1 2 DR. MAREN PREUSS (Orcid ID : 0000-0002-8147-5643) 3 DR. HEROEN VERBRUGGEN (Orcid ID : 0000-0002-6305-4749) 4 DR. GIUSEPPE C. ZUCCARELLO (Orcid ID : 0000-0003-0028-7227) 5 6 7 Article type : Regular Article 8 9 10 The organelle genomes in the photosynthetic red algal parasite Pterocladiophila 11 hemisphaerica (Florideophyceae, Rhodophyta) have elevated substitution rates and 12 extreme gene loss in the plastid genome 1 13 14 15 Maren Preuss2 16 School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, 17 6140, New Zealand 18 19 Heroen Verbruggen 20 School of BioSciences, University of Melbourne, Parkville, VIC 3010, Australia 21 and 22 Giuseppe C. Zuccarello 23 School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, 24 6140, New Zealand 25 26 27 Author Manuscript 28 Running title: Pterocladiophila organelle genomes 29 This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/JPY.12996-20-002 This article is protected by copyright. All rights reserved 30 1 Received Accepted ___________ 31 2 Corresponding author: [email protected] 32 33 34 Editorial Responsibility: M. Coleman (Associate Editor) 35 36 37 ABSTRACT 38 Comparative organelle genome studies of parasites can highlight genetic changes that occur 39 during the transition from a free-living to a parasitic state. Our study focuses on a poorly 40 studied group of red algal parasites, which are often closely related to their red algal hosts and 41 from which they presumably evolved. Most of these parasites are pigmented and some show 42 photosynthetic capacity. Here, we assembled and annotate the complete organelle genomes of 43 the photosynthetic red algal parasite, Pterocladiophila hemisphaerica. The plastid genome is 44 the smallest known red algal plastid genome at 68,701 bp. The plastid genome has many 45 genes missing, including all photosynthesis-related genes. In contrast, the mitochondrial 46 genome is similar in architecture to that of other free-living red algae. Both organelle 47 genomes show elevated mutation rates and significant changes in patterns of selection, 48 measured as dN/dS ratios. This caused phylogenetic analyses, even of multiple aligned 49 proteins, to be unresolved or give contradictory relationships. Full plastid datasets 50 interfered by selected best gene evolution models showed the supported relationship of P. 51 hemisphaerica within the Ceramiales, but the parasite was grouped with support as sister to 52 the Gracilariales when interfered under the GHOST model. Nuclear rDNA showed a 53 supported grouping of the parasite within a clade containing several red algal orders including 54 the Gelidiales. This photosynthetic parasite which is unable to photosynthesize with its own 55 plastid, due to the total loss of all photosynthesis genes, raises intriguing questions on 56 parasite-host organelle genome capabilities and interactions. 57 58 Key index words: dN/dS; General Heterogenous Evolution On a Single Topology (GHOST) Author Manuscript 59 model; heterotachy; mitochondrial genome; parasitism; reduced plastid genome; positive 60 selection; relaxed selection 61 62 Abbreviations: CTAB, cetyl trimethylammonium bromide THIS ARTICLE IS PROTECTED BY COPYRIGHT. ALL RIGHTS RESERVED 63 INTRODUCTION 64 Organelle (mitochondrial and plastid) genomes in parasites are useful to understand 65 evolutionary changes occurring during the transition from free-living to parasitic modes of 66 life. Comparative plastid genome studies in plants showed extensive gene loss after loss of 67 photosynthesis (Wicke et al. 2013, Barrett et al. 2018, Schneider et al. 2018) and no clear 68 convergent gene loss patterns between parasitic taxa (Wicke and Naumann 2018). Generally, 69 genes loss is correlated with a decrease in genome size and an increase in AT content 70 (Delannoy et al. 2011, Wicke et al. 2013, Petersen et al. 2015, Cusimano and Wicke 2016, Su 71 et al. 2019). In contrast, changes in mitochondrial genome architecture in parasites in 72 comparison to free-living taxa are less clear (Burger et al. 2003), as the size of mitochondrial 73 genomes in free-living taxa can vary greatly (Sloan et al. 2012, Petersen et al. 2015, Su et al. 74 2019). Comparative mitochondrial studies indicate that parasite mitochondrial genomes have 75 rather conserved architecture compared to their free-living relatives (Hancock et al. 2010, Fan 76 et al. 2016). Few studies investigate and characterize mitochondrial and plastid genomes 77 together and similarities between these organelle genomes in gene loss and selection might be 78 overlooked. 79 One ideal group to study these organelle evolution patterns in parasites are red algal 80 parasites, which are red algae parasitic on other red algae. These parasites are highly diverse, 81 with over 123 species (Preuss et al. 2017, Preuss and Zuccarello 2017, 2019a) and many 82 independent transitions to parasitism (Goff et al. 1996, Blouin and Lane 2016, Preuss et al. 83 2017). A majority of these parasites are at least partially pigmented (Preuss et al. 2017) and at 84 least one species (Pterocladiophila hemisphaerica) is able to photosynthesize independently 85 when removed from its host (Preuss and Zuccarello 2019b). While parasitic red algae are 86 diverse, they are poorly studied. 87 Red algal parasites mostly infect members of the same family (Goff et al. 1996, Preuss 88 and Zuccarello 2014, 2017) or occasionally different families within the same order 89 (Zuccarello et al. 2004). The close relationships between many red algal parasites and their 90 hosts led to the proposition that red algal parasites evolved from their host (Setchell 1918, 91 Goff et al. 1997). Phylogenetic analyses demonstrated that some parasites and their host are 92 more closely related to each other than to other species in the same host genus (Goff et al. 93 1997, Zuccarello etAuthor Manuscript al. 2004, Preuss and Zuccarello 2017), whereas other parasites are more 94 distantly related to their host species, possibly due to host switching (Kurihara et al. 2010, 95 Preuss and Zuccarello 2014, 2017). THIS ARTICLE IS PROTECTED BY COPYRIGHT. ALL RIGHTS RESERVED 96 In addition, red algal parasites exhibit a unique organelle transfer mechanism of 97 infection by forming a connection between a parasite and a host cell, called a secondary pit 98 connection (Goff and Coleman 1984, 1985), leading to cells containing cellular components 99 of both species (“heterokaryons”) within the host and parasite thallus. Some studies have 100 suggested that the heterokaryon, in some cases, transformed into a parasite cell, reduces host 101 nuclei but keeps host plastids (Goff and Zuccarello 1994, Goff and Coleman 1995). Newly 102 formed parasite cells would then produce reproductive structures that contained parasite 103 nuclei but host plastids (Goff and Coleman 1995). While earlier evidence suggested that 104 parasites only contained host plastids (Goff and Coleman 1995, Zuccarello et al. 2004); a 105 reduced plastid (i.e., lacking all photosynthetic genes) was found in the unpigmented parasites 106 Choreocolax polysiphoniae (Salomaki et al. 2015) and Harveyella mirabilis (Salomaki and 107 Lane 2019). Currently, it is unknown if a similar highly reduced plastid is present in other red 108 algal parasites, especially ones that are pigmented. 109 For this study, we choose the pigmented red algal parasite Pterocladiophila 110 hemisphaerica from New Zealand. This parasite is able to photosynthesize independently 111 when removed from its host Pterocladia lucida (Gelidiales; Preuss and Zuccarello 2019b), 112 and its taxonomic position in the order Gracilariales is still based solely on morphological 113 similarities with two other red algal parasite genera, Holmsella and Gelidiocolax (Fredericq 114 and Hommersand 1990) placed in this order. Our general aims of this study are to: (i) 115 compare organelle genome architecture and characteristics between the parasite P. 116 hemisphaerica and its host species, (ii) analyze selection patterns on individual genes of 117 organelle genes in this parasite, and (iii) investigate the phylogenetic placement of this 118 parasite. 119 120 MATERIALS AND METHODS 121 Algal specimens, DNA extraction and partial gene sequencing. 122 Red algal specimens of Pterocladia lucida and its parasite Pterocladiophila hemisphaerica 123 were collected from shore (drift) at Akitio Beach (40°37'25'' S, 176°24'39'' E) in November 124 2011 and Kairakau Beach (39°56'30'' S, 176°55'50'' E) in May 2013, or by SCUBA in August 125 2016 at Princess Bay, Wellington, New Zealand (41°20'46'' S, 174°47'26'' E). Drift specimens 126 were dried in silicaAuthor Manuscript gel and used to amplify single genes, whereas scuba collections were 127 freshly ground in liquid nitrogen and used for High-Throughput-Sequencing (HTS). 128 Parasite pustule was held with forceps and cut off with a razor blade at the base with 129 as much distance from the host-parasite contact area as possible. Some pustules were THIS ARTICLE IS PROTECTED BY COPYRIGHT. ALL RIGHTS RESERVED 130 cystocarpic but spores were not released in culture. All samples were extracted using a 131 modified CTAB protocol (Zuccarello and Lokhorst 2005). Extracted DNA of drift specimens 132 were used to amplify partial cox1, LSU and SSU rDNA following established PCR 133 conditions, purification and sequencing (Preuss and Zuccarello 2017). Cox1 sequences were 134 used to identify the clade of Pterocladia host in New Zealand, as three cryptic species exist 135 (Boo et al. 2016). 136 137 Sequencing, assembly, and annotation of organelle genomes. 138 Library preparation and sequencing for the parasite Pterocladiophila hemisphaerica (n=~100) 139 and one uninfected specimen of Pterocladia lucida (host, n=1) of fresh material from Princess 140 Bay were performed separately using Illumina TruSeq DNA nano by Macrogen Inc. (Seoul, 141 Korea).