Steinhart Aquarium biologists at the California Academy of Sciences found that a shark egg case dropped by an adult bamboo shark showed signs of healthy development.
The scientists were surprised because the aquarium's female brownbanded bamboo sharks had spent 45 months in complete isolation from males.
When one viable egg resulted in the birth of a healthy pup, Academy scientists set out to examine this unprecedented example of sharks' long-suspected ability to store sperm over long periods of time.
"Long-term sperm storage - where a female can delay fertilisation for months or even years after mating - is a remarkable adaptation that helps promote genetic diversity," said Dr Luiz Rocha, Academy Curator of Ichthyology.
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"In contrast, asexual reproduction produces offspring with very little genetic variation," Rocha said.
The Academy's scientific investigation began in 2010, when biologist Nancy Sinai transferred several brownish egg cases found in the aquarium's Shark Lagoon exhibit into a separate incubator on public display, where two of the eggs showed signs of healthy embryonic development.
Two guesses were more likely: either a female adult shark reproduced asexually in a process called 'parthenogenesis' (as has been observed in four different shark species), or it had stored sperm from its last mating event several years before the fertilised egg appeared in the Lagoon.
Aquarium records showed that the Academy's three female bamboo sharks - each a possible mother to the new pup - had no contact with compatible males since their 2007 residence at the Aquarium of the Pacific in Southern California.
The shark pup displayed comparable results with each female on a particular genetic test that looks at genetic variation within an individual.
If the mother had reproduced asexually through parthenogenesis, the offspring would have shown less genetic variation than that of its mother.
The pup also displayed genetic material - in the form of 32 alleles, or parts of genes - absent from all three adult females.
Study authors concluded that the young shark most likely inherited this 'mystery' genetic material from its father - an unknown male from the long-ago tank at the Aquarium of the Pacific.