Morphology of Obligate Ectosymbionts Reveals Paralaxus Gen. Nov., a New

bioRxiv preprint doi: https://doi.org/10.1101/728105; this version posted August 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Harald Gruber-Vodicka 2 Max Planck Institute for Marine Microbiology, Celsiusstrasse 1; 28359 Bremen, 3 Germany, +49 421 2028 825, [email protected] 4 5 Morphology of obligate ectosymbionts reveals Paralaxus gen. nov., a 6 new circumtropical genus of marine stilbonematine nematodes 7 8 Florian Scharhauser1*, Judith Zimmermann2*, Jörg A. Ott1, Nikolaus Leisch2 and Harald 9 Gruber-Vodicka2 10 11 1Department of Limnology and Bio-Oceanography, University of Vienna, Althanstrasse 12 14, A-1090 Vienna, Austria 2 13 Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, 14 Germany 15 16 *Contributed equally 17 18 19 20 Keywords: Paralaxus, thiotrophic symbiosis, systematics, ectosymbionts, molecular 21 phylogeny, cytochrome oxidase subunit I, 18S rRNA, 16S rRNA, 22 23 24 Running title: “Ectosymbiont morphology reveals new nematode genus“ 25 Scharhauser et al. 26 27 bioRxiv preprint doi: https://doi.org/10.1101/728105; this version posted August 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Scharhauser et al. 2 28 Abstract 29 Stilbonematinae are a subfamily of conspicuous marine nematodes, distinguished by a 30 coat of sulphur-oxidizing bacterial ectosymbionts on their cuticle. As most nematodes, 31 the worm hosts have a simple anatomy and few taxonomically informative characters, 32 and this has resulted in numerous taxonomic reassignments and synonymizations. 33 Recent studies using a combination of morphological and molecular traits have helped 34 to improve the taxonomy of Stilbonematinae but also raised questions on the validity 35 of several genera. Here we describe a new circumtropically distributed genus 36 Paralaxus (Stilbonematinae) with three species: Paralaxus cocos sp. nov., 37 P. bermudensis sp. nov. and P. columbae sp. nov.. We used single worm metagenomes 38 to generate host 18S rRNA and cytochrome oxidase I (COI) as well as symbiont 39 16S rRNA gene sequences. Intriguingly, COI alignments and primer matching analyses 40 suggest that the COI is not suitable for PCR-based barcoding approaches in 41 Stilbonematinae as the genera have a highly diverse base composition and no 42 conserved primer sites. The phylogenetic analyses of all three gene sets however 43 confirm the morphological assignments and support the erection of the new genus 44 Paralaxus as well as corroborate the status of the other stilbonematine genera. 45 Paralaxus most closely resembles the stilbonematine genus Laxus in overlapping sets 46 of diagnostic features but can be distinguished from Laxus by the morphology of the 47 genus-specific symbiont coat. Our re-analyses of key parameters of the symbiont coat 48 morphology as character for all Stilbonematinae genera show that with amended 49 descriptions, including the coat, highly reliable genus assignments can be obtained. bioRxiv preprint doi: https://doi.org/10.1101/728105; this version posted August 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Scharhauser et al. 3 50 Introduction 51 The identification of many nematode genera and species is difficult based on 52 morphological characters alone (Sudhaus & Kiontke 2007, de-León & Nadler 2010, 53 Derycke et al. 2008, Palomares-Rius et al. 2014). A prime example for this problem are 54 the Stilbonematinae, a subfamily of the Desmodoridae that have experienced several 55 changes in the classification of species and even genera in the past (Tchesunov 2013; 56 Ott et al. 2014). The exclusively marine Stilbonematinae are common members of the 57 interstitial meiofauna in sheltered intertidal and subtidal porous sediments and have 58 been found worldwide (Ott et al. 2004, Tchesunov et al. 2013). Their diversity and 59 abundances are highest in subtropical and tropical shallow-water sands, but some 60 species were also described from higher latitudes (e.g. Gerlach 1950, Wieser 1960, 61 Platt & Zhang 1982, Riemann et al. 2003, Tchesunov 2012), deep-sea sediments (Van 62 Gaever et al. 2004, Tchesunov et al. 2012, Leduc 2013), near shallow hydrothermal 63 vents (Kamenev et al. 1993, Thiermann et al. 1997) and methane seeps (Dando et al. 64 1995). 65 To date, a total of 10 different stilbonematine nematode genera and more than 50 66 different species are described (reviewed by Tchesunov 2013; Leduc 2013, Armenteros 67 et al. 2014b, Ott et al. 2014a,b, Leduc and Sinniger 2018). Most species and genera still 68 lack molecular data, but recent taxonomic work has started to integrate molecular 69 data using the 18S rRNA gene (18S) (Ott et al. 2014a, b; Armenteros et al. 2014ab; 70 Leduc and Zhao 2016, Leduc and Sinniger 2018) and the mitochondrial cytochrome 71 oxidase subunit I (COI) gene (Armenteros et al. 2014a, b). The phylogenetic signal of 72 the two marker genes was however not consistent and called morphological genus and 73 species identifications into question (Armenteros et al. 2014b). While the monophyly 74 of the subfamily Stilbonematinae has strong support based on 18S data (Kampfer et al. 75 1998, Bayer et al. 2009, van Megen et al. 2009, Ott et al 2014a and b; Leduc & Zhao 76 2016), it was questioned in a recent study by Armenteros et al. (2014b) which used 77 both 18S and COI. 78 bioRxiv preprint doi: https://doi.org/10.1101/728105; this version posted August 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Scharhauser et al. 4 79 A trait that unites all stilbonematine nematodes is the conspicuous ectosymbiosis with 80 sulphur-oxidizing Gammaproteobacteria that cover major parts of the cuticle 81 (reviewed by Ott et al. 2004). Stilbonematine symbionts have diverse morphologies, 82 including rod, coccus, coccobacillus, filament, corn-kernel or crescent shapes, and each 83 host species harbours a single morphotype (Ott & Novak 1989, Polz et al. 1992; Polz et 84 al. 1994; Ott et al., 2004; Bayer et al. 2009, Pende et al 2014, Ott et al. 2014a, b). In 85 phylogenetic analyses of the symbiont 16S rRNA gene (16S) sequences, all 86 stilbonematine symbionts fall into a monophyletic clade of Gammaproteobacteria 87 related to Chromatiaceae, which has been described as Candidatus Thiosymbion (from 88 here on ‘Thiosymbion’) (Bayer et al. 2009, Bulgheresi et al. 2011, Heindl et al. 2011, 89 Zimmermann et al. 2016). Based on phylogenetic analyses of host 18S and symbiont 90 16S sequences closely related stilbonematine nematode species of the same genus 91 consistently harbour closely related and genus specific ‘Thiosymbion’ symbionts (Ott et 92 al. 2004, Zimmermann et al. 2016). ‘Thiosymbion’ encompasses not only the 93 ectosymbionts of stilbonematine nematodes, but also the primary symbionts of gutless 94 phallodriline oligochaetes and siphonolaimid nematodes (Zimmermann et al. 2016). 95 Comparative analyses of stilbonematine nematodes and their symbionts showed a 96 high degree of phylogenetic congruence that indicates a long-term co-diversification 97 between ‘Thiosymbion’ and the Stilbonematinae (Zimmermann et al. 2016). 98 Recent 18S-based phylogenetic analyses suggested a novel, morphologically 99 uncharacterized stilbonematine genus from the Caribbean Sea (Belize) and the 100 Australian Great Barrier Reef (Heron Island) (Zimmermann et al. 2016). The genus level 101 of this clade was supported by the distinct 16S sequences of their symbionts that also 102 formed a new phylogenetic clade in the study. Here we combine morphological and 103 molecular data to describe this recently detected host clade as ‘Paralaxus’ gen. nov.. 104 We use single worm metagenomes to provide host 18S and COI gene sequences as 105 well as symbiont 16S sequences from nine Paralaxus specimens, and 20 additional 106 specimens covering six stilbonematine genera. We place Paralaxus in a unified 107 taxonomy of Stilbonematinae and formally describe the new genus Paralaxus with a 108 Caribbean type species P. cocos sp. nov. In addition, we describe two new species, bioRxiv preprint doi: https://doi.org/10.1101/728105; this version posted August 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Scharhauser et al. 5 109 P. bermudensis sp. nov. and P. columbae sp. nov. from Bermuda and Florida, and 110 report on members of this new genus from localities in the Western and Central Pacific 111 Ocean. While characterizing the key anatomical features of Paralaxus, we identified 112 the high taxonomic value of its symbiont coat. We extend this concept and evaluate 113 coat and symbiont morphology as additional traits for all stilbonematine genera. 114 In addition, we substantially improve the molecular dataset of Stilbonematinae full- 115 length 18S and COI marker gene sequences to answer the following questions: (1) Are 116 Stilbonematinae monophyletic? (2) At which taxonomic level are 18S and COI valuable 117 phylogenetic markers for Stilbonematinae? (3) Can we find suitable COI PCR priming 118 sites that are conserved across the subfamily? 119 bioRxiv preprint doi: https://doi.org/10.1101/728105; this version posted August 7, 2019.

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