochondrion gene losing function and becoming a pseudogene due to a frameshift mutation, and again, pinpointing the exact location of the mutation is difficult. By manually manipulating the sequence and deleting a nucleotide from a sting of 43 T's and 7 C's between 23,501 and 23,550 basepairs into the published sequence, an ORF that is highly conserved with other Florideophyceae TatC genes containing an ATT initiation codon is observed. Due to high levels of variation in length and sequence of Florideophyceae TatC genes, we continue to recognize the Ceramium japonicum TatC gene as a pseudogene until firm evidence contradicts this.
However, based on our findings that all frameshift mutations previously discussed in this manuscript were the result of sequencing error or downstream analysis, it seems likely that is again the case here. Resequencing of this region is essential before considering TatC (SecY) as a true loss in Ceramium japonicum and the addition of RNA sequence data would help to confirm or reject this hypothesis.

Some Genes Have Degraded Into Pseudogenes
Even though secondary analysis of published sequences combined with subsequent PCR and resequncing efforts have found many of the genes that have been reported missing, this is not the case for all losses. The rpl20 gene seems to blur the lines of deciphering when a gene is lost, and it appears to be the least conserved gene in Florideophyceae mitochondria. Interestingly, aside from its presence in red algal mitochondria, the only other lineage of eukaryotes reported to maintain rpl20 are the jakobids (Burger & Nedelcu 2012). Retaining up to 67 genes, the most of any known mitochondria, Jakobid mitochondria are considered to most closely resemble the alpha-proteobacteria endosymbiont that became the contemporary mitochondrion (Gray et al. 2004;Burger & Nedelcu 2012;Burger et al. 2013). In the Florideophyceae, rpl20 has been reported missing or a pseudogene in 11 species including the two new additions from this study.
Annotation of rpl20 has been complicated because, in addition to ATG, which is the most commonly used initiation-codon for Florideophyceae mitochondrion genes, it appears that ATA may serve as an initiation-codon for rpl20 in Ahnfeltia plicata, and ATT in Rhodymenia pseudopalmata and Sporolithon durum (Table 4).
Without these alternative initiation-codons, rpl20 is likely a pseudogene in A. plicata, R. pseudopalmata, and S. durum. In addition to the aforementioned species, a conserved copy of rpl20 using the ATG start codon was located in Schimmelmannia schousboei (previously not annotated) and Schizymenia dubyi (previously annotated as a hypothetical protein CDS).
In Ceramium japonicum, Ceramium sungminbooi, Choreocolax polysiphoniae, Gelidium elegans, Gelidium vagum, Hildenbrandia rubra, and Vertebrata lanosa, the 3' region of rpl20 gene remains somewhat conserved, however the 5' end of the sequence is laden with stop codons, or appears to be missing entirely. Therefore rpl20 is considered a pseudogene in these species. No substantial region in the Dasya binghamiae mitochondrial genome appears to be homologous to the rpl20 gene.
Furthermore, rpl20 is annotated as a gene/coding sequence (CDS) in the Coeloseira compressa mitochondrial genome, however the 3' region is slightly truncated and not highly conserved with other rpl20 copies, suggesting that perhaps this also is a pseudogene. This wide variability in rpl20 initiation codons and conservation cause annotation to be extremely difficult. Confirming the presence or absence of a functional rpl20 localized in the mitochondrion is difficult and will likely require RNA sequencing and nuclear genome sequencing to identify possible cases of EGT.
The only unique Florideophyceae mitochondrion gene loss that appears to stand up to further scrutiny also encodes a ribosomal protein. Based on the published sequence of the Sporolithon durum mitochondrion, rps11 has degraded to a pseudogene. In all other Florideophyceae, rps11 is found adjacent to the 3' end of nad3, however in S. durum, this region contains no ORFs that can be attributed to a full-length copy of rps11. As in other genes, a frameshift mutation appears to be initially responsible for rps11 becoming a pseudogene. However, in all previously discussed frameshift derived pseudogenes, it was apparent that the insertion or deletion of a nucleotide or two would 'repair' the gene and result in a conserved copy, that could then subsequently be confirmed by PCR. In the case of the Sporolithon durum rps11, artificially 'fixing' the gene could restore a conserved 3' end of the gene, however a 6 residue gap upstream of this 'fix' remained in translated alignments adding further support that rps11 is no longer functional in S. durum. RNA and nuclear genome sequencing work remains necessary to identify whether this is a complete loss or a case of EGT from mitochondrion to the nucleus.

The Importance of Nomenclature
Identifying gene losses in Florideophyceae mitochondrial genomes has been further complicated by the use of two different names for a homologous gene. In Yang et al. (2015), the ymf39 gene was reported as the most widely lost gene in Florideophyceae mitochondria, and was noted as being absent in six species: Ceramium japonicum, Gracilariopsis andersonii, Hildenbrandia rubra, Kappaphycus striatus, Schizymenia dubyi, and Sebdenia flabellata. Furthermore, this gene is annotated only as a hypothetical protein CDS in Gracilariopsis chorda. Resequencing of the Gracilariopsis andersonii (KX687878) and reanalysis of the published Hildenbrandia rubra data reveals that the ymf39 gene is present in both mitochondria.
Interestingly, the other four species lacking ymf39 are also the only Florideophyceae mitochondria with an annotated atp4 gene, which is found between the cox3 and cob genes, the same location as ymf39 in other Florideophyceae mitochondria . According to Burger et al. (2003), ymf39 encodes subunit b of mitochondrial F 0 F 1 -ATP synthase and should formally be designated as atp4.
Although it has not led to reports of gene loss, it is of note that three names have been applied to the TatC gene in Florideophyceae mitochondria. In Chondrus crispus the gene currently annotated as ymf16 was initially described as a gene of unknown function called ORF 262 (Leblanc et al. 1995). In the publication of the Porphyra purpurea mitochondrial genome it was noted that ymf16 is recognized as a homolog of E. coli TatC encoding a protein in the Sec-independent protein translocation pathway   Additionally, it is recommended that SecY and ymf16 annotations be changed to TatC.

The Use of Alternative Initiation-Codons
The most widespread initiation-codon in Florideophyceae mitochondrion genes is ATG, though some exceptions have been previously proposed (Table 4). For example, in the Grateloupia angusta mitochondria the use of ATT, TTA, or TTG as initiation-codons was reported for 9 genes (Kim et al. 2014). Further examination of the published G. angusta mitochondrial genome revealed that 7 of the genes reported with an alternative initiation-codon (atp4 (as ymf39), atp6, cox2, orf-Gang5, rps11, sdhB, and sdhC), an ORF starting with ATG could be found within a few basepairs of the previously annotated gene, and the current G. angusta atp4 (as ymf39) gene annotation on GenBank does use an ATG start-codon. The reasoning behind the decision to opt for an alternative codon rather than ATG at the beginning of the gene was not described in the genome announcement.
Alternative start-codons have been suggested in a few other Florideophyceae mitochondrion genes besides Grateloupia angusta. For example, in Asparagopsis taxiformis the atp4 gene is annotated with the initiation-codon ATT, yet 6 basepairs (2 amino acid residues) away in the same reading frame is an ATG, which could also serve as the initiation-codon (Table 4). The Grateloupia angusta and Sporolithon durum copies of atp6 are both annotated to start with ATT codons that are 9 and 3 basepairs (3 and 1 amino acid residues), respectively, upstream of an ATG (Table 4).
A complete assessment of Florideophyceae mitochondrion genes that have been annotated using non-ATG protist mitochondrion initiation-codons and their proximity to a potential ATG start-codon is shown in Table 4.
Even though some of the alternative start-codon usage is questionable, though not necessarily incorrect, there appear to be several Florideophyceae mitochondrion genes that likely are using alternative start-codons (Table 4). In several of these cases the alternative hypothesis is that the genes are severely truncated and have been rendered non-functional. For example, it seems much more reasonable to believe that  Table 4).
The Chondrus crispus TatC (annotated as ymf16) gene is also likely reliant on an alternative start codon. Currently the Chondrus crispus TatC gene is annotated with a GTT initiation codon. However, GTT has not been used as an initiation codon in any other Florideophyceae mitochondrion gene, nor is it one of the start codon options in translation table 4 (Protozoa Mitochondrion). Four other ORFs in the same reading frame that use either ATA or TTA as a start codon for TatC gene are found from 12 -39 nucleotides downstream of the GTT codon. All Chondrus crispus ORFs that can be reasonably attributed to TatC use a start-codon other than ATG, suggesting that this is another reliable instance for invoking an alternative, however the use of GTT is questionable.

Why Were Genes Reported Missing?
There are several technical and biological reasons that could explain the previous results of missing genes in Florideophyceae mitochondrial genomes. Each cell maintains numerous mitochondria and some of these may in fact maintain the frameshift mutations that have led to gene losses being reported in published literature Yang et al. 2015). Preferential amplification of these mitochondrial genomes, or segments of the genome when using targeted PCR, would lead to the aforementioned findings even in cases where other mitochondria in the cell remain fully functional. The first four Florideophyceae mitochondrial genomes to be sequenced were completed primarily using nuclease digestions or PCR amplification and Sanger sequencing methods to assemble the genome at ~2x depth (Leblanc et al. 1995;. The advances in sequencing technologies and reduction in costs since the Chondrus crispus mitochondrion was first sequenced over 20 years ago have enabled much greater sequencing depths. For example the Vertebrata lanosa mitochondrial genome published here has average read coverage of 391x. This increased depth allows for the correction of 'errors' either in the biology or technical aspects of sequencing by utilizing the deeper coverage of sequencing reads when forming a consensus sequence. It is noteworthy that all frameshift mutations that have been reported and led to missing genes, pseudogenes or the inversion of the Gracilariopsis andersonii rps11 were found in long homopolymer regions. Assembling sequences containing long regions of low-complexity, often dominated by a single nucleotide, has been recognized as a major complication for sequencing (Kieleczawa 2006;Laehnemann et al. 2016) and was especially difficult for the 454 FLX technology used in   (Gilles et al. 2011;Loman et al. 2012).
RNA data can sometimes help identify problematic annotation or assembly.
The apparent atp8 and sdhC pseudogenes observed in their Gracilariophila oryzoides DNA data were confusing to the authors as they noted that both genes were still being transcribed based on RNA sequencing efforts . However, transfer to the nucleus was invoked at the time as a possible explanation. In retrospect, the RNA data was a strong indication to reexamine the data assembly. The sequencing errors reported in the first few published red algal mitochondrial genomes formed the foundation that was used as a reference for the annotation of subsequently sequenced Florideophyceae mitochondrial genomes. The apparent flexibility of mitochondrial genomes based on early sequencing efforts set a precedent for gene loss in Florideophyceae mitochondria. Building on a flawed foundation has allowed for the gene loss to be overstated without a deeper reanalysis of results. This is in no way meant as a criticism of the researchers themselves and it is plausible that results shown in data being published with current technology will be revised with future advances.

Conclusions
A detailed investigation of previously reported gene losses in Florideophyceae mitochondria reveals that losses are much less common and widespread than the published literature indicates. Prior to this study there genes had been either described as lost, or annotations were overlooked from 18 of the 30 published mitochondrial genomes (Tables 1 & 2). Thoroughly examining each loss using the available published sequence data, in combination with resequencing those specimens that we could obtain material from, has positively identified 23 of the 'missing' genes.
Overwhelmingly, the 'missing' genes or pseudogenes were the result of overlooked ORFs in the available sequence data and artificial frameshift mutations that resulted from sequencing and/or downstream assembly and analysis. In light of these findings, it is essential to thoroughly investigate results that indicate genes are degrading into pseudogenes or being lost entirely.
The C. japonicum mitochondrion was described as having lost five genes (atp4, rpl20, sdhC, sdhD, & TatC) of which three were identified here. Additionally, the translation of the existing annotation of the nad3 gene shares no homology with any other gene, though that homology is restored through the manual deletion of a 'T' in a low complexity region of that gene. Furthermore a gap in the sequence is annotated between cob and nad6. Resequencing this mitochondrion to confirm the presence or absence of the rpl20 and TatC, and close the gap is essential prior to inferring biological relevance resulting from these losses. However it does seem likely that at least rpl20 remains absent from the C. japonicum mitochondrion considering it has also been truncated in all other Ceramiales mitochondrial genomes.
It is logical that gene losses would be rare in red algal mitochondria since the core genes encoded are essential for cellular respiration and oxidative phosphorylation. The loss of genes involved in these processes would interfere with the organisms' ability to produce cellular energy and would likely be a lethal mutation. The ribosomal proteins rps11 and rpl20 have been lost in the mitochondria of other lineages (Burger & Nedelcu 2012) and may be examples of gene transfer from the mitochondrion to the nucleus. Additional red algal genome data will allow for the identification of nuclear-encoded, mitochondrial target proteins.
Hildenbrandia rubra 22,165 22,150 15 (5) nad4L - Ahnfeltia plicata a Grateloupia angusta a  (9) a Indicates examples where other non-ATG initiation codons from translation table 4 are also possible locations for the gene to start although no ATG codon is found within 30 nucleotides (10 amino acid residues) upstream or downstream from the start of the currently annotated gene.
b The Hildenbrandia rubra sdhC gene annotation is longer than other copies of sdhC and the beginning of the gene overlaps with a tRNA. Starting annotation at ATG makes the gene much more similar in length to other Florideophyceae copies of sdhC.
c Gene not previously annotated in GenBank d The Chondrus crispus TatC (ymf16) gene is currently annotated with a GTT initiation codon, which is not found for any other Florideophyceae mitochondrion gene nor is it a start codon in translation table 4 (Protozoa Mitochondrion). Four other ORFs in the same reading frame that use either ATA or TTA as a start codon for TatC gene are found from 12 -39 nucleotides downstream of the GTT codon.

Abstract:
Parasitism is a life strategy that has repeatedly evolved within the Florideophyceae. Until recently, the accepted paradigm of red algal parasite evolution was that parasites arise by first infecting a close relative and, either through host jumping or diversification, adapt to infect more distant relatives. The terms adelphoparasite and alloparasite have been used to distinguish parasites that are closely related to their hosts from those more distantly related to their hosts, respectively. Phylogenetic studies have cast doubt on the utility of these terms as data show that even alloparasites predominately infect with the same family. All adelphoparasites that have been investigated have lost a native plastid and instead hijack and incorporate a copy of their hosts' plastid when packaging spores. In contrast, a highly reduced plastid that has lost all genes involved with photosynthesis was sequenced from the alloparasite Choreocolax polysiphoniae, which indicates that it did not pass through an adelphoparasite stage. In this study we investigate whether other species in the Choreocolacaceae, a family of alloparasites, also retains its native plastid, as well as test the hypothesis that alloparasites can arise and subsequently speciate to form monophyletic clades that infect a range of hosts. We present the plastid genome for Harveyella mirabilis which, similar to that of C. polysiphoniae, has lost genes involved in photosynthesis. The H. mirabilis plastid shares more synteny with free-living red algal plastids than that of C. polysiphoniae. Phylogenetic analysis identifies a well-supported monophyletic clade of parasites in the Choreocolacaceae, which retain their own plastid genomes, within the Rhodomelaceae. We therefore transfer genera in the Choreocolacaceae to the Rhodomelaceae.

INTRODUCTION:
Since the 1845, when Microcolax botryocarpa Hooker & Harvey, became the first formally described parasitic red alga, the known biodiversity of red algal parasites has been steadily increasing Setchell , 1923Preuss, Nelson, and Zuccarello 2017). Red algal parasites exclusively infect other rhodophytes and are predominately unpigmented, appearing as galls or irregular growths on their free-living red algal hosts. Recent counts have identified 121 distinct species of red algal parasites distributed across eight different orders of the Florideophyceae Preuss, Nelson, and Zuccarello 2017). Florideophyceae seem to be prone to adopting parasitism, in large part due to their ability to form direct cell-to-cell connections called secondary pit connections between adjacent, non daughter cells (Wetherbee and Quirk 1982;. With few exceptions [e.g. Choreonema thuretii (Broadwater and LaPointe 1997)], red algal parasites leverage their ability to form secondary pit connections as a means of infecting their host .
Two main hypotheses have been proposed for the origin of red algal parasites. Setchell (1918) initially proposed that parasites arose as spores from their host, which had mutated to no longer be capable of photosynthesis. Sturch (1926) later proposed that parasites evolved from epiphytes that penetrated and, over time, became nutritionally reliant on the host.  later added to Sturch's hypothesis by suggesting that epiphytes that are closely related to their hosts are more likely to succeed in forming secondary pit connections and therefore increase the likelihood of successfully establishing a parasitic relationship.
Despite their diminutive habit, parasitic red algae share morphological characteristics with other close relatives, and thus were assigned to tribes or families at the time of their initial discovery (Reinsch 1875, Feldmann and. Historically parasites that have infected close relatives have been considered 'adelphoparasites', while those more distantly related to their hosts are called 'alloparasites' . Approximately 80% of the described red algal parasite diversity is considered to be adelphoparasitic, while the remaining 20% is alloparasitic . Based initially on morphological observations, and later coupled with molecular data, it was proposed that parasites evolve sympatrically with their hosts, as adelphoparasites, and over time diversify or adapt to infect new and more distantly related hosts, becoming alloparasites . Sturch (1926) initially described the family Choreocolacaceae as a family in the Gigartinales, which consisted of morphologically reduced parasites lacking chlorophyll including members of the genera Choreocolax, Harveyella, and Holmsella. The Choreocolacaceae was the subject of a thorough phylogenetic analysis of alloparasites to confirm whether the family was truly a monophyletic clade of parasitic red algae . This study supported previous morphological observations  confirming that the genus Holmsella was a member of the Gracilariaceae and questioned the legitimacy of recognizing a family of red algal parasites.
In the few other cases where molecular tools have been applied to assess evolutionary histories of red algal parasites, data suggests that red algal parasites arise though independent evolutionary events ). In addition to phylogenetic analyses, molecular tools have also been applied to investigate the parasite-host dynamics throughout parasite development. Analyses of the adelphoparasites Gardneriella tuberifera Kylin,

Gracilariophila oryzoides
Setchell & H. L. Wilson demonstrated that, although the parasites maintain a native mitochondrion, they have lost their native plastid and instead 'hijack' a host plastid when packaging their own spores . To date, all red algal parasites examined maintain a fully functional mitochondrion . All adelphoparasites that have been investigated have lost their native plastid Salomaki and Lane unpublished). In contrast, a highly reduced native plastid was sequenced from the alloparasite Choreocolax polysiphoniae Reinsch, which has lost genes involved in photosynthesis, yet maintains functions including fatty acid and amino acid biosynthesis . The lack of plastids in adelphoparasites, in combination with finding a native plastid in the alloparasite C. polysiphoniae, demonstrates that not all parasites pass through an adelphoparasite stage and that there are multiple paths to parasitism in red algae.
In their study examining relationships in the Choreocolacaceae,  found that Holmsella pachyderma and Holmsella australis form a monophyletic clade within the Gracilariaceae. Additionally they identify that parasite genera Choreocolax, Harveyella, and Leachiella are members of the Rhodomelaceae, though have fairly low support for their relationships to each other. Using molecular data we investigate the relationships of Choreocolax, Harveyella, and Leachiella, and test the hypothesis that alloparasites can arise and subsequently speciate, forming monophyletic clades that infect a range of hosts. Furthermore, we set out to determine if another alloparasite besides C. polysiphoniae retains its own native plastid.

Sample Collection and DNA Extraction
Choreocolax polysiphoniae, found on its host, Vertebrata lanosa (Linneaus) collected in a 1.5 mL microcentrifuge tube. The parasite tissue was hand-ground using a Corning Axygen® PES-15-B-SI disposable tissue grinder pestle in a 1.5 mL microcentrifuge tube while submerged in 100µL of DNA extraction buffer . DNA was extracted from specimens using a standard phenol/chloroform extraction with all ratios adjusted for an initial buffer volume of 100µL . Additionally, lyophilized DNA from specimen GWS021225 (Saunders 2014) was acquired from the Saunders lab and rehydrated in 20µL of 5mM Tris/HCl pH 8.5.

Molecular analyses
A 664 bp fragment at the 5-prime end of the mitochondrial cytochrome oxidase 1 gene (COI-5P), which has been used extensively for barcoding red algal species (Saunders 2005), was PCR amplified using the GWSFn (Le Gall and Saunders 2010) and GWSRx (Saunders and Moore 2013) primer pair according to protocols in

Plastid Genome Annotation
A 90,654 bp contig was identified as the plastid genome of Harveyella mirabilis from the previously described assembled Illumina MiSeq data. Open reading frame (ORF) prediction on the H. mirabilis plastid was done in Geneious Pro v6.1 and the resulting ORFs were manually annotated using GenBank and Pfam (Finn et al. 2010(Finn et al. , 2015 databases. Functional annotations were assigned from the UniProt (The UniProt Consortium 2017) and KEGG databases (Kanehisa et al. 2016). Genes found in red algal plastid genomes that were missing from the H. mirabilis plastid were searched for using BLAST, against the plastid sequence and the genomic assemblies to verify their absence and check for evidence of transfer to another genetic compartment. The plastid genome sequence was submitted to the tRNAscan-SE online server v1.21  for identification of tRNA sequences and to MFannot (http://megasun.bch.umontreal.ca/cgibin/mfannot/mfannotInterface.pl) to identify rRNA sequences and confirm manual gene annotations.

Plastid Genome Comparative Analysis
The  .

Identification of a Cryptic Parasite
A maximum likelihood phylogenetic analysis of 823 Rhodomelaceae COI-5P sequences was completed using RAxML. This analysis provided weak support (bootstrap support of 23) for a clade containing the alloparasites Choreocolax polysiphoniae, Harveyella mirabilis, Leachiella pacifica, as well as 2 sequences labeled Rhodomela sp1Cal voucher GWS021225 (KM254767) and Rhodomela sp1Cal voucher GWS021347 (KM254267). Interestingly, these two GenBank sequences had bootstrap support of 100 as being sister to the sequence of Leachiella pacifica generated in this study (data not shown). Based upon these results, the specimens were reexamined and parasite galls were found on the host, which was subsequently identified as Polysiphonia paniculata based upon rbcL sequence data.

Monophyletic Alloparasite Clade
Although identification of parasites on the samples GWS021225 and GWS021347 was a direct result of resolving a clade of parasite sequences, bootstrap support for a monophyletic clade of alloparasites was weak and a subsequent Bayesian phylogeny failed to recover the same clade (data not shown). Based upon this original analysis, COI-5P data from a reduced dataset of the parasites and 28 of their closest relatives was subjected to maximum likelihood analysis, which again recovered a poorly supported monophyletic alloparasite clade (data not shown). To resolve the issue of low statistical support, thirteen taxa that were continually allied to the alloparasite clade were selected based upon availability of published comparative data.

Taxonomic Considerations
Choreocolax polysiphoniae was initially described by  from the Atlantic coast of North America as parasitic on Polysiphonia fastigiata (now Vertebrata lanosa). Specimens used the phylogenetic analysis presented here ( Figure   1) were collected at Beavertail State Park in Jamestown, RI, USA infecting Vertebrata lanosa and are a strong match to the type description . As the type collection cannot be located, we formally lectotypify C. polysiphoniae on image #49 accompanying the description in . Based upon the molecular analyses here, which resolved a monophyletic clade of parasitic red algae within the Rhodomelaceae (Figure 1), we formally transfer Choreocolax to the Rhodomelaceae and recognize Choreocolacaceae as a synonym of this family. To adhere to the principle of monophyly, the genus Harveyella, based on the type and only species H.
mirabilis and included in our analyses (Figure 1), is also transferred to the Rhodomelaceae.

Harveyella mirabilis Plastid Genome
The plastid genome of Harveyella mirabilis was assembled as a 90,654 kb circular molecule with 322x coverage. The plastid genome has an overall AT content of 76.5% and contains 84 protein coding genes, 3 rRNAs, and 23 tRNAs (Figure 2).
Similar to the Choreocolax polysiphoniae plastid , all genes related to photosynthesis have been lost with the exception of petF which has been demonstrated to be involved in electron transport in other metabolic pathways ). Genes involved in transcription/translation and fatty acid, amino acid, protein, isoprene biosynthesis remain conserved. As in the C. polysiphoniae plastid, gltB appears to be a vanishing pseudogene. BLAST similarity searches are able to find conserved homology, however the presence of stop-codons throughout the region suggests that the gene is likely no longer capable of being completely translated.

Plastid Genome Comparisons
A whole genome MAUVE alignment of the H. mirabilis with C. polysiphoniae and nine representative free-living Florideophyceae plastid genomes identified 13 locally collinear blocks in the H. mirabilis genome that aligned with the free-living plastids ( Figure 3). There were no rearrangements or inversions in the H. mirabilis plastid genome when compared to photosynthetic Rhodomelaceae taxa. When aligning H.
mirabilis to the C. polysiphoniae plastid genome, 11 locally collinear blocks are identified, and several genome inversions and rearrangements are evident ( Figure 4).
Additionally, gene content varies slightly between the two parasite plastid genomes.

Origin of Parasites
With 121 described parasites occurring across eight Florideophyceae orders Preuss, Nelson, and Zuccarello 2017), red algae appear more able to transition from autotrophy to parasitic lifestyles than any other eukaryotic lineage. The terms adelphoparasite and alloparasite have traditionally been used to describe parasites that infect hosts within their tribe/family, or in different tribes/families, respectively. The use of these terms has been questioned as molecular data have revealed that alloparasites, like adelphoparasites, infect close relatives rather than distantly related species . Defining red algal parasites using taxonomy alone is no longer appropriate (See conclusion chapter). Phylogenetic analysis using data presented here place Choreocolax polysiphoniae, Harveyella mirabilis, and two species of Leachiella firmly within the same family as their hosts further supporting abandonment of these terms for differentiating two types of red algal parasites.
The Demise of Exclusively Parasitic Families Sturch (1926) initially described the Choreocolacaceae as a family of holoparasites, containing the genera Choreocolax, Harveyella, and Holmsella.
However, more recent morphological investigation considered that the genus Holmsella was related to the parasites Gelidiocolax and Pterocladiophila, and it was moved to the family Pterocladiophilaceae in the Gracilariales . Their observations were subsequently supported by molecular data generated with the specific aim of testing the phylogenetic affinities of parasites that Sturch had assigned to the Choreocolacaceae. This work demonstrated that Holmsella australis and Holmsella pachyderma formed a well-supported monophyletic clade within the Gracilariaceae .
Their molecular data also indicated that Choreocolax and Harveyella belong in the Ceramiales, leading the authors to question whether the Choreocolacaceae should continue to be recognized . However, their use of 18S sequence data was insufficient to resolve the issue of a monophyletic clade for these parasites. Other authors have also noted that species in Choreocolax, Harveyella, and Leachiella have features aligning them to the Ceramiales, but again, taxonomic affinities among the parasite species and within this order remained uncertain Kugrens 1982;. Our data confirm the findings of , placing Choreocolax, Harveyella and Leachiella, within the Rhodomelaceae. Furthermore, the phylogeny utilizing additional molecular markers provides strong support for a monophyletic clade containing Choreocolax, Harveyella and Leachiella (Figure 1), and supports the placing the Choreocolacaceae in synonymy with the Rhodomelaceae.

Cryptic Species
Until recently, the dogma behind red algal parasite evolution was the notion that parasites arise sympatrically as adelphoparasites and over time evolve and adapt to infect more distant hosts becoming alloparasites . Based on molecular data demonstrating that alloparasites also infect members of their own family , we now consider that these terms are not suitable for distinguishing parasites.
The recognition of multiple monophyletic parasite clades, the Holmsella clade , and the Rhodomelaceae clade uncovered here containing Choreocolax, Harveyella, and Leachiella (Figure 1), supports the idea of a sympatric origin of alloparasites with subsequent speciation as parasites adapt to their hosts, without passing through an adelphoparasite-like stage.
Previously, Leachiella pacifica was described from multiple hosts including members of the genus Polysiphonia and Pterosiphonia (Kugrens 1982;Zuccarello and West 1994). In their study on L. pacifica host specificity, Zuccarello and West (1994) found that parasite spores isolated from one host genus were unable to infect members  (Zuccarello and West 1994;. Molecular data here identify two distinct species of Leachiella that infect separate hosts but are otherwise difficult to distinguish. Due to the highly reduced morphology of these parasites, it seems likely that host identity may be the easiest way to identify parasites in the genus Leachiella to species. Some parasitic red algae are reportedly capable of infecting a range of hosts [e.g., Asterocolax -see ], however molecular data are contradicting that notion. Rather, it appears that red algal parasite species have higher host specificity than previously believed, and instead, we have underestimated the amount of parasite diversity as a result of their reduced morphology. Morphology based studies report Leachiella pacifica infecting at least seven unique host species (Zuccarello and West 1994), further molecular analyses will surely uncover additional species in the genus Leachiella.

Parasite Plastids
The link between plastid origin in red algal parasites and their evolutionary relationship to their hosts may be central to our understanding of red algal parasite evolution. The Harveyella mirabilis plastid genome (Figure 2) represents the second red algal parasite demonstrated to retain a reduced native plastid. Similar to Choreocolax polysiphoniae, the H. mirabilis plastid genome remains conserved for functions including amino acid, fatty acid, and protein biosynthesis, but has lost genes involved in building the light harvesting apparatus, photosystems I and II, and other photosynthesis related genes. Identifying a monophyletic clade of parasites (Figure 1) in which the two earliest branching members identified so far retain plastids, strongly suggests that plastids are also retained in species of Leachiella. Preliminary data generated in the Lane lab supports that hypothesis though work remains to completely sequence plastids from Leachiella species.
The H. mirabilis plastid gene order, with the exception of the missing photosynthesis genes, is conserved when compared to plastids of free-living Rhodomelaceae species (Figure 3). In contrast, we find that the C. polysiphoniae plastid has undergone greater gene loss than the H. mirabilis plastid, and a substantial amount of genome reorganization (Figure 4). Harveyella mirabilis retains argB and carA, which are involved in arginine biosynthesis processes, rpoZ, which promotes RNA polymerase assembly, nine genes involved in building ribosomes, and an uncharacterized hypothetical protein, all of which have been lost from the C.
polysiphoniae plastid (Table 2). However, C. polysiphoniae does retain copies of dnaB, which is involved in DNA replication, and fabH, which is involved in fatty acid biosynthesis, both of which have been lost in H. mirabilis. Analysis of additional plastid genomes in this clade will provide greater insights into patterns of plastid genome evolution in red algal parasites.
Two competing hypotheses have been proposed for the origin of red algal parasites. Setchell (1918) suggested that a mutation in a spore causes parasites to arise sympatrically, while Sturch (1926) postulated that parasitic red algae start out as epiphytes that over time become endophytes and increasingly rely on the host for nutrition. It seems plausible that Setchell's origin hypothesis could explain the rise of the so-called adelphoparasites, which comprise the majority of known red algal parasite biodiversity. By evolving from their hosts, adelphoparasites could easily incorporate a copy of a genetically similar plastid as their own. Replacing a functionally reduced or non-functional native plastid with a host-derived plastid seems to be an easy mechanism for survival. Unfortunately, reliance on the host plastid also starts the newly evolved parasite down a path towards inevitable extinction. In order for a non-photosynthetic parasite to survive it still must retain compatibility for other plastid functions including amino acid and fatty acid biosynthesis. Because the hostderived plastid is newly acquired during each new infection, the host plastid experiences one set of evolutionary pressures while the parasite evolves and accumulates mutations of its own. Eventually the parasite will inevitably lose the ability to communicate with the host plastid as the parasite and host increasingly become genetically distinct. This leaves the parasite with two possibilities for survival, either to find another host with a compatible plastid or go extinct. Alternatively, the success of an alloparasite may be explained by Sturch's hypothesis. By evolving from a closely related epiphyte that is able to create secondary pit connections, the parasite may retain its own plastid and therefore enable its longevity and the opportunity to even speciate as the parasite adapts to new hosts. Therefore, what were previously viewed as competing hypotheses to explain the evolution of red algal parasites, may each explain how different types of parasites arise.
Although organisms have transitioned from photosynthesis to other methods of nutrient acquisition numerous times across the tree of life, support for total plastid loss is rare (Gornik et al. 2015) with most cases resulting in highly reduced plastids retained for core functions (e.g. isoprenoid and fatty acid biosynthesis) de Koning and Keeling 2006) or in extreme cases the retention of the apicoplast in Apicomplexans . Red algal parasites traditionally considered adelphoparasites, like Gracilariophila oryzoides and Gardneriella tuberifera, still require a copy of the host plastid for some function that, to date, remain undetermined. Interestingly, in the Gracilariophila oryzoides genome and transcriptome, photosynthesis related nuclear-encoded plastid-targeted genes remain conserved while those same genes are absent from the Choreocolax polysiphoniae transcriptome (Salomaki and Lane, unpublished).
Data presented here further highlight the need to abandon the notion that taxonomy defines alloparasites. By placing the family Choreocolacaceae in synonymy with the Rhodomelaceae we are making steps to remove the artificial appearance of parasites infecting hosts in different families. Furthermore, we recognize that a major distinction between types of red algal parasites is the origin of the parasite plastids.
While some parasites, including Harveyella mirabilis and Choreocolax polysiphoniae, retain a native plastid that evolves in concert with the parasite, others including Gracilariophila oryzoides and Gardneriella tuberifera, incorporate a copy of a host plastid when packaging spores, but will inevitably evolve and become incompatible with the host plastid and blink out of existence. It is predicted that investigations of parasites that have traditionally referred to as alloparasites, like Holmsella and Gelidiocolax, will also provide evidence of plastid retention and monophyletic clades of parasites. Translation; structural constituent of the 50S ribosome rpl17 Translation; structural constituent of the 50S ribosome rpl24 Translation; structural constituent of the 50S ribosome rpl32 Translation; structural constituent of the 50S ribosome rpl33 Translation; structural constituent of the 50S ribosome rpl34 Translation; structural constituent of the 50S ribosome rpoZ Transcription; DNA binding rps18 Translation; structural constituent of the 30S ribosome rps20 Translation; structural constituent of the 30S ribosome ycf21 Uncharacterized hypothetical protein   basepairs and contains 84 protein coding genes (Green), the 5S, 16S, and 23S rRNAs (Red), and 23 tRNAs (Pink). All genes involved with photosynthetic functions, except petF, have been lost. The ftsH gene is truncated but may still be transcribed, however gltB is a non-functional pseudogene (Yellow).

Harveyella mirabilis
Plastid Genome 90,654 bp  The first parasitic red alga was described in 1845 with twenty-one additional species being described before the turn of the century. Today, more than 120 red algal parasites have been described from eight different orders within the Florideophyceae. parasites. Based upon these observations, we propose the terms archaeplastic parasites and neoplastic parasites to distinguish parasitic red algae based on a biological characteristic rather than relying on terms derived from a taxonomic framework.

A History of Red Algal Parasites
The first red algal parasite described was Microcolax botryocarpa (Hooker & Harvey) F. Schmitz in 1845 (Schmitz andFalkenberg 1897, Preuss et al. 2017) and over the next 55 years, another twenty-one red algal parasites were described, largely as a result of work by Reinsch & Schmitz (Reinsch 1875, Schmitz and Falkenberg 1897). Compared to their free-living relatives, parasitic red algae are greatly reduced in size and generally lacking pigmentation. Despite their diminutive nature, parasitic red algae were taxonomically assigned to families based upon morphological features as early as the late 19 th and early 20 th century (Reinsch 1875, Schmitz andFalkenberg 1897). Due to their relative lack of morphological characteristics, the use of molecular data has provided a new lens through which to examine phylogenetic affiliations of parasitic red algae (Chapter 4). The most recent counts identify 121 described species of red algal parasites Lane 2015, Preuss et al. 2017), however molecular data has demonstrated crypsis is common (see independent evolutions of Asterocolax gardneri in Goff et al. 1997, and Chapter 4) and most parasites have not been examined with molecular methods.
Molecular and morphological analyses indicate that multiple independent evolutionary events have given rise to parasites throughout Florideophyceae ). In the early 20 th century, two hypotheses arose to explain the origins of red algal parasites. First, Setchell (1918) noted that approximately 80% of the parasites he was studying infected hosts in the same taxonomic family. Based upon his observations, he proposed that parasites originated as carpospores or tetraspores of their host, which had undergone a mutation causing it to have reduced photosynthetic capabilities (Setchell 1918). Later, Sturch (1926) proposed that rather than evolving sympatrically, parasitic red algae started out as small epiphytes of their hosts that eventually would penetrate cortical cells of the host, becoming an endophyte. Once established as an endophyte, the alga would adopt mechanisms to obtain nutrients from the host, becoming increasingly reliant on its host, solidifying an irreversible path towards parasitism (Sturch 1926  , however the possibility that not all parasites arise through the same mechanisms has also been suggested  and perhaps the Setchell and Sturch hypotheses both hold true. As previously noted (e.g. Setchell 1918), the  recognized a distinction between parasites that were closely related to their hosts and those that infected hosts outside of their family. Based upon their observations, the term adelphoparasite (adelpho-is Greek for 'kin') was applied to those parasites that infect hosts within their same family/tribe and the term alloparasite (allo-is Greek for other) was adopted to describe parasites that infect hosts from other families . These terms were widely accepted as a means of discussing parasites and, to date, the ratio of adelphoparasites to alloparasites (5:1) has remained quite similar to Setchell's initial observations (1918). Goff and colleagues later proposed that all red algal parasites begin as adelphoparasites, and over time diversify and/or adapt to infect new hosts as they develop into alloparasites .

Redefining Red Algal Parasites
Since their adoption by , the terms adelphoand alloparasite have been used to distinguish red algal parasites. Phylogenetic studies have cast doubt on the utility of these terms as data show that even alloparasites predominately infect with the same family . However, differences between these two groups of parasites remain, including their developmental patterns as they infect their hosts, and the origins of organelles. Plastids remain essential for the survival of red algal parasites however the origin of their plastid differs and appears to be intimately linked with other developmental characteristics discussed here. Therefore, we recommend using the term archaeplastic parasite to refer to those parasites that retain a native plastid (formerly alloparasites), and neoplastic parasite (formerly adelphoparasites) to discuss those that hijack a host plastid rather than retain their own copy.
It remains plausible that an archaeplastic parasite will be found infecting a close relative or even a sister species, especially if they originate via sympatric speciation, as Setchell (1918) hypothesized. Rather than distinguishing types of parasites by their evolutionary relationships to their hosts, it seems more meaningful to use biological distinctions between groups that can be easily examined using widely available tools. Many developmental characteristics separate the two groups of parasites including the ability to vegetatively grow between host cells , the location of parasite DNA replication (Goff and Coleman 1984, and in a phylogenetic construct, the ability to successfully speciate (Zuccarello et al. 2004, Chapter 4). However, there are flaws in each of these options including the difficulty to assess parasite vegetative growth and location of DNA replication as well as the need for appropriate comparative data for phylogenetic analyses.

Developmental differences
As technology progressed, new tools became available to investigate the cellular interactions between red algal parasites and their hosts. The association between parasitic red algae and their hosts is facilitated, at least in part, by the ability of red algae to form cell-to-cell fusions between adjacent, non-daughter cells (Sturch 1899, 1926. With modern microscopy, the structure and formation of cell fusions between parasites and their hosts was determined , Goff and Cole 1976a, Wetherbee and Quirk 1982a. Furthermore, these cell-to-cell fusions were proposed to serve as the mechanism by which nutrients are transported from the host to parasite cells . With the use of epifluorescence microscopy, Goff and colleagues went on to establish their importance in the infection process by demonstrating that red algal parasites are able to transfer their nuclei and organelles into host cells via cell fusions (Goff and Coleman 1984. In addition to recognizing the role of cell fusions in the infection process, sophisticated microscopy advanced our understanding of how parasitic red algae spread throughout their hosts and characterized the host responses. In a groundbreaking series of manuscripts, Goff and Cole described in great detail the biology of the archaeplastic parasite Harveyella mirabilis (Reinsch) F. Schmitz et Reinke, including its development, structure, and nutrient acquisition from its host Odonthalia flocossa (Esper) Falkenberg (Goff and Cole 1973, 1976a, 1976b, 1979a, 1979b. These investigations provided a framework to more intimately understand the development and interactions of a range of red algal parasites.
Aside from the formation of cell fusions that initiate host infection, important differences have been observed between neoplastic parasites and archaeplastic parasites regarding their developmental patterns subsequent spread throughout their hosts (reviewed in Lane 2014, Freese andLane 2017). First, archaeplastic parasites including Choreocolax, Harveyella, and Holmsella spread throughout their hosts by producing mitotically dividing rhizoidal filaments that grow between host cells (Sturch 1899, 1926, Goff and Cole 1976a. In contrast, the neoplastic parasites Gracilariophila oryzoides, Gardneriella tuberifera, and Janczewskia gardneri infect their hosts directly and spread from hosts cell to host cell rarely creating their own rhizoidal filaments Coleman 1987a, Goff and. Additionally, while neoplastic parasites appear to transform infected host cells and undergo nuclear replication solely within host cells , archaeplastic parasites appear to only undergo DNA replication in their rhizoidal filaments and infected host cells maintain a 1:1 ratio of parasite nuclei to cell fusions (Goff and Coleman 1984. These key developmental differences have furthered support for distinguishing types of parasites. However, the terms adelpho-and alloparasite, which specifically refer to taxonomic relatedness to their hosts, does not reflect the differing biology of the organisms.

Organellar Origins
The combination of microscopy with molecular tools has substantially advanced our understanding of red algal parasites and their interactions with their hosts. In a study investigating the origins of organelles from three neoplastic parasites, Gracilariophila oryzoides, Gardneriella tuberifera, and Plocamiocolax pulvinata,  demonstrated that the parasites retain their own mitochondria, however all three had lost their native plastid and instead incorporate a dedifferentiated host plastid when packaging their spores. Molecular investigations including deep genomic sequencing, have confirmed that the neoplastic parasites Faucheocolax attenuata, Gracilariophila oryzoides, Gardneriella tuberifera, Janczewskia gardneri, and Plocamiocolax pulvinata do not retain a native plastid (Goff and Coleman 1995, Salomaki and Lane, unpublished).
Recently, plastid genomes have been completely sequenced from the archaeplastic parasites Choreocolax polysiphoniae and Harveyella mirabilis. Both plastids are greatly reduced in coding capacity compared to the plastids of free-living photosynthetic red algae, having lost all genes related to photosynthesis (Salomaki et al. 2015, Chapter 4). Interestingly, genes involved in amino acid and fatty acid biosynthesis, iron-sulfur cluster synthesis, as well as transcription and translation are conserved (Salomaki et al. 2015, Chapter 4). Aside from the loss of photosynthesis genes, the H. mirabilis plastid retains a high level of synteny with plastids of closely related free-living red algae, however the C. polysiphoniae plastid has experienced extensive genome reorganization (Figure 1). Furthermore, the discovery that some parasites retain functionally reduced plastids, while others rely on a host-derived plastid, provides another physical characteristic for differentiating between two types of parasites rather than relying on evolutionary relationships.
Phylogenetic investigations have definitively demonstrated that archaeplastic parasites rarely infect hosts in a different family or tribe . A recent analysis recognizes that the archaeplastic parasites C.
polysiphoniae, H. mirabilis, and two species of Leachiella, form a monophyletic clade in which all species retain their native plastid (Chapter 4). Plastid origin remains a remarkable difference between types of parasites and is easy to investigate with molecular tools these days. Furthermore, plastid origin is a meaningful biological characteristic that can distinguish between types of red algal parasites regardless of relationship to their host. In light of these results, the terms alloparasite and adelphoparasite, as originally described by , create more confusion than they allay .
Aside from the previously discussed developmental differences including the location of parasite DNA replication and mechanism of spreading throughout the host, plastid origin appears to be central to differences observed in red algal parasite evolution. One hypothesis is that the observed developmental differences between the two groups of parasites are linked to disparities in their plastid origin. Parasites that maintain a native plastid are in essence, a complete red alga in their own right, whereas those that incorporate a host derived plastid are borrowing parts of another organism they remain compatible with. The retention of native organelles means the plastid remains under the same selective pressures as the rest of the parasite genome, and therefore, retains its ability to function for amino acid, fatty acid, protein, and isoprene biosynthesis while losing genes for photosynthesis. Those parasites that retain their own plastid are capable of evolutionary processes typical of other formerly photosynthetic parasites, such as Apicomplexans. A native plastid would therefore enable those parasites maintain control over organellar evolution and presumably be more successful adapting to new hosts and speciating to form monophyletic clades.
This hypothesis can be tested in future studies by investigating the organellar origins of parasites in the genus Holmsella.
Holmsella pachyderma was previously placed in the Choreocolacaceae based upon its developmental patterns within its host, until 1990 when it was transferred to the Gracilariaceae along with Holmsella australis, based upon morphological characteristics . A subsequent phylogenetic analysis including H. australis and H. pachyderma found the species to form a monophyletic clade within the Gracilariaceae ). To date no work has been completed on the plastid origin of members of the genus Holmsella, however if they do retain a native plastid, Holmsella would represent a second successful evolution of parasites retaining a native plastid that forms mitotically dividing rhizoidal filaments as a means of vegetative growth throughout their hosts, and forms a monophyletic clade of parasites. Further investigation is warranted to determine the taxonomic affiliation of parasite genera Gelidiocolax and Pterocladiophila, which have also been placed within the Gracilariaceae based on morphological characteristics.
The origin of parasites that have lost their native plastid and instead hijack a plastid from their host, though well studied, remains uncertain. The ability of florideophycean algae to easily form cellular fusions with adjacent cells would enable the spore with a non-functional plastid to germinate and utilize genetically similar plastids. By exploiting a host plastid, the newly evolved parasite is rescued from what otherwise would have been a lethal mutation. The parasite nucleus and mitochondrion could continue spreading from cell-to-cell of its newly acquired host via cell fusions, transforming the host cells as described in detail by Goff and colleagues . While providing a means of short-term survival for the newly transitioned parasite, co-opting a host plastid as their own would also establish these parasites as evolutionary dead ends. All red algae rely on a plastid for essential functions beyond photosynthesis including amino acid and fatty acid biosynthesis. By retaining a new plastid from its host in each new infection cycle, the parasite looses control of organelle evolution. While the parasite nucleus and mitochondrion will remain under a unique selective pressures, the host and host plastid will be experiencing different selective pressures. Over time this will render the parasite incapable of communicating with the host plastids, rendering it unable to grow and reproduce in a diverging host.
In addition to the organelles themselves, nuclear-encoded, organellar-targeted, genes are also the subjects of ongoing investigations in the evolution of parasitism in red algae. Comparative analyses of the transcriptomes from the neoplastic parasite, Gracilariophila oryzoides and the archaeplastic parasite Choreocolax polysiphoniae, revealed distinct differences in the expression of nuclear-encoded plastid-targeted photosynthesis genes involved in the carotenoid biosynthesis pathway. This research indicates that all genes with the exception of geranylgeranyl phosphate synthase (GGPS) are not being transcribed in the C. polysiphoniae transcriptome, while genes in the carotenoid biosynthesis pathway are transcribed by G. oryzoides (Figure 2) (Salomaki & Lane, unpublished). It is reasonable that C. polysiphoniae would no longer maintain selective pressure on photosynthesis related genes since it retains a plastid that is no longer capable of photosynthesizing. However, the finding that G.
oryzoides not only retains functional copies, but also is expressing plastid-targeted genes involved in the carotenoid biosynthesis pathway suggests that selection is acting to conserve these genes. Their expression indicates the proteins are being targeted to the host-derived plastid as a means of controlling and utilizing the host plastid as its own. Analyses remain ongoing to determine the nature and extent of nuclear-encoded, plastid-targeted, genes in both parasites, and this research direction appear to hold promise in further explaining the interactions between red algal parasites and their hosts.