SG1 belongs to the class Mollicutes, a group of bacteria within the phylum Mycoplasmatota that includes some of the smallest and simplest known cellular organisms. Until now, SG1 had been documented exclusively within the scorpion family Vaejovidae, specifically in the genera Vaejovis and Mesomexovis, and only in Mexican populations. Its absence from other major scorpion families, including Caraboctonidae, Euscorpiidae, Diplocentridae and Buthidae, had suggested a highly specialized host-symbiont relationship. The new discovery in A. phaiodactylus, a member of the distantly related family Anuroctonidae, upends that assumption and forces researchers to reconsider the evolutionary boundaries of this unusual bacterial association.

The key to the discovery lies in a technological shift. Previous studies of the scorpion venom microbiome relied on short-read sequencing platforms, particularly Illumina, which excels at producing vast numbers of reads but struggles with certain sequence regions. In their earlier work using the same venom samples, the researchers analyzed approximately 2.5 million Illumina reads and failed to detect SG1 at all. When they aligned the newly recovered Anuroctonidae SG1 haplotypes against the Illumina primer sequences, they found a seven base pair mismatch in the reverse primer relative to the SG1 region. That small difference was likely sufficient to prevent primer binding and amplification entirely, meaning the symbiont was effectively invisible to the short-read approach.

To overcome this limitation, the team turned to Oxford Nanopore Technology, a long-read sequencing platform capable of reading much longer stretches of DNA in a single pass. They sequenced the 16S rRNA gene — a standard molecular marker for bacterial identification — from the venom of two scorpion species: Paruroctonus becki, a vaejovid, and A. phaiodactylus, an anuroctonid. Both species occur farther north than previously sampled scorpion hosts and are among the few that extend into the Great Basin Desert, a colder desert system not represented in earlier SG1 studies. The researchers hypothesized that SG1 would be detected in P. becki, extending its known distribution to another vaejovid host, but not in A. phaiodactylus, given its more distant evolutionary relationship.

The results were the opposite of what they expected. SG1 was undetected in all 14 P. becki venom samples, though the researchers caution that the low read count — just 768 reads across all samples — may have limited the detection of rare sequences rather than reflecting a true biological absence. In contrast, SG1 was present in 6 out of 16 A. phaiodactylus samples, a frequency of 38 percent. From 19,890 total reads, 137 mapped to SG1, roughly one in every 145 reads. Nine distinct SG1 haplotypes were recovered, with read lengths ranging from 959 to 1,551 base pairs and averaging 1,469 base pairs, covering the majority of the 16S rRNA gene. All sequences from A. phaiodactylus showed percent identities above 97 percent when compared to SG1 from vaejovid scorpions, indicating similarity at the genus level.

To place these new sequences in evolutionary context, the researchers constructed a phylogenetic tree using Bayesian inference, combining the nine newly generated haplotypes with 26 previously published SG1 sequences from vaejovid scorpions. The resulting phylogeny revealed two major clades. Most strikingly, all A. phaiodactylus SG1 sequences formed a distinct clade that was unique and highly divergent from the previously characterized Vaejovidae SG1 lineages. This relationship was strongly supported by a posterior probability of 1.0. A complementary haplotype network analysis, built using the median-joining approach, confirmed the pattern, showing the Anuroctonidae haplotypes organized separately from the vaejovid sequences with substantial mutational distance between the two groups.

The placement of one particular sequence complicated a simple story of host-symbiont cospeciation. An SG1 sequence previously recovered from Mesomexovis aff. oaxaca did not fall within the main vaejovid cluster but instead grouped closer to the A. phaiodactylus haplotypes in both the phylogeny and the network. As a result, the sampled SG1 sequences from Vaejovidae are not monophyletic with respect to the A. phaiodactylus lineage. This pattern suggests that SG1’s history is likely more complex than a strict host-symbiont cospeciation model. The researchers note that additional processes may be involved, including ancient SG1 diversity, incomplete sorting of symbiont lineages, or host switching. Broader sampling across vaejovids, anuroctonids and other scorpion families will be needed to distinguish among these alternatives.

The amount of mutational difference between the Anuroctonidae and Vaejovidae SG1 lineages suggests an ancient divergence rather than a recent host-switching event. This supports the idea that SG1 may have had a long-term, stable association with the Anuroctonidae host lineage, persisting through millions of years of scorpion evolution. The researchers also note that the reduced frequency of SG1 in A. phaiodactylus compared to previous reports from Vaejovis smithi — where SG1 was found in 94 percent of samples — suggests that SG1 may not be a major component of A. phaiodactylus venom. Alternatively, SG1 may be more frequent than the current estimate and simply difficult to detect. The limited venom volumes, ranging from about 3 to 20 microliters per sample, and the resulting low DNA yields likely constrained sequencing depth.

The researchers took several steps to rule out contamination as an explanation for their findings. Negative controls lacking venom were processed through all DNA extraction steps, and none produced 16S rRNA PCR products. Results were also consistent across duplicate sequencing runs: of six samples sequenced in duplicate, one repeatedly showed SG1 sequences while SG1 was consistently undetected in the other five. Furthermore, the unique haplotypes recovered across samples, even those with low SG1 read counts, indicate that these sequences likely represent reliable biological variation rather than cross-sample artifacts. Their prior work on the same specimens had already demonstrated, through beta diversity metrics, a statistically significant difference between the microbial composition of the venom and exterior controls.

The discovery underscores how biological structure can go unnoticed depending on the methodology employed. A lineage that remained hidden through millions of Illumina reads became visible with a shift to long-read sequencing. More broadly, the findings suggest that additional SG1 diversity may still be hiding in unsampled or overlooked scorpion lineages, waiting to be revealed. Whole genome sequencing of the M. aff. oaxaca SG1 and the A. phaiodactylus SG1 would be particularly interesting, the researchers note, to compare any potential functional adaptations or metabolic differences that may allow SG1 to survive across taxonomically distant scorpion hosts. As sampling expands and sequencing technologies evolve, the true evolutionary story of this remarkable symbiont — and the scorpions that carry it — is only beginning to come into focus.

Subject of Research: Discovery of the symbiotic bacterium Scorpion Group 1 in the venom of Anuroctonus phaiodactylus and the expansion of its known host range through long-read sequencing

Article Title: Scorpion Group 1 discovered in the venom of Anuroctonus phaiodactylus

Article References: Heidorn, A., Graham, M. R., & Murdoch, B. (2026). Scorpion Group 1 discovered in the venom of Anuroctonus phaiodactylus. Frontiers in Arachnid Science, 5, Article 1858835. https://doi.org/10.3389/frchs.2026.1858835

Image Credits: AI Generated

DOI: 10.3389/frchs.2026.1858835

Keywords: Scorpion Group 1, Anuroctonus phaiodactylus, Mollicutes, Oxford Nanopore sequencing, venom microbiome, symbiosis, Vaejovidae, Anuroctonidae, 16S rRNA, scorpion microbiota, Scorpion, Group