Charles Darwin once remarked, “It is hardly an exaggeration to say that Nature tells us, in the most emphatic manner, that she abhors perpetual self-fertilization.” And yet, Kobe University botanist SUETSUGU Kenji knows of a few islands in Japan where orchids reproduce without ever opening their flowers. He says: “I’ve long been captivated by Darwin’s skepticism about plants that rely entirely on self-pollination. When I found those non-blooming orchids, I felt this was a perfect chance to directly revisit this issue. The apparent defiance of evolutionary common sense made me wonder what precise conditions — both environmental and genetic — would allow a purely self-pollinating lifestyle to emerge, let alone persist.”

On the Northern Ryukyu Islands of Kuroshima, Takeshima and Yakushima exist the only wild populations of plants known to reproduce solely by self-pollination. “Our group spent over ten years working with local plant enthusiasts, monitoring more than a hundred individual plants across several islands, so we can say with certainty that these orchids never open their flowers in their natural habitats,” explains Suetsugu. He and his team decided to subject these populations to genetic analysis that can detect minute differences even between closely related individuals, which allowed them to track gene flow and relatedness.

In the journal Proceedings of the Royal Society B, the Kobe University-led team now reports that the extreme genetic uniformity between the plants in each species proves that they are truly purely self-pollinating. In addition, they also found that the two species each arose from insect-pollinated species that already have a very low degree of genetic variation in this geographic region. The variation is so low, in fact, that even though pollen might get transported from one plant to the next, it’s nearly identical to self-pollination.

This is only made worse by another observation Suetsugu made: The cross-pollinating relatives on these islands rely on fruit flies with limited flight ability. The animals thus only pollinate, if they pollinate the plants at all, flowers of the same plant or of those that live very close by, even further reducing the benefit of insect pollination. Suetsugu explains: “Darwin’s statement was motivated by the idea that a purely self-pollinating lineage would accumulate harmful mutations and eventually face an evolutionary dead end. Yet our findings show that for the relative species with open flowers, the real genetic payoff for outcrossing might be marginal, giving the self-pollinating orchids, which are more successful at producing fruit, an evolutionary edge.”

As it turns out, Darwin’s skepticism might not have been unfounded. Another result of the Kobe University study is that even with conservative estimates, these self-pollinating species are at most 2,000 years old. Given that there are no other known examples of purely self-pollinating plants in the wild, such species might be ephemeral. “The fact that these orchids truly never outcross raises intriguing questions about their long-term viability, especially under pressures like habitat fragmentation and climate change,” says Suetsugu. He nevertheless is proud of his team’s findings, saying, “Each new data point, each newly described species, brings me closer to grasping the full spectrum of evolutionary possibilities.”

This research was funded by the Japan Society for the Promotion of Science (grants JP15K18470, JP17H05016 and JP18K06408), the Japan Science and Technology Agency (grant JPMJPR21D6), the Ministry of the Environment, Japan (grant 4-2001) and the Alexander von Humboldt Foundation. It was conducted in collaboration with researchers from Tohoku University, Osaka Metropolitan University, Fukushima University, and the National Institute of Genetics.

The large-scale study analysed more than 1,000 stool samples from people with diarrhoeal illness to harness two cutting edge tools.

Diarrhoea, a common symptom of infectious intestinal disease, affects an estimated 18 million people each year in the UK. However, despite its prevalence, traditional lab tests often fail to recognise its cause, especially when infections are caused by unidentifiable or emerging pathogens. This newly published study used metagenomic (DNA-based) and metatranscriptomic (gene or RNA-based) sequencing. Unlike traditional methods, these techniques do not rely on growing organisms in a lab. Instead, they detect and analyse the genetic material directly from patient samples.

Co-lead author, Dr Edward Cunningham-Oakes, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool said: “This is the UK’s largest study to compare traditional diagnostics with these next-generation tools. We not only found infections missed by standard tests, but we could see what the bugs were doing inside the gut — something standard diagnostics just can’t show.”

The work captures, for the first time, a comprehensive snapshot of the Salmonella gene expression directly from a human stool sample. The transcriptomic data provides new insights into how the bacteria survive and adapt after leaving the human gut. As Salmonella remains a priority diarrhoeal pathogen in the UK, the knowledge will be invaluable for helping scientists to target this dangerous pathogen.

The study’s key findings highlight the power of RNA tests in detecting hidden infections — including elusive parasites and RNA viruses — while also identifying which genes are active during infection. Remarkably, RNA remained stable in stool samples even without preservatives, suggesting it is more robust than previously thought. Notably, the ratio of RNA to DNA helped differentiate true infections from harmless gut microbes. By combining both DNA and RNA data, researchers gained the clearest and most accurate picture of the infection process.

Co-lead Professor Alistair Darby, Co-Director of the University’s Centre for Genomic Research said: “This study shows how genetic tools can revolutionise how we identify and understand intestinal infections. By understanding not just what’s there, but what it’s doing, we can improve public health responses, particularly around foodborne outbreaks.”

The findings could significantly impact how diarrhoeal diseases are diagnosed, managed, and studied in the UK and beyond — especially in light of the growing need for rapid, accurate diagnostics that don’t rely on outdated culturing methods.

The research also highlights the strategic role of Liverpool’s Centre for Genomic Research (CGR) and the new Microbiome and Infectious Disease (MaID) initiative, which is part of the Liverpool City Region’s Life Sciences Innovation Zone.

Professor Alistair Darby continued: “This is about more than diagnosing infections — it’s about building a platform for innovation in healthcare. Our previous work has shown that healthcare professionals are open to this. By making our data open-access, we hope to help other researchers, NHS labs, and public health agencies build on our work.”

Dr. Edward Cunningham-Oakes added: “Our results show that RNA, once thought too fragile to use in stool testing, can actually give us powerful insights into how infections work. That opens up new possibilities for diagnosing and treating these illnesses more effectively.”

The research, led by scientists at the University of Liverpool, including Salmonella experts Dr Blanca Perez-Sepulveda and Professor Jay Hinton, was funded by the NIHR Health Protection Research Unit in Gastrointestinal Infections (HPRU-GI), a collaboration between the University of Liverpool, UK Health Security Agency, and other partners.