Parallel Evolution in the Integration of a Co-obligate Aphid Symbiosis
Authors: Marie Bunker
E Toby Kiers
Jacintha Ellers
Raphaella Jackson
David Monnin
Lee M Henry
Journal: Current Biology (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Buchnera aphidicola; Serratia symbiotica; aphid; co-obligate; evolution of dependence
Affiliations: School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK., Department of Ecological Science, Vrije Universiteit Amsterdam, Amsterdam 1081 HV, the Netherlands., Biology Department, University of Puget Sound, 1500 N. Warner St., Tacoma, WA 98416, USA., School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK. Electronic address: .

Abstract:  Insects evolve dependence-often extreme-on microbes for nutrition. This includes cases in which insects harbor multiple endosymbionts that function collectively as a metabolic unit [1-5]. How do these dependences originate [6], and is there a predictable sequence of events leading to the integration of new symbionts? While co-obligate symbioses, in which hosts rely on multiple nutrient-provisioning symbionts, have evolved numerous times across sap-feeding insects, there is only one known case in aphids, involving Buchnera aphidicola and Serratia symbiotica in the Lachninae subfamily [7-9]. Here, we identify three additional independent transitions to the same co-obligate symbiosis in different aphids. Comparing recent and ancient associations allow us to investigate intermediate stages of metabolic and anatomical integration of Serratia. We find that these uniquely replicated evolutionary events support the idea that co-obligate associations initiate in a predictable manner-through parallel evolutionary processes. Specifically, we show how the repeated losses of the riboflavin and peptidoglycan pathways in Buchnera lead to dependence on Serratia. We then provide evidence of a stepwise process of symbiont integration, whereby dependence evolves first. Then, essential amino acid pathways are lost (at ∼30-60 mya), which coincides with the increased anatomical integration of the companion symbiont. Finally, we demonstrate that dependence can evolve ahead of specialized structures (e.g., bacteriocytes), and in one case with no direct nutritional basis. More generally, our results suggest the energetic costs of synthesizing nutrients may provide a unified explanation for the sequence of gene losses that occur during the evolution of co-obligate symbiosis. 

Retracted: No

PubPeer comments:
 In the present work, Monnin _et. al._ investigate aphid (Hemiptera: Aphididae) co-obliagte nutritional symbiosis between the primary obligate beneficial enodsymbiont _Buchnera aphidicola_ and the secondary endosymbiont _Serratia symbiotica_. For this purpose, the authors focused on a set of species they determined to have _Se. symbiotica_ as apparently fixed: a set of _Periphyllus_ spp. (Aphididae: Chaitophorinae: Chaitophorini) and two species feeding on the common nettle (Urtica dioica) _Aphis urticata_ (Aphididae: Aphidinae: Aphidini), and _Microlophium carnosum_ (Aphididae: Aphidinae: Macrosiphini). Cited evidence for _Se. symbiotica_ being fixed in this species comes from two main sources: the authoru2019s own collection efforts and subsequent 16S PCR amplification and sequencing (Table S1), and previous 16S amplicon high-throughput sequencing (HTS) of different aphids (doi:10.1111 1758-2229.12795, namely accessions SRX6899781 [_Ap. urticata_] and SRX6899792 [_Mi. carnosum_]). To investigate more in depth these putative co-obligate associations, the authors performed curing experiments (i.e. antibiotic treatment targeting the _Se. symbiotica_ symbiont); whole genome sequencing of three selected species of _Periphyllus_, _Ap. urticata_, and _Mi. carnosum_; and FISH microscopy using labelled probes targetting the two symbionts. The authors convincingly show that a co-obligate associations have been established in the analysed _Periphyllus_ species, and that the dependence of _Buchnera_ and the aphid host likely evolved in the common ancestor of _Periphyllus_ aphids. This would in fact represent an independent acquisition of secondary co-obligate endosymbionts from that observed across Lachninae subfamily aphids (Gosalbes _et. al._, 2008; Lamelas _et. al._, 2011; Manzano-Maru00edn _et. al._, 2016, 2018; Manzano-Maru00edn Latorre, 2014; Manzano-Maru0131u0301n _et. al._, 2020; Meseguer _et. al._, 2017). After thoroughly reviewing the manuscript and associated data I have major concerns regarding the alleged co-obligate status _Se. symbiotica_ in both Aphidinae species analysed: _Ap. urticata_ and _Mi. carnosum_. I will detail these concerns below separating them by the evidence that is claimed to support these _Buchnera_-_Se. sybmiotica_ co-obligate endosymbioses. **Evidence for the alleged fixed status of _Se. symbiotica_ in _Ap. urticata_ and _Mi. carnosum_** To identify aphids that hosted putative co-obligate _Buchnera_-_Se. symbiotica_ symbioses, the authors relied on a previous study (Henry _et. al._, 2015) and symbiont identification in their own sampling. Henry _et. al._ (2015) analysed a number of aphid populations from different species by diagnostic PCR (Table S1, Appendix S1) targetting three facultative symbiont lineages (_Se. symbiotica_, _Hamiltonella defensa_, and _Regiella insecticola_) and 16S amplicon HTS sequencing of circa 10 individuals from what can be understood to be a population of each species. For some aphid species that were collected from multiple plant species, more than one sample was analysed (Appendix S2). The authors in the previously mentioned study state in Table S2 that for both _Ap. urticata_ and _Mi. carnosum_, the relative abundance of aphids harbouring _Se. symbiotica_ was 1 (meaning all aphids do carry the symbiont). From looking at Appendix S1, a number of _Mi. carnosum_ populations (almost all from Oxford and the surrounding areas) were analysed by PCR and only 17 23 (73.9%) were found to harbour _Se. symbiotica_. The populations sampled from _Solanum tuberosum_, _Sonchus oleraceus_, _Cirsium_ sp., _Rubus fruticosus_, and _Circaea lutetiana_ were not found to harbour the symbiont, and only the populations on _So. tuberosum_ and _Cirsium_ sp. were associated to another symbiont (_Re. insecticola_). On _U. dioica_, 100% of the populations were found to harbour the secondary endosymbiont _Se. symbiotica_. In regards to _Ap. urticata_, from all populations sampled (also mostly from Oxford) 6 8 (75.0%) were found to harbour the symbiont. Particularly, the two samples that were not found to harbour neither _Se. symbiotica_, _H. defensa_, nor _Re. insecticola_ were those collected from species other than _U. dioica_ (i.e. _Urtica_ sp. and _Cirsium_ sp.). In addition, a previous study looking at symbiont diversity of four populations of four Aphidinae species found that out of 101 _Mi. carnosum_ individuals sampled from _U. dioica_, 95 (94.06%) harboured _Se. symbiotica_ (referred to as R-type) and 72 were infected with _Re. Insecticola_ (Haynes _et. al._, 2003). The _Ap. urticata_ and _Mi. carnosum_ sampling of Monnin _et. al._ (2020) consisted of several individuals (10m apart) from what can only be assumed to be the two geographically isolated populations (one from the UK and one from the Netherlands, further details not provided) growing all on the same host plant species, _U. dioica_. PCR screening of individuals from these populations revealed that 100% of the _Ap. urticata_ (9 9 UK and 6 6 NL) and _Mi. carnosum_ were indeed infected by a _Se. symbiotica_ strain (20 20 UK and 7 7 NL) (Table S1). _Ap. urticata_ and _Mi. carnosum_ feed mainly on _U. dioica_, the former only formerly described growing on _U. dioica_ (Blackman Eastop, 2006, 2020). _Mi. carnosum_ has also been recorded on other _Urtica_ spp. and _Giardinia cuspidata_. _Ap. urticata_ has a wide geographical distribution range (Europe, Middle East, Central Asia, Pakistan, and North America) just as _Mi. carnosum_ does (Europe, Asia east to Pakistan and Mongolia, Africa [Burundi, Rwanda], North America, and Chile). Looking at all the evidence, two things become clear: First, the presence of _Se. symbiotica_ strains is not universal neither in _Ap. urticata_ nor in _Mi. carnosum_, as evidenced by the two previous studies, and particularly the cited Henry _et. al._ (2015). Second, even across geographically close populations, _Se. symbiotica_ does not seem to be universally present, but fixed in aphids growing on _U. dioica_. While the authors did find an infection rate of 100% of their sampled and tested aphids, these seem to come from a single geographical population, at best closely related populations of around 10m apart from two European countries. From studies on other aphids, we know that a specific facultative symbiont can be highly prevalent in a population, and even fixed, depending on, for example, environmental conditions challenges (probably due to a rapid rise after exposure) or even host plant (Brady _et. al._, 2014; Ferrari _et. al._, 2012; Henry _et. al._, 2013; Leclair _et. al._, 2016; Peccoud _et. al._, 2015; Qian _et. al._, 2018; Simon _et. al._, 2003; Tsuchida _et. al._, 2002, 2004; Weldon _et. al._, 2013). Thus, the evidence for _Se. symbiotica_ being actually fixed, and thus being putatively co-obligate, in these two aphid species (_Ap. urticata_ and _Mi. carnosum_) remains unconvincing. **Curing experiments (i.e. antibiotic treatment)** In order to assess experimentally the dependence of both _Ap. urticata_ and _Mi. carnosum_, the Monnin _et. al._ (2020) conducted antibiotic treatment experiments on the UK and NL aphids, including two _Periphyllus_ spp., _Acyrthosiphon pisum_ (facultative _Se. symbiotica_), and _Macrosiphoniella artemisiae_ (no _Se. symbiotica_) (Figure 1, S1, and Data S1A). Figure 1 shows a reduction of lifetime fecundity for _Ap. urticata_, _Mi. carnosum_, and both _Periphyllus_ spp; while no apparent reduction in was observed for _Ac. pisum_ and _Ma. artemisiae_. However, closer inspection of the supplementary material and data (Figure S1 and DataS1A) it becomes immediately evident that the figure presented in the main article can be deceiving, since results for one of the control species _Ac. pisum_ (allegedly harbouring _Se. symbiotica_ strains) are contradictory between the UK and NL experiments. _Ma. Artemisae_ curing experiments were only conducted in the UK. In the UK experiment a significant reduction in fecundity was observed, while in the NL experiment, no significant reduction was observed (but rather a mean increase). Additionally, antibiotic treatments were not performed in the same way, given that in the UK the treatment lasted five days and in NL it lasted three days. These two experiments should not have been mixed, they should have been represented separately in the main article and clearly specified in the main text. This fact reflects that variation is observed when conducting experiments at least for different time periods, and that even species or populations where _Se. symbiotica_ strains are facultative, its clearing can lead to either a statistically significant reduction in lifetime fecundity or no significant change at all. Previous curing experiments have indeed been conducted in a couple of aphid species. For _Ac. pisum_, selective elimination of facultative strains of _Se. symbiotica_ resulted in differences in fitness consequences between aphid strains, mainly in regards to longevity (Koga _et. al._, 2007). Most importantly, and similarly to Monnin _et. al._ (2020), the authors did recorded offspring from G1s of some of their antibiotic treatment experiments, and both G1 and their offspring were subjected to diagnostic PCRs to confirm presence absence of the Serratia symbiont. Some lines were followed for up to 10 generations. This follow up of the offspring was apparently not done by Monnin _et. al._ (2020). This shortcoming becomes of great importance, given that if the symbiont is indeed a co-obligate one it would be expected that the aphids would either all become completely sterile, as is commonly observed when eliminating _Buchnera_ (Koga _et. al._, 2007), or would not be able to go further than a small number of generations (with additional testing for the presence of the alleged eliminated symbiont in each of the successive generations). Given the authorsu2019 discrepancy in the curing results for _Ac. pisum_ and the clearly different mean lifetime fecundities observed between the UK and NL experiments on _Ap. urticata_ (mean lifetime fecundity in NL-antibiotic(+) very similar to UK-antibiotic(-), Figure S1), more replicates (i.e. more geographically distant populations) should have been tested, accompanied by a symbiont genotyping to ensure that the same strain of the secondary symbiont was being tested, since different populations of the same species can harbour different strains of the same endosymbiont species (Brady _et. al._, 2014; Ferrari _et. al._, 2012; Haynes _et. al._, 2003) (which could then have different effects on their hosts). In at least the case of _Mi. carnosum_, it could be hypothesised that a _Se. symbiotica_ endosymbiont is possibly only required for growing in _U. dioica_, since populations collected on different host plants have previously tested negative for the presence of this bacterial taxa (Henry _et. al._, 2015). In the same publication, the authors also reported a negative presence of _Se. symbiotica_ on the only two populations of _Ap. urticata_ collected on species other than _U. dioica_, and thus if _Ap. urticata_ can indeed grow on other species, it could also imply that _Se. symbiotica_ is solely needed to grow on its most commonly recorded host species, _U. dioica_. **Genome sequencing and metabolic inference** One important factor when assigning the u201cco-obligateu201d stamp to an endosymbiont, especially when other lines of evidence are not available or are not conclusive is to perform a genome-based metabolic inference. This has been used as corroborating evidence for a number of studies looking at co-obligate associations in insects (Bennett Moran, 2013; Husnik McCutcheon, 2016; Manzano-Maru00edn Latorre, 2014; Meseguer _et. al._, 2017). For this purpose, the author selected three species of _Periphyllus_, _Ap. urticata_ and _Mi. carnosum_. By looking at the complementation of a comprehensive set of essential nutrients that are normally provided by _Buchnera_ in mono-symbiotic (_Buchnera_-only) aphids (Figure 3, Data S1D). There are a couple of issues with this figure. First, the alleged lack of _yigL_ (coding for a haloacid dehalogenase-like hydrolase, the putative 5-amino-6-(5-phospho-D-ribitylamino)uracil phosphatase from _Buchnera_) in the genome of _Buchnera_ from Aphis glycines is simply wrong. The _yigL_ gene (IX46_00150) is located between the genes _glmU_ and _metR_-_metE_. This genomic context is conserved across _yigL_-bearing _Buchnera_. Second, the alleged lack of a _ribD _gene (coding for a bifunctional enzyme) in the _Buchnera_ from _Mi. carnosum_ is also untrue. The _ribD_ gene is found split into two open reading frames (ORFs) each one containing one of the its two catalytic domains: Deaminase (G4A98_02295, EC:3.5.4.26) and reductase (G4A98_02300, EC:1.1.1.193). This is not uncommon across _Buchnera_ from mono-symbiotic Aphidinae, as it is observed in _Ap. glycines_ (BAg, CP009253.1), _Ac. pisum_ (LL01, CP002300.1; LSR1, ACFK01000001.1), and _Rhopalosiphum padi_ (Rpa, CP034858.1), to name a few. Additionally, experimental evidence from the RibD (or RibG) protein in _Bacillus subtilis_ has demonstrated that the enzymatic activity of each domain is preserved when expressed as two separate proteins (Richter _et. al._, 1997). Both _ribD_ genes of _B. subtitlis_ and _Escherichia coli_ have the same domain structure, and both retain a putative ribosome binding site right before an internal start codon. _Buchnera_ with split _ribD_ genes, including that of _Mi. carnosum_, retain such a Shine-Dalgarno sequence before the start codon (u201cAGGTGu201d in _Mi. carnosum_). Thus, the split bifunctional enzymes found in several _Buchnera_ from Aphidinae can be assumed to be functional and it does not imply the need of a co-obligate symbiont. Regarding the pseudogenisation of _murF_ in _Ap. glycines_ (Data S1E), it is not uncommon to see a frameshift in one or two low complexity regions even within isolates from the same aphid species (e.g. LL01 and APS from _Ac. pisum_, u201cCTATTA-AAAAAAAAAATATAGu201d and u201cTTAAAGAAAAAAAAA-TAAu201d). This happens across _Buchnera_ strains (including those of _Mi. carnosum_ and _Ap. urticata_ compare strains available in GenBank) from different aphid species, which could arise from either sequencing assembly errors or real frameshifts that could likely be surpassed by ribosomal frameshifting. Similar cases are those of _ddlB_, _trpS_, among others. _rmpF_ missing in _Buchnera_ from _Peripyllus acericola_ is likely simply missing from the annotation, since its flanking geneu2019s 5u2019-end runs off a contig end. Other ribosomal proteins such as RplC and RplT are not actually missing from _Buchnera_ from _Cinara tujafilina_ (locus tags BCTU_352 and BCTU_083). This examples reflect a lack of manual curation of the genes (or reference genome sequence) that are mentioned and highlighted in the manuscript. In brief, at least the annotations presented in terms of presence absence (key to the interpretation of results and discussion) should have been further manually curated. Other genes like _panB_ and _panC_ are commonly pseudogenised or lost in different Aphidinae species (Manzano-Maru00edn _et. al._, 2018). **FISH microscopy** One concern regarding the FISH experiments, using the PASSisR probe (Koga _et. al._, 2003) targetting _Se. symbiotica_ of _Ac. pisum_ (not all species since it does not match well smaller strains from Lachninae), is the interpretation of the location of this symbiont in _Ap. urticata_ in a u201csmall bacteriomeu201d or u201cin a single small bacteriomeu201d. The authors should take into account that the bacteriome is in fact a three-dimensional structure, not as typically depicted showing two bands on each side of the aphidu2019s abdomen. So, depending on the layer that is currently being visualised in the microscope, it could look like, what I call, a butterfly-like structure (as in Figure 4 for _Ac. pisum_ and _Mi. carnosum_), a U-shaped or oval-like structure if is is tilted to one side (Figure 1A in Tsuchida _et. al._, 2005) or even a smaller continuous Y structure (if visualising a plane closer to the dorsal side of an aphid or towards the juncture of both u201csidesu201d of the bacteriome, e.g. Figure 3F or S1I Manzano-Maru00edn _et. al._, 2017). Thus, the interpretation of the central cluster of cells in Figure 4 (_Ap. urticata_) from Monnin _et. al._ (2020) is incorrect. That cluster of cells are actually towards the junction of both u201csidesu201d of the bacteriome. I would point out Figure 1B from Tsuchida _et. al._, (2014), where the facultative endosymbiont _Rickettsiella viridis_ can locate to such a cluster of cells within the bacteriome or the co-obligate _Sodalis_-like endosymbionts of _Eulachnus rileyi_ (Figures 4B C, S1O) and _Cinara_ (_Schizolachnus_) _obscurus_ (Figure 4E) (Manzano-Maru00edn _et. al._, 2017). Lastly, both facultative endosymbionts, as well as early co-obligate ones can infect bacteriocytes, and sometimes they can even be found co-infecting along with _Buchnera_ (Manzano-Maru00edn _et. al._, 2017; Manzano-Maru00edn Latorre, 2014; Moran _et. al._, 2005; Tsuchida _et. al._, 2010, 2014). In summary, the authors make a compelling case and show through different lines of evidence that co-obligate _Se. symbiotica_ endosymbionts are indeed present in _Periphyllus_ spp. and that this dependence likely evolved in the common ancestor of this genus. Thus this would represent a second independently evolved case of co-obligate endosymbiotic systems in aphids. However, the authors fail to provide evidence supporting the claim of co-obligate _Buchnera_-_Se. symbiotica_ systems in _Ap. urticata_ and _Mi. carnosum_. It becomes apparent that this conclusion was reached by Monnin _et. al._ (2020) because of five main reasons: **(i)** An inadequate analysis of the previously reported data from previous studies, **(ii)** incorrect experimental design that did not include several different geographically-distant populations of _Ap. urticata_ and _Mi. carnosum_, **(iii)** inadequate experimental design and mixture of different antibiotic treatment methods when plotting statistical analysis in the main text, **(iv)** insufficient manual curation of annotations and genome-based metabolic inference of symbionts biosynthetic capabilities, and **(v)** an incorrect interpretation of the FISH microscopy results, namely for _Ap. urticata_. **Minor comments corrections**  While co-obligate symbioses, in which hosts rely on multiple nutrient-provisioning symbionts, have evolved numerous times across sap-feeding insects, there is only one known case in aphids, involving _Buchnera aphidicola_ and _Serratia symbiotica_ in the Lachninae subfamily This is simply incorrect. Co-obligate associations with _Buchnera_ have been described in Lachninae aphids for _Sodalis_, _Sodalis_-like, _Erwinia_, _Hamiltonella_, and _Fukatsuia_ (Manzano-Maru00edn _et. al._, 2017, 2018; Meseguer _et. al._, 2017). The authors state in the discussion that the _Se. symbiotica_ symbiont of _Periphyllus lyropictus_ likely constitutes an event of symbiont replacement within this lineage. Why did the authors not attempted phylogenetic reconstructions to test this?  Furthermore, our findings indicate that genomic integration may occur in advance of anatomical integration. This has actually already been documented in the Lachninae aphid _Cinara tujafilina_, where the co-obligate symbiont _Se. symbiotica_ has a distribution in the bacteriome resembling that of the facultative lineage(s) found in _Ac. pisum_ (Manzano-Maru00edn _et. al._, 2017; Manzano-Maru00edn Latorre, 2014). **REFERENCES** Bennett GM Moran NA. (2013). 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