The all-rounder sodalis: A new bacteriome-associated endosymbiont of the lygaeoid bug henestaris halophilus (heteroptera: Henestarinae) and a critical examination of its evolution

The All-Rounder Sodalis: A New Bacteriome-Associated Endosymbiont of the Lygaeoid Bug Henestaris halophilus (Heteroptera: Henestarinae) and a Critical Examination of Its Evolution Diego Santos-Garcia 1, *, Francisco J. Silva 2,3 , Shai Morin 1 , Konrad Dettner 4 , and Stefan Martin Kuechler 4, * 1 Department of Entomology, The Hebrew University of Jerusalem, Rehovot, Israel 2 Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de Vale`ncia, Spain 3 Institute for Integrative Systems Biology (I2SysBio), Universitat de Vale`ncia-CSIC, Spain 4 Department of Animal Ecology II, University of Bayreuth, Germany *Corresponding authors: E-mail: diego.sa[email protected].il; [email protected]. Accepted: September 25, 2017 Data deposition: This project has been deposited at ENA of EMBL-EBI under the accession number PRJEB12882. Abstract Hemipteran insects are well-known in their ability to establish symbiotic relationships with bacteria. Among them, heteropteran insects present an array of symbiotic systems, ranging from the most common gut crypt symbiosis to the more restricted bacteriome-associated endosymbiosis, which have only been detected in members of the superfamily Lygaeoidea and the family Cimicidae so far. Genomic data of heteropteran endosymbionts are scarce and have merely been analyzed from the Wolbachia endosymbiont in bed bug and a few gut crypt-associated symbionts in pentatomoid bugs. In this study, we present the first detailed genomic analysis of a bacteriome-associated endosymbiont of a phytophagous heteropteran, present in the seed bug Henestaris halophilus (Hemiptera: Heteroptera: Lygaeoidea). Using phylogenomics and genomics approaches, we have assigned the newly characterized endosymbiont to the Sodalis genus, named as Candidatus Sodalis baculum sp. nov. strain kilmister. In addition, our findings support the reunification of the Sodalis genus, currently divided into six different genera. We have also conducted comparative analyses between 15 Sodalis species that present different genome sizes and symbiotic relationships. These analyses suggest that Ca. Sodalis baculum is a mutualistic endosymbiont capable of supplying the amino acids tyrosine, lysine, and some cofactors to its host. It has a small genome with pseudogenes but no mobile elements, which indicates middle-stage reductive evolution. Most of the genes in Ca. Sodalis baculum are likely to be evolving under purifying selection with several signals pointing to the retention of the lysine/tyrosine biosynthetic pathways compared with other Sodalis. Key words: Lygaeoidea, endosymbiosis, taxonomy, comparative genomics, metabolism, molecular evolution. Introduction Most insects have established specific associations with bacterial symbionts. These associations show a broad range of symbiotic interactions, ranging from parasitism to mutualism. Bacterial symbionts can be found on the surface of the insects but also inside their bodies (e.g. the gut system). Often, mutualistic symbionts and insects establish a more intimate relationship, where the symbionts are maintained inside specialized host cells, called bacteriocytes, that can form an organ-like termed bacteriome (Buchner 1965). These intracellular symbionts (hereafter endosymbionts) are usually defined as primary, or obligate, if the insect requires the symbiotic relationship for survival, and secondary, or facultative, if the relationship is not essential for its survival. However, in some cases, a secondary endosymbiont can act as a coprimary one, if its presence is also essential for the insect or the primary endosymbiont (Sudakaran et al. 2017). Although different bacterial lineages are capable of establishing a stable ßThe Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permis[email protected] Genome Biol. Evol. 9(10):2893–2910. doi:10.1093/gbe/evx202 Advance Access publication September 25, 2017 2893 GBE Downloaded from https://academic.oup.com/gbe/article/9/10/2893/4237403 by Consejo Superior de Investigaciones Cientificas (CSIC) user on 01 December 2023 endosymbiotic relationship with insects, representatives of the Bacteroidetes as well as Alpha-, Beta-, and Gammaproteobacteria, especially Enterobacteriaceae,are the most prominent groups (Moya et al. 2008;Moran et al. 2008;Sabree et al. 2009;Husn ık et al. 2011;Sudakaran et al. 2017). Among others, species of the Sodalis group (Gammaproteobacteria:Enterobacterales:Pectobacteriaceae) offer a spectrum of various types of endosymbiosis. The eponymous strain was originally described as a secondary endosymbiont of the tsetse fly Glossina morsitans (Dale and Maudlin 1999). Because then, numerous different Sodalis or Sodalis-allied species were found in several insect groups, such as weevils (Heddi et al. 1999;Oakeson et al. 2014), hippoboscid louse fly (Nov akov aandHyp  sa 2007; Chrudimsk y et al. 2012), chewing lice (Fukatsu et al. 2007; Smith et al. 2013)andseallice(Boyd et al. 2016). In addition, hemipteran insects such as aphids (Burke et al. 2009), psyllids (Sloan and Moran 2012;Arp et al. 2014), scale insects (Gruwell et al. 2010;Husn ık and McCutcheon 2016), spittlebugs (Koga and Moran 2014), and stinkbugs (Kaiwa et al. 2010,2011;Matsuura et al. 2014;Hosokawa et al. 2015) frequently harbor Sodalis endosymbionts. Recently, a Sodalis-allied bacterial strain was also isolated from a human wound infection (Clayton et al. 2012), possibly representing a free-living ancestral state of Sodalis. This Sodalis, named Sodalis praecaptivus, and the one from G. morsitans are the only species cultivable so far. Based on their pattern of occurrence in different ecological niches and insects, the “characteristics” of each Sodalis species and their specific effects on their hosts are quite diverse. For example, Sodalis species are often described as facultative endosymbionts, but have also been found in insect bacteriocytes, showing strict mutualistic obligatory relationship with their weevil hosts (Oakeson et al. 2014), or as copartners, complementing missing metabolic functions of an obligatory endosymbiont in the Carsonella-psyllid system (Sloan and Moran 2012). This illustrates that representatives of the genus Sodalis, or allied bacteria, cover a broad spectrum, ranging from free-living species, over facultative commensals to obligate mutualists of insects. The phylogeny and taxonomy of Sodalis-allied symbionts, mainly derived from analyses of their 16S rRNA and few other gene sequences, present several inconsistencies produced by events of horizontal transmission and new hosts acquisition (Dale et al. 2001;Snyder et al. 2011;Smith et al. 2013). Numerous primary and secondary endosymbiotic bacteria, and hosts’ structures that harbor them, were described in stinkbugs or true bugs (Heteroptera) (Buchner 1965). Sodalis-allied endosymbionts were also detected in some members, for the first time in the superfamily Pentatomoidea (Heteroptera: Pentatomomorpha), more specifically in the families Acanthosomatidae, Pentatomidae, Scutelleridae, and Urostylididae (Kaiwa et al. 2010,2011, 2014;Matsuura et al. 2014;Hosokawa et al. 2015). It is generally argued that Sodalis endosymbionts do not play an essential role in the biology of most of their heteropteran hosts, although such functions could not be completely excluded in urostylidid stinkbugs, due to the high infection rates in these species (Kaiwa et al. 2014;Hosokawa et al. 2015). Until present, no Sodalis symbiont has been found in the superfamily Lygaeoidea (reviewed in Sudakaran et al. 2017). The reason for this is not clear, because most lygaeoid bugs also harbor a broad range of endosymbiotic bacteria accommodated in specific structures, ranging from midgut crypts to bacteriomes, depending on the (sub)families (Kuechler et al. 2010, 2011, 2012;Kikuchi et al. 2011;Matsuura et al. 2012). One of these bacteriome-associated endosymbiosis was also described in Henestaris halophilus, a member of the lygaeoid subfamily Henestarinae (Heteroptera: Lygaeoidea: Geocoridae), but has not been analyzed in detail so far (Kuechler et al. 2012). The subfamily Henestarinae is mainly distributed in southern Palearctic and African regions and contains about 19 species placed in 3 genera (Schuh and Slater 1995). All species, mainly characterized by their stalked eyes, live in saline-affected habitats both inshore and inland. The genus Henestaris is phytophagous and H. halophilus mainly feeds on seeds and infructescence of halophytes, like Plantago maritima,Artemisia maritima,Aster trifolium or Atriplex spp., (Wachmann et al. 2007), but occasionally also on grasses, especially Puccinella distans (Hiebsch 1961). In the present work, we provide the first detailed description of the bacteriome-associated endosymbiont of H. halophilus, identified as a member of the Sodalis group, including molecular characterization, ultrastructural morphology and localization and transmission route. We also present the endosymbiont’s complete genomic sequence which is characterized by a reduced genome size and a very low coding density. Our metabolic reconstruction analysis suggests that the main contribution of the endosymbiont to its insect host involves processes related to cuticle hardening and the production of vitamins. Finally, several Sodalis-allied species were compared at both the metabolic and sequence levels, and the taxonomic status of the whole Sodalis group was revisited. Materials and Methods Insect Material Adults and larval stages of Henestaris halophilus were collected from their natural habitat in Talamone (Italy) and Su¨ lldorf (Saxony-Anhalt, Germany). Live individuals were brought to the laboratory and maintained at 25 Cunder long day conditions (16:8 h) on sunflower seeds and distilled water enriched with 0.05% ascorbic acid. Laid eggs were carefully collected and allowed to develop at 25 C. Developing eggs were extracted for fixation (eggs were dissected in 90% [vol/vol] ethanol to remove chorion and Santos-Garcia et al. GBE 2894 Genome Biol. Evol. 9(10):2893–2910 doi:10.1093/gbe/evx202 Advance Access publication September 25, 2017 Downloaded from https://academic.oup.com/gbe/article/9/10/2893/4237403 by Consejo Superior de Investigaciones Cientificas (CSIC) user on 01 December 2023 vitelline membrane) followed by whole mount fluorescence in situ hybridization (wFISH) analysis. Insect bacteriomes and ovaries were dissected in Ringer’s solution (8.0 g NaCl, 0.4 g KCl, 0.4 g CaCl 2 , and 1.0 g Hepes per liter, pH 7.2). Microscopy Analysis For wFISH, freshly dissected bacteriomes, ovaries, and embryos were incubated overnight at room temperature in Carnoy’s solution (ethanol:chloroform:acetate, 6:3:1) and then washed in an ascending ethanol series (70%, 90%, and 2100%). After washing, the fixed samples were stored at 20 C until use. Afterwards, all samples were washed with PBSTw [PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 ,1.5mMKH 2 PO 4 , pH 7.4) containing 0.3% Tween 20] three times for 10 min. After thorough washing, the samples were equilibrated with hybridization buffer [30% (vol/vol) formamide, 0.02 M Tris–HCl (pH 8.0), 0.9 M NaCl, 0.01% SDS] three times for 10 min, followed by overnight incubation at 28 C in hybridization buffer containing 1% of 10 nmol/ml symbiont specific probe Hen500 (5’-Cy3CCATTGTCTTCTTCTCCGCC-3’) and helper probe Hen500_H1 (5’-GAAAGTGCTTTACAACCCTAAGG-3’). Next, the samples were incubated for 20 min at 42 Cin hybridization buffer without probe. The samples were washed again with PBSTw three times for 15 min, and then incubated with 1% (vol/vol) SYBR Green I (1:10,000). The staining was stopped by washing in PBSTw. At the final step, the samples were mounted onto glass slides using antifade solution (citifluor) and glycerol (1:1) containing medium. The samples were examined under an SP5 confocal laserscanning microscope (Leica). Electron microscopy was performed as described by Kuechler et al. (2012). DNA Extraction, Sequencing, and Genome Annotation A pool of bacteriomes dissected from 25 females was utilized for total genomic DNA extraction using the PureLink Genomic DNA Mini Kit (Invitrogen). Six independent whole-genome amplification reactions (GenomiPhi v2, GE Healthcare) were performed following manufacturer instructions. Because chimera formation seems to be a random process, samples were mixed to maintain possible chimeras at a low ratio relative to the amplified nonchimeric DNA. Amplified DNA was used for sequencing by the Illumina HiSeq2000 (350-bp paired-end library and 2100 bp) platform at Macrogen, Inc. Genome assembly and annotation procedures are presented in Supplementary Material online. Genome and Metabolism Comparisons Several Sodalis genomes and allied-species genomes were downloaded from NCBI and other sources (see table 1). Pantoea ananatis LMG 5342 (NC_016816) was used as an outgroup to allow topology comparisons (Husn ık and McCutcheon 2016). The proteomes of the above species were used as input for OrthoMCL v2.0.9 (1.5 inflation value) using USEARCH v9.1 (ublast -id 0.5 -maxhits 10,000 -acceptall -evalue 1e5 -accel 1 -weak_evalue 0.1) (Li et al. 2003; Edgar 2010). The orthologous clusters of proteins (hereafter OCPs) output from OrthoMCL (supplementary files: phylogenomics) was used to calculate the number of clusters composing the core genome, pangenome, pairwise shared clusters and strain specific clusters in Python (supplementary table S1, Supplementary Material online). Cluster of Orthologous (COG) and KEGG groups were assigned to each species using DIAMOND v0.8 (e-value 1e5, Buchfink et al. 2015) and MEGAN6 Community Edition (Huson et al. 2016) using the RefSeq database (accessed: July 5, 2016) clustered at 98% identity with CD-HIT v4.6 (Fu et al. 2012). Pathway tools v19 (Karp et al. 2002) was used to reconstruct, and compare, the metabolism of each Sodalis endosymbiont (supplementary files: pathway-tools-databases). TyrA Protein Analysis Tridimensional structure plays a crucial role in protein activity. To predict if TyrA protein of Henestaris endosymbiont is likely to be still functional, its tridimensional (tertiary) structure was modeled with the I-TASSER server (Yang et al. 2015). Putative dimerization (quaternary structure) of TyrA was modeledwiththeCOTHserver(Mukherjee and Zhang 2011). Pdb files were viewed, aligned, and compared with UCSF Chimera v1.11.2 (Pettersen et al. 2004) (supplemen tary files: tyrA_analysis). Phylogenomic Analysis A core set of 153 single copy proteins were codon-aligned with a Perl wrapper using MAFFT v7.215 (Katoh et al. 2002), Transeq (EMBOSS: 6.6.0.0, Rice et al. 2000), PAL2NAL v14 (Suyama et al. 2006), and Gblocks v0.91b (codon data with no gaps allowed) (Castresana 2000). Alignments with more than 70% of the columns present in all the species were selected and screened for the saturation of the phylogenetic signal with a custom R script (R Core Team 2016). Briefly, saturation was measured using the correlation coefficient between raw genetic distances and the corrected distances (K80) (supplementary files: phylogenomics). Only protein alignments that showed a coefficient greater than 0.7 at the codon-level were selected for further analysis (77 proteins). Maximum-Likelihood (ML) phylogenetic tree reconstruction was performed on IQ-TREE v1.5.5 (Nguyen et al. 2015) using ModelTest (Kalyaanamoorthy et al. 2017) with seven partition schemes: 1) a single partition (concatenated alignment), 2) fully partitioned (each protein as a partition) with each partition having its own evolutionary model, 3) as 2) with different branch lengths, 4) as 2) but allowing partition mixing, 5) as 3) but allowing partition mixing, 6) a single partition A New Bacteriome-Associated Endosymbiont of the Lygaeoid Bug Henestaris halophilus GBE Genome Biol. Evol. 9(10):2893–2910 doi:10.1093/gbe/evx202 Advance Access publication September 25, 2017 2895 Downloaded from https://academic.oup.com/gbe/article/9/10/2893/4237403 by Consejo Superior de Investigaciones Cientificas (CSIC) user on 01 December 2023 with JTT þCAT20 (profile mixture models), and 7) partitions obtained in 2) but with CAT20. In addition, a Bayesian posterior consensus tree was inferred with MrBayes v3.2.2 (4 chains, 2,000,000 generations, 1,000 sampling frequency, 1,000 burn-in) (Ronquist et al. 2012). The standard deviation of split frequencies was below 0.01 in the four chains and their convergence was checked with Tracer v1.6. The approximately unbiased (AU) test (Shimodaira 2002) implemented in IQ-TREE was used to select the best possible tree under three partitions model: a single partition, full partitioned and partitioned with a mixing strategy. The selected tree was plotted with Figtree v1.4.3 and modified with InkScape v0.92. Averaged Nucleotide Identity/Averaged Amino Acid Identity Values Calculation The aforementioned genomes (table 1) plus some phylogenetically related genomes, including some endosymbionts, were downloaded (Dickeya,Pantoea,Serratia,Brenneria, Pectobacterium,Erwinia,Wigglesworthia,andBlochmannia; see supplementary table S2, Supplementary Material online) were used for calculating the averaged nucleotide identity (ANI) and averaged amino acid identity (AAI). Some Wolbachia strains were used as representatives of a non Gammaproteobacteria endosymbiont genus. ANI values were calculated with JSpecies v1.2.1 (Richter and Rossell oM ora 2009). AAI values were obtained with the enveomics toolbox using USEARCH v9.1 (ublast -id 0.1 -maxhits 1,000 - acceptall -evalue 1e5 -accel 1) as alignment algorithm (Edgar 2010;Rodriguez-R and Konstantinidis 2016). Heatmaps and hierarchical clustering (Euclidean distances and complete clustering) were produced with the gplots package from R (R Core Team 2016). Molecular Evolution within the Sodalis Genus Codeml from PAML v4.7 package (Yang 2007)wasusedto calculate dS, dN, and their omega ratio (x) values in the different OCPs. Divergence times between different Sodalis species were standardized using a triplet approach, which utilized the species of interest, one reference Sodalis (S. glossinidius or S. praecaptivus) from the opposite branch of the species selected (see fig. 4 for more details) and Pantoea ananatis as an outgroup. This set-up allowed us to fix the time, in the common branch, from P. ananatis to the Sodalis last common ancestor, making the time because divergence of the Sodalis species equal (e.g. S. melophagi—S. glossinidius— P. ananatis or Sodalis of Heteropsylla cubana—S. praecaptivus—P. ananatis). For each orthologous group in each triplet, three branch models were computed: m0 (one x), m1 (free xratios in each branch) and m2 (two xsetting the species of interest the foreground branch). Each model was computed three times and the iteration with the greater likelihood was Table 1 Genome Features of Several Sodalis Symbionts Ordered by Genome Size Organism Host Short Name Accession Contigs Genome (Mb) GC (%) CDSjwCDS (%) rRNAsj tRNAsj ncRNAs Ca. Mikella endobia Mealybug MiEn LN999831 1 0.35 30.6 273j7 75, 5 3j41j6 Ca. Moranella endobia PCVAL Mealybug MoEn NC_021057 1 0.54 43.5 411j15 76, 2 5j41j1 Ca. Moranella endobia PCIT Mealybug MoPC CP002243 1 0.54 43.5 406j29 77 5j41j0 Ca. Hoaglandella endobia Mealybug HoEn LN999835 1þ20.64 c 42.8 517j16 80, 4 3j41j10 Ca. Doolittlea endobia Mealybug DoEn LN999833 1þ10.85 c 44.2 568j99 59, 8 3j41j11 Ca. Gullanella endobia Mealybug GuEn LN999832 1 0.94 28.9 461j29 48, 1 3j39j7 S-endosymbiont of Heteropsylla cubana Psyllid SoHc NC_018420 1 1.12 28.9 532j19 47, 3 3j38j2 Sodalis-like symbiont of Philaneus spumarius PSPU Froghopper SoPS b BASS01000000 562 1.38 54.1 1400jNA NA 4j39j44 S-endosymbiont of Ctenarytaina eucalypti Psyllid SoCe NC_018419 1 1.44 43.3 758j21 47, 9 3j40j2 P-endosymbiont of Henestaris halophilus True bug SoBa a PRJEB12882 1 1.62 44.5 713j166 37, 3 3j42j10 Sodalis-like endosymbiont of Proechinophthirus fluctus str. SPI-1 Seal louse SoPf LECR01000000 92 2.18 50 695j683 NA 16j40j2 Sodalis glossinidius str. “morsitans” Tsetse fly SoGl NC_007712-15 1þ34.29 c 54.7 3177j1280 52, 9 22j72j1 Ca. Sodalis pierantonius str. SOPE Weevil SOPE CP006568 1 4.51 56 2309j1771 46, 2 9j55j3 Ca. Sodalis melophagi Hippoboscid louse fly SoMe b http://users.prf.jcu. cz/novake01/ d 236 4.57 50.8 4545jNA NA NA Sodalis praecaptivus Human wound SoHS NZ_CP006569-70 1 4.16 c 57.5 4429j25 81 23j76j1 a The acronym refers to the proposed name Ca. Sodalis baculum sp. nov. strain kilmister see below. It is introduced here to have a consistent abbreviationineachpart. b No annotation available, annotation was done using prokkav1.12 with default parameters plus gram negative and metagenome options (Seemann et al. 2014). c Including plasmids. d Last accessed September 29, 2017. Santos-Garcia et al. GBE 2896 Genome Biol. Evol. 9(10):2893–2910 doi:10.1093/gbe/evx202 Advance Access publication September 25, 2017 Downloaded from https://academic.oup.com/gbe/article/9/10/2893/4237403 by Consejo Superior de Investigaciones Cientificas (CSIC) user on 01 December 2023 stored. The best model was selected using the likelihood ratio test (LTR) and comparing first the m1 against the m2, and the winner against the m0. P-values of LTR tests were corrected using a Bonferroni method (two tests). Python and related scripts are presented in the supplementary files: dNdS_analysis. COG groups were assigned using the output from MEGAN6. All statistical tests were performed in R. In general, statistical tests were performed on OCPs with xvalues below 1, as most of the genes were evolving at this ratio. Only few genes had x values greater than 1. Some of these values should be taken cautiously, as they can represent alignment artifacts (e.g. open reading frames from fragmented genes in draft genomes). Briefly, raw and log transformed data were checked for normality (Shapiro’s test and QQ-plots) and heteroscestaticity (Levene’s test). Parametric tests were used on normal (or close to normal) and homoscedastic data while nonparametric tests were used in case of heteroscedasticity data. Ordinary Linear Modeling (OLM) was used to detect significant correlation in single Sodalis symbionts. Phylogenetic generalized least squares (PGLS) was used to detect significant correlations across species as it accounts for phylogenetic autocorrelations. All the statistical analyses are presented as an Rmd file (supple mentary files: dNdS_analysis). Results Bacteriome Characterization All dissected individuals of Henestaris halophilus (fig. 1A)possessed a pair of elongated, tubular-shaped, red-colored bacteriomes, located on either side of the abdomen adjacent to the gonads (fig. 1B). The bacteriomes extended in adults from the second to the fourth abdominal segment and were subdivided into three sections, not completely separated from each other. Male individuals often presented slender bacteriomes. Fluorescence in situ hybridization (FISH) was used for localization of the H. halophilus endosymbionts. A specific endosymbiont signal was detected in the tubular-shaped bacteriomes (fig. 1C). In addition, fluorescent activity was detected in the ovaries (fig. 1D), where several bacteriocytes formed an infection zone, and in the developing embryos. At the beginning of the embryonic development (36 h), a symbiont mass, in general described as a “symbiont ball,” was observed on the anterior pole side of the egg (fig. 1E). After embryonic katatrepsis, the developing bacteriomes were recorded at the same position in the abdomen as described for adults (fig. 1F). Initially, bacteriomes were of spherical shape, but were extended to their final tubular shape during the postkatatrepsis embryonic development (data not shown). These observations strongly indicate that the described endosymbiont is transferred to offspring via vertical maternal transmission. Ultrastructural examinations by electron microscopy (TEM) revealed that the bacteriocytes present a single nucleus and are densely filled with rod-shaped bacteria, presenting the typical gammaproteobacterial structure and three membranes (the bacteria cell wall and a host-derived one) (fig. 2Aand B). Endosymbiont Identification A 1.5 kb 16S rRNA bacterial gene fragment was amplified by PCR from DNA samples of H. halophilus bacteriomes, derived from geographically distant localities. Cloning and sequencing indicated that all nucleotide sequences are nearly identical (99.6–100%). Comparison with GenBank databases indicated that the bacteriome-associated endosymbiont of H. halophilus is related to the gammaproteobacterial Sodalis cluster (supplementary fig. S1, Supplementary Material online). The 16S rRNA sequence showed the highest similarity (94–95%) to sequences of Sodalis-allied endosymbionts of scale insects from the Coelostomidiidae family and Sodalisallied endosymbionts of stinkbugs and weevils. The complete 16S rRNA sequence of the H. halophilus bacteriomeassociated endosymbiont was obtained by genome sequencing (see below). Sequences of two additional bacteria, Ca. Lariskella arthropodarum and Rickettsia sp. were also detected in the Illumina genomic reads, but with very low coverage. However, no FISH signals of Lariskella and Rickettsia were detected in the analyzed bacteriomes and ovaries (data not shown), suggesting that these endosymbionts might have sporadic appearance or that they are present in H. halophilus in very low amounts. Comparative Genomics of H. Halophilus Endosymbiont and Related Species The genome of the bacteriome-associated endosymbiont of H. halophilus was assembled as a single closed circular chromosome with a coverage of 527. The genome was found to be of intermediate size (1.62 Mb), showed no AT enrichment (45% GC content) and displayed low coding density (37.3%) (table 1). In addition, it presented a reduced number of coding genes (713), pseudogenes (166), no active mobile elements, a single rRNA operon, and a reduced set of tRNA genes (42). Among the pseudogenes, several transcription factors (11), cell wall and transporter genes (29), genes encoding enzymes involved in amino acid and cofactors metabolism (19) or genes related to the replication, transcription and translation machinery (50) were identified (supplementary fig. S2 and table S3, Supplementary Material online). Comparisons against 14 related Sodalis and Sodalis-allied endosymbionts genomes suggested an intermediate to advanced stage of reductive evolution (supplementary table S1, Supplementary Material online). A New Bacteriome-Associated Endosymbiont of the Lygaeoid Bug Henestaris halophilus GBE Genome Biol. Evol. 9(10):2893–2910 doi:10.1093/gbe/evx202 Advance Access publication September 25, 2017 2897 Downloaded from https://academic.oup.com/gbe/article/9/10/2893/4237403 by Consejo Superior de Investigaciones Cientificas (CSIC) user on 01 December 2023 FIG.1.—The endosymbiotic system of Henestaris halophilus (A) Adult female. (B) Dissected bacteriome (b) of tubular shape on the right side of the abdomen. The paired bacteriomes are slightly separated into three parts by contractions. (C) Fluorescence in situ hybridization (FISH) of the Sodalis endosymbiont inside the bacteriome, stained with the specific probe Hen500 (Cy3; green) and SYBR Green I (blue). (D) Extensive signals were also detected in the ovaries. The symbionts are located in ovarial bacteriocytes forming an “infection zone” (iz), where from symbionts are transferred into the developing oocyte by an emerging “symbiont ball” (sb). (E) During early embryogenesis (36 h after egg deposition), the symbiont ball is attached to abdomen, followed by infection of the embryo. (F) After katatrepsis, an embryonic back flip within the egg, the symbionts are already located inside the bacteriome. Santos-Garcia et al. GBE 2898 Genome Biol. Evol. 9(10):2893–2910 doi:10.1093/gbe/evx202 Advance Access publication September 25, 2017 Downloaded from https://academic.oup.com/gbe/article/9/10/2893/4237403 by Consejo Superior de Investigaciones Cientificas (CSIC) user on 01 December 2023 Three duplicated segments, remnants of two duplication events, of 12, 10, and 2 kb, including the groS–groL operon among other genes, were found. For most of these duplicated genes, one of the duplicated copies is pseudogenized while the other (or the two others, in the case of groS and groL) retains the functionality. Orthologous clusters of proteins (OCPs) were computed for the Sodalis endosymbiont of H. halophilus, the 14 Sodalis-allied species and P. ananatis (supplemen tary table S1, Supplementary Material online). The Sodalis core genome, mainly driven from the most reduced Sodalis, harbors 166 OCPs, 75% of them belonging to the J, K, L, and O COG categories (translation, transcription, replication, and post-translational machinery, respectively). Among the other categories, three OCPs were classified as E (amino acid metabolism). From them, two were related to the Fe–S sulfur cluster protein biosynthesis (IscS, SufS) and one to the chorismate pathways (AroK). Three OCPs were classified as H (coenzyme metabolism), including LipA and LipB that compose the complete salvage lipoate pathway, and RibE/H, which is an intermediate reaction in the riboflavin pathway. The rest of OCPs were found to belong to other COG categories (21) or remained without an ascription to a specific COG (12). The Sodalis endosymbiont of H. halophilus presented 146 strain-specific OCPs, but only three of them were annotated as nonhypothetical proteins: HBA_0606 (DeaD division protein), HBA_0622 (a duplicated GroS), and HBA_0766 (secretion monitor precursor SecM). Most of the hypothetical proteins were short proteins (60 amino acids in average) without hits in the databases used for their annotation (see Supplementary Material online). Also, these proteins were not classified to a COG category. One possibility is that these proteins are open reading frames (ORFs) derived from unrecognizable pseudogenes or small proteins with an unidentified function. Taxonomy of the Sodalis Clades A phylogenomic tree, based on 77 single copy core proteins belonging to all 15 analyzed Sodalis species, was obtained. The tree clearly indicated the presence of two main clades, with the two cultivable species of Sodalis,S. glossinidius,and S. praecaptivus, being associated with clade A and clade B, respectively. The Sodalis endosymbiont of H. halophilus was placed in clade B (fig. 3). In addition, no clear association was observed between the phylogeny of the Sodalis species and the taxonomy of their insect hosts (fig. 3). The presence of the two cultivable species of Sodalis in different clades made us question the taxonomic status of the other 13 Sodalis-allied species, by utilizing a genome comparison approach (using ANI and AAI methods). As a reference, free-living and endosymbiotic bacterial species, belonging to eight additional genera of Gammaproteobacteria (which are phylogenetically related to Sodalis) and one Alphaproteobacteria (outgroup) were considered. Multiple comparisons indicated that a restrictive threshold of 80% (75-81%) AAI, discriminates well between the eight genera used as reference. When clustering analysis was applied to the ANI/AAI matrices, one large cluster including almost all the Sodalis-allied species was recovered for both, with the exception of the three fast-evolving lineages (Mikella,Gullanella,andS. of H. cubana)(fig. 4). AAI values among the five Sodalis species with the largest genomes: S. glossinidius,Sodalis of P. spumarius (clade A), S. praecaptivus,S. pierantonius,andS. melophagi (clade B), ranged between 85 and 96%. Moreover, the averaged AAI values between the aforementioned five Sodalis species and the remaining Sodalis-allied species (except Mikella and the symbiont of H. cubana),werehigherorclose to 80%, clearly suggesting that all analyzed species belong to one Sodalis genus. In comparison, AAI values between the Sodalis group and the reference genera showed a range of values lower than 70% AAI. FIG.2.—Transmission electron microscopy (TEM) micrographs of the Sodalis endosymbiont of Henestaris halophilus.(A) Overwiew of a bacteriocyte completely filled by rod-shaped endosymbiont (S). (B) Enlarged image of dividing endosymbionts showing the characteristic gammaproteobacterial cell structure. Nucleus (N). A New Bacteriome-Associated Endosymbiont of the Lygaeoid Bug Henestaris halophilus GBE Genome Biol. Evol. 9(10):2893–2910 doi:10.1093/gbe/evx202 Advance Access publication September 25, 2017 2899 Downloaded from https://academic.oup.com/gbe/article/9/10/2893/4237403 by Consejo Superior de Investigaciones Cientificas (CSIC) user on 01 December 2023 Applying the 95% ANI threshold (Konstantinidis and Tiedje 2005), the Sodalis endosymbiont of H. halophilus was identified as a new Sodalis species. It also confirmed the strain status of the two M. endobia species and indicated that although S. praecaptivus,S. pierantonius,andS. melophagi are likely to be undergoing a speciation process within their different hosts, they can still be considered strains of the same species. Based on our phylogenomics and the ANI/AAI results, we propose the name Candidatus Sodalis baculum sp. nov. strain kilmister for the newly described endosymbiont of H. halophilus. The species epitheton refers to the structure of the bacteriome. The slender, tubular-shaped appearance is similar to a baculum (penis bone) that can be found in many placental mammals. The strain name is proposed in honor of the musician Ian “Lemmy” Fraser Kilmister (1945–2015). Metabolic Capabilities of Candidatus Sodalis Baculum The full metabolism of Ca. Sodalis baculum (hereafter abbreviated as SoBa) was reconstructed (fig. 5). Despite the low coding density of its genome, SoBa still harbors a complete glycolytic pathway and a functional pentose phosphate pathway that produces several intermediate metabolites and reducing agents (NADPH). Furthermore, SoBa is capable of producing its own cell wall, reflected by its rod shaped cell appearance (fig. 2), which is comparable to free-living related species. In contrast, the SoBa genome does not contain all the required pathways for the synthesis of nucleotides. The genes encoding for enzymes synthesizing inosine monophosphate from 5-phosphoribosyl 1-pyrophosphate (PRPP) have been lost or pseudogenized, while the genes involved in the synthesis of uridine monophosphate from uracil using PRPP have been retained. Consequently, the capability of synthesizing pyrimidines importing/using only uracil is still present, while purines have to be imported from the insect host. Furthermore, SoBa has lost most of the genes encoding enzymes required for amino acid biosynthesis, limiting its capabilities to the production of five amino acids (alanine, glycine, lysine, serine, and tyrosine). Alanine may be produced in a single step, probably as a byproduct of the transfer of sulfur to tRNAs, from imported cysteine (ABC transporter CydD). The presence of the enzyme glycine/serine hydroxymethyltransferase (encoded by glyA) might reflect an ability to produce glycine from serine or vice versa, but also to produce tetrahydrofolate, which serves as a one-carbon carrier of the biosynthesis of purines and other compounds. The tyrosine and lysine biosynthetic pathways are present in SoBa (fig. 5). The tyrosine pathway is partially shared by the phenylalanine and tryptophan pathways, but the loss of one phenylalanine and several tryptophan biosynthetic genes significantly reduces the possibility that SoBa is capable of synthesizing these amino acids. The essential amino acid lysine is synthesized using aspartate, which is likely imported from the hosts’ cytosol by the glutamate/aspartate transporter GltP. Although the argD gene encoding succinyldiaminopimelate transaminase is missing, the specific catalytic reaction might be performed by phosphoserine aminotransferase (SerC) as reported in Escherichia coli (Lal et al. 2014). The synthesis of L-homoserine is theoretically possible, but the conservation of this pathway is more likely to be associated with the fact that the thrA and asd genes encoded enzymes are required also in the lysine biosynthetic pathway. FIG.3.—Phylogenomic tree of several Sodalis and Sodalis-allied species. The two clades used in subsequent analysis are denoted by the letters A (blue) and B (green). Only the best topology found by the AU-test is displayed: ML tree with a single partition schema under JTTþCAT20 model. Node legends denote ML boostrap and Bayesian posterior probabilities; * in Bayesian posterior probabilities denotes alternatives topologies found in MrBayes partitioned reconstruction (S-endosymbiont of H. cubana together with Mikella endobia and Sodalis-like of P. fluctus as a basal clade of S-endosymbiont of C. eucalypti). Santos-Garcia et al. GBE 2900 Genome Biol. Evol. 9(10):2893–2910 doi:10.1093/gbe/evx202 Advance Access publication September 25, 2017 Downloaded from https://academic.oup.com/gbe/article/9/10/2893/4237403 by Consejo Superior de Investigaciones Cientificas (CSIC) user on 01 December 2023 In addition to the biosynthesis of intermediate metabolites and amino acids, SoBa preserves the complete pathways for the biosynthesis of several cofactors and vitamins, such as acetyl-CoA, lipoate, NAD, riboflavin and its derivatives, pyridoxal 5-phosphate (vitamin B6), thiamin diphosphate (TDP), ubiquinol-8, S-adenosyl-L-methionine (SAM), and tetrahydrofolate (vitamin B9). Finally, the SoBa genome also contains the whole Fe–S biosynthesis pathway cluster and is capable of producing glutathione. Metabolic Pathways Comparisons among Sodalis Amino acid and cofactors biosynthetic potential of each Sodalis-allied species was explored at the pathway level (fig. 6). With respect to the ability to synthesize essential amino acids, we found that tryptophan can be produced by all of the Sodalis-allied species of hosts that feed exclusively on plant sap. In hosts feeding on other diets, tryptophan can probably be obtained in other ways, as indicated by the loss of the pathway in SoBa, Sodalis endosymbiont of Proechinophthirus fluctus and S. pierantonius. The lysine biosynthetic pathway, which is present in SoBa, was lost in all Sodalis-allied species present in mealybugs, the psyllid H. cubana and the louse P. fluctus.TheSodalis from psyllids, mealybugs and the froghopper retain some genes that complement the essential amino acid production of their hosts’s primary endosymbionts. Sodalis of P. fluctus cannot produce any essential amino acid, but is still able to produce several vitamins. Only the recently acquired Sodalis maintain the ability to produce most (8 or more) of the 10 essential amino acids, including tryptophan and lysine. The analysis of the synthesis of nonessential amino acids in Sodalis indicated that Sodalis-allied species with reduced genomes only rarely synthesize these amino acids. Moreover, the nonessential amino acids that are produced are probably byproducts of essential pathways for the symbiotic relationship. This phenomenon could be explained by the settlement of these endosymbionts in the host environment, acquiring most of the amino acids from their hosts’ cytosol. Tyrosine biosynthesis was found to be conserved only in SoBa, while all the other Sodalis-allied species with reduced genomes have lost this ability. In addition, a functional FIG.4.—Hierarchical clustering of pairwise Average Nucleotide Identity (ANI, left) and Average Amino Acid Identity (AAI, right). Clusters containing Sodalis-allied species are highlighted in blue. SoBa is highlighted in purple. Values greater than 95% start at blue in the color scale. A New Bacteriome-Associated Endosymbiont of the Lygaeoid Bug Henestaris halophilus GBE Genome Biol. Evol. 9(10):2893–2910 doi:10.1093/gbe/evx202 Advance Access publication September 25, 2017 2901 Downloaded from https://academic.oup.com/gbe/article/9/10/2893/4237403 by Consejo Superior de Investigaciones Cientificas (CSIC) user on 01 December 2023 Supplementary Material Supplementary Material includes supplementary Material and Methods, supplementary figures S1 to S3 and supplementary tables S1 to S3 and is are available at Genome Biology and Evolution online. Supplementary Files Supplementary Files can be found at http://dx.doi.org/10.17632/n38gkjry35.1 Authors’ Contributions S.M.K. conceived the study, collected the insect material, designed, and performed the microscopic experiments and characterized the endosymbiont. F.J.S. designed the molecular evolutionary analysis. D.S-G. performed the bioinformatic analysis. D.S-G. and S.M. designed the statistical analysis. S.M.K., F.J.S., and D.S.-G. analyzed the data and wrote the manuscript with inputs from S.M. 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