Biodiversity, evolution and microbiome of the New Zealand Psylloidea (Hemiptera: Sternorrhyncha)

Psyllids, also known as jumping plant lice, belong to the superfamily Psylloidea (Hemiptera: Sternorrhyncha) and globally are divided into almost 4000 described species. Psyllids are phloem-feeders, with a number of species considered economic pests including through the vectoring of phloem-restricted plant pathogens, and others used as biological control agents against invasive plants. In recent years some economically important plant diseases have emphasised the role of highly polyphagous psyllids in pathogen epidemiology, for example of Candidatus Liberibacter spp. vectored by Bactericera cockerelli causing zebra chip disease, and by Diaphorina citri causing Huanglongbing disease. This has generated substantial interest in the biology of such psyllids. However, it has also highlighted the lack of information on other psyllid species, many of which are poorly studied and are difficult to identify or completely undescribed. This can confound accurate species diagnosis for both ecological and biosecurity applications, and undermines any understanding of their potential role in maintaining disease-causing bacteria in the environment. The psyllid fauna of New Zealand provides a cross section of the superfamily Psylloidea, with species representatives in the families Aphalaridae, Calophyidae, Homotomidae, Liviidae, Psyllidae and Triozidae, including pests and bio control agents. However, despite almost 100 known species there, information about the endemic fauna, which has representatives across three families, is scarce and many taxa still await description. Documented knowledge on New Zealand psyllids is now outdated as a result of new taxonomic classifications and new arrivals. Furthermore, the recent introduction of B. cockerelli and the spread of the zebra chip disease raised a number of questions on the role of other psyllid species in its horizontal transmission and also presence of any other pathogens that might already exist. This study aimed to understand which psyllid species are present in New Zealand and their evolutionary relationships, and to develop the first information on the composition of their natural, internal bacterial community. This will not only enable new psyllid species arrivals to be recognised, but also allow interrelationships across psyllid taxa, their microflora and host plants to be understood. In turn, hypotheses as to the potential for native psyllids to also transmit introduced pathogens can be advanced. Field-collected specimens from almost 600 locations around New Zealand, Australia and United States of America were used to generate (a) an up-to-date list of the New Zealand Psylloidea, based on a morphological-molecular integrative taxonomy concept; (b) a phylogenetic analysis of the psyllid collection using sequences of cytochrome oxidase subunit 1 [COI] DNA barcode region plus partial 18S ribosomal DNA, and including a region of elongation factor 1-alpha (EF-1α) for a species subset; and (c) a partial 16S metabarcode next generation sequencing (MiSeq, Illumina) bacterial inventory. Morphological and genetic analysis, together with distribution and host plant associations, resulted in the identification of 90 different taxa of psyllids in New Zealand; this was in addition to another 30 species known to be present in this region but not collected. The collection included one newly introduced species from Australia and 20 novel undescribed native species including a number of morphologically cryptic taxa. The phylogenetic study performed on these species revealed an evolutionary structure that was congruent with the current taxonomy. Furthermore, the position of the genus Atmetocranium was clarified and re-attributed to the family Aphalaridae, confirming an original placement. The presence of likely six ancestral arrivals (for the psyllids included in this work) has been proposed together with the different evolutionary strategies that led to the present psyllid fauna of New Zealand. These include a number of host switches for the species of the genus Trioza, that likely happened when the insect colonized the host plant, and a relatively more strict psyllid-plant association for the genera Ctenarytaina and Psylla. Subsequent partial 16S metabarcode analysis of 220 individual psyllids from 65 species across the six New Zealand families confirmed the universal presence of the primary symbiont Candidatus Carsonella rudii; this included some unexpected species-level variation (>4% divergence) according to the operational taxonomic units (OTUs) defined by the VSEARCH pipeline. A prevalence of symbionts belonging to the family Enterobacteriaceae was also revealed, but species-level assignment was not possible with the partial 16S r DNA region used. Nevertheless, the Mantel and partial Mantel tests confirmed that, the microbial composition is highly correlated (almost 40%) to the genetic distance between insects after accounting for the host plant variation. On the other hand, inverting the variables, host plant associations are responsible for just 15% of the microbial composition after accounting for the psyllid genetic distance. These observations are consistent with the idea that the psyllid microbial composition is mostly influenced by the psyllids species and not the plant. Furthermore, potential coevolution between psyllids and some secondary symbionts is proposed. The pathogen-containing bacterial genera Liberibacter and Phytoplasma were detected with BLAST indications from the 16S sequences as to species previously not recorded in New Zealand. The range of curated specimens and the molecular framework generated here supplies a substantial resource for further taxonomic and ecological enquirey. This work provides a valuable dataset enabling comparisons between both species native to New Zealand and between these and other psyllid taxa from all over the world. In turn this provides fundamental taxonomic and biodiversity information that subsequently can be exploited as outcomes for plant health bio-protection and biosecurity