Effect of Oceanic Islands on an Insect Symbiont Genome in Transition to a Host-Restricted Lifestyle

Abstract

Islands offer unique opportunities to study adaptive radiations and their impacts on host genome evolution. In Hawaiian Pariaconus psyllids, all species harbor the ancient nutritional symbiont Carsonella, while only free-living and open-gall species on younger islands host a second stable cosymbiont, Makana. In contrast, a third cosymbiont, Malihini, appears to be in an early stage of host restriction and genome degradation, making it a valuable model for understanding symbiont evolution during island radiations. Here, we examine Malihini genome evolution across multiple Pariaconus lineages using 16S rRNA sequencing, metagenomics, phylogenetic reconstruction, and microscopy. We find that Malihini is codiversifying with its hosts on the oldest island Kaua'i (kamua group; open- and closed-gall makers) and on the younger islands only in free-living species (bicoloratus group). Comparison of five Malihini genomes-including three newly assembled in this study-shows ongoing genome reduction from a large-genome ancestor (>3,900 protein-coding genes), likely driven by relaxed selection, vertical transmission bottlenecks, and island dispersal over the past 5 million years. On Kaua'i, the galling psyllids appear to depend more heavily on cosymbiont (Malihini) for the biosynthesis of amino acids and B-vitamins than galling species on younger islands-especially closed-gall species, which only have Carsonella. Surprisingly, free-living psyllids on younger islands with all three symbionts show metabolic reliance similar to Kaua'i gall makers. Together, our results demonstrate that island biogeography and host plant ecology shape symbiont losses and codiversification patterns. Malihini represents an early stage of symbiont genome degradation during host restriction, in sharp contrast to its more stable coresidents, Carsonella and Makana.

Keywords: Malihini; Pariaconus; Hawaiian Islands; adaptive radiation; genome evolution; symbiosis.

Fig. 1.

Schematic of sampled psyllids from the Hawaiian Pariaconus species radiation. Cladogram represents the relationships of the 21 species collected from the four species groups kamua, minutus, bicoloratus, and ohialoha and subjected to 16S rRNA or metagenomic (bold) sequencing. Samples sequenced in this study are underlined. The cladogram was modified from Percy (2017) and Percy et al. (2018). See Tables S5 and S6 for more details on the new samples. Generalized host–plant ecologies and symbiont repertoires are shown for five subgroups of species along with their islands of origin. Host plant ecologies are abbreviated as FL, OG, or CG. Dating of the islands is presented as millions of years ago (Ma) and is based on dating from Bonacum et al. (2005).

Fig. 2.

ASV similarity matrices for Hawaiian Pariaconus psyllids. Heatmaps of ASV nucleotide similarity for Pariaconus symbionts a) Makana, b) Malihini, and c) Wolbachia recovered from 16S rDNA tag sequencing (n = 440 nt). Note that heatmap color ranges for each plot correspond with their included key for each panel. Colored bars adjacent to the species names represent the psyllid species groups minutus (black), bicoloratus (red), kamua (yellow), and ohialoha (blue). dor, dorsostriatus; haw, hawaiiensis; mon, montgomeri; pro, proboscideus; pyr, pyramidalis; kup, kupua; mau, mauiensis; oah, oahuensis.

Fig. 3.

Whole-mount FISH confocal microscopy images of Malihini within an immature specimen of P. crassiorcalix. a) Image showing the dorsal view of a cleared specimen, P. crassiorcalix, fifth instar cropped and rotated from Percy et al. (2018) (Creative Commons License). b and c) Merged confocal FISH images of the dorsal view of an immature specimen of P. crassiorcalix at different magnifications and depths (Z-stacks). Arrows indicate 16S rRNA probe AL555 binding specifically to Malihini DNA (red) in and around the bacteriome. All DNA is counterstained with DAPI (blue), which primarily shows host DNA. Note that the whole insect is orientated similarly to the specimen in a). d) Merged fluorescence image of a second, unstained P. crassiorcalix immature specimen (no DAPI, no AL555) displays minimal background fluorescence at either wavelength.

Fig. 4.

Gene loss and inactivation are key drivers of Malihini genome differences. a) Venn diagram of intact Malihini CDSs reveals a highly reduced core genome with 18% to 35% of CDS in an individual genome that is uniquely intact in that genome. Genome-encoded pseudogenes (ψ) were accounted for, and b) the history of gene inactivations and losses was reconstructed using parsimony rules on the Malihini species phylogeny. Adjacent to each extant genome, the total number of intact CDS (blue) and the numbers that are unique (blue in parentheses) and the number of total pseudogenes (red) and the numbers that are unique (red in parentheses) are presented. Note that the number of unique CDS differs from a) since some CDSs were found as pseudogenes in one or more of the other genomes. The tree represents maximum likelihood phylogeny of 373 conserved core proteins analyzed with RAxML-NG and FastTree. RAxML-NG bootstrap scores are shown above, and statistical support values from FastTree are shown below. Starting from the reconstructed ancestral genome, each doughnut plot at each node represents the estimated proportion of genes that remained intact, those that were pseudogenized and those that were lost along that branch. The doughnut diameters are scaled by the relative number of genes.

Fig. 5.

Malihini genomes of the kamua species group encode distinct amino acid and vitamin pathways. Heatmaps represent presence, absence, or pseudogenziation (ψ) of biosynthesis pathways and transport systems (t) for amino acids and vitamins in the Malihini genomes. The number of genes involved is shown to the right, and colored boxes to the left represent the essential (pink) and nonessential (green) amino acids, or the distinct vitamins. Two multisubstrate transporters are shown in black; aromatic = Phe, Tyr, Trp, and His and polar = His, Arg, Lys, Glu, Gln, and Asp. Pathways or transporters with ≥1 detected pseudogene are indicated with a ψ symbol.

Fig. 6.

Hawaiian Pariaconus psyllids have a strict pattern of cospeciation and loss. Core gene phylogenies for Carsonella, Makana, and Malihini can be perfectly overlayed, illustrating their histories of strict vertical inheritance and cospeciation. Host species names include the lifestyle (FL, free-living; FL*, putative free-living; OG, open-gall; CG, closed-gall), and the species group is shown to the right (m, minutus). Adjacent to each node, in a color matching the symbiont, statistical support values ≥ 50 from FastTree are shown above and RAxML-NG bootstrap scores are shown below.

Fig. 7.

Hawaiian Pariaconus psyllid hosts receive variable benefits from their symbionts. Radiation and speciation of Pariaconus psyllids have resulted in different symbiont configurations that in turn have the potential to contribute distinct pools of vitamins and essential amino acids to their hosts.