DOI: 10.5061/dryad.gxd2547gs
title: Genetic variability, community structure, and horizontal transfer of endosymbionts among three Asia II‐Bemisia tabaci mitotypes in Pakistan

description: Endosymbionts associated with the whitefly Bemisia tabaci cryptic species
 are known to contribute to host fitness and environmental adaptation. The
 genetic diversity and population complexity were investigated for
 endosymbiont communities of B. tabaci occupying different
 micro-environments in Pakistan. Mitotypes of B. tabaci were identified by
 comparative sequence analysis of the mitochondria cytochrome oxidase I
 (mtCOI) gene sequence. Whitefly mitotypes belonged to the Asia II- 1, -5,
 and -7 mitotypes of the Asia II major clade. The whitefly-endosymbiont
 communities were characterized based on 16S ribosomal RNA Operational
 Taxonomic Unit (OTU) assignments, resulting in 43 OTUs. Most of the OTUs
 occurred in the Asia II-1 and II-7 mitotypes (r2=0.9, p&lt;0.005)
 while the Asia II-5 microbiome was less complex. The microbiome OTU groups
 were mitotype-specific, clustering with a basis in phylogeographical
 distribution and the corresponding ecological niche of their whitefly
 host, suggesting mitotype-microbiome co-adaptation. The primary
 endosymbiont Portiera was represented by a single, highly homologous OTU
 (0-0.67% divergence). Two of six Arsenophonus OTUs were uniquely
 associated with Asia II-5 and -7, one occurred exclusively in Asia II-1,
 two only in Asia II-5, and one in both Asia II-1 and -7. Four other
 secondary endosymbionts, Cardinium, Hemipteriphilus, Rickettsia, and
 Wolbachia OTUs were found at ≤29% frequencies. The most prevalent
 Arsenophonus OTU was found in all three Asia II mitotypes (55% frequency),
 whereas the same strain of Cardinium and Wolbachia were found in both Asia
 II-1 and -5, and a single Hemipteriphilus OTU occurred in Asia II-1 and
 -7. This pattern is indicative of horizontal transfer, suggestive of a
 proximity between mitotypes sufficient for gene flow at overlapping
 mitotype ecological niches.
description: The <i>16S rDNA</i> (~1500 bp) sequence was
 PCR-amplified using ‘universal’ primers 27F (5'-AGAGTTTGATCMTGGCTCAG)
 and 1513R (5'-ACGGYTACCTTGTTACGACTT) (0.4 μM) [64, 65]. The PCRs were
 set as explained above using 20ng of whitefly DNA. Cycling parameters were
 an initial denaturation at 94°C for 2 min, and 35 cycles of 94°C for 30 s,
 55°C for 30 s, and 72°C for 2 min, with a final extension at 72°C for 10
 min. Amplicon sizes were verified by agarose gel electrophoresis and
 cloned. Twelve clones per whitefly were selected by colony PCR
 amplification, and sequenced. 
description: Figure S1. Bayesian phylogeny of the Bemisia tabaci mitotypes
 from Pakistan based on the 3’-end fragment of the mitochondrial cytochrome
 oxidase I gene (COI) (725 bp). Acronyms are lineages designations by De
 Moya et al. (2019). Numbers at nodes are posterior
 probabilities. Figure S2. Distribution of a Portiera, b
 Arsenophonus, c Cardinium, d Hemipteriphilus, e Rickettsia, and f
 Wolbachia in twelve districts of Pakistan. Bars represent the relative
 abundance of endosymbionts per district. Bemisia tabaci mitotypes Asia
 II-5 and -7 were only recorded from Lahore, whereas mitotype Asia II-1 was
 widely distributed across cotton growing districts. Blue lines in map
 represent rivers, dotted lines indicate provincial limits and the polygon
 shows the cotton growing area of Pakistan. Figure S3.
 Rarefaction curves for individual whitefly samples. Red lines are whitefly
 samples belonging to Asia II-7, black lines are Asia II-1 individuals and
 blue are whitefly samples from the Asia II-5 group. The curves were
 generated in mothur v1.43.0 based on the observed richness with 1000
 iterations. Table S1. Collection information,
 phylogenetic affiliation and endosymbiotic composition of individual
 whitefly samples from Pakistan. The presence and absence of the
 endosymbiont is given by the + and – signs, respectively. Capital letters
 were used to denote the endosymbiont genus as follows:  P= Portiera, A=
 Arsenophonus, C= Cardinium, H= Hemipteriphilus, R= Rickettsia, W=
 Wolbachia Table
 S2. Taxonomic classification, accession numbers of <i>16S ribosomal
 RNA</i> (<i>16S rRNA</i>) sequences and host
 mitochondrial <i>cytochrome oxidase I</i>
 (<i>mtCOI</i>), sample location, whitefly host plant, and
 relative frequency per mitotype of operational taxonomic units (OTUs)
 detected in whitefly samples from Pakistan. Table S3. Absolute frequencies of
 bacterial classes and families based on 1084 bp the 16S ribosomal RNA gene
 (16S rRNA), for the three Bemisia tabaci mitotypes identified in Pakistan,
 Asia II-1, -5, and -7. The 16S rDNA sequences were taxonomically
 classified using the SILVA taxonomy database. Approximately 90% of the
 sequences clustered within the Bacteroidaceae, Enterobacteriaceae, and
 Halomonadaceae families.  Table S4. The OTU abundance table
 per whitefly sample, taxonomic classification and whitefly mitotypic group
 designation.