Genetic Rescue of the Northern White Rhino
People are taking eclectic approaches to saving rhinos from poachers.
Tracking devices on the animals detect an increase in heart rate when danger approaches, like a FitBit wearer encountering a dog that’s sprung it’s invisible fence.
A concoction of rhino keratin (the protein that forms the horn) made in recombinant yeast and rhino DNA (to mark its authenticity) offers a substitute that may keep poachers away.
The Rhino Rescue Project captures rhinos, injects dye into their horns, then releases them, the stain rendering the appendage less desirable to hunters. Dehorning is another approach.
Such efforts may appear to be too late for the brink-of-extinction northern white rhino, but results of a new study published in Genome Research offer hope: genome sequences of nine northern white rhinos reveal a genetic diversity that may provide a way to save them. “Our study demonstrates the emerging role for whole genome sequencing analysis to evaluate the potential for population recovery,” said Cynthia C. Steiner, from the San Diego Zoo Institute for Conservation Research and director of the study.
When the last male northern white rhinoceros, Sudan, died from an infection on March 19 at age 45 at the Ol Pejeta Conservancy in Kenya, only Najin and her daughter Fatu were left, and they’ve been unable to have offspring. But genomes from stored rhino cells at the San Diego Frozen Zoo may rejuvenate at least a small founding population. The Frozen Zoo houses more than 10,000 cell cultures, eggs, sperm, and embryos representing nearly 1,000 types of organisms.
Of the five living species of rhinoceros, the black, Javan, and Sumatran species are considered critically endangered, and the greater one-horned rhino deemed vulnerable. The white rhino Ceratotherium simum is split, the southern subspecies (C. simum simum) near-threatened, but the northern subspecies (C. simum cottoni) much worse off, called critically endangered.
The southern white rhino population is now 20,000 strong, thanks mostly to conservation efforts after poachers whittled the group down to fewer than 50 animals in the early 20th century. Most of these rhinos live in South Africa.
The threat isn’t shrinking habitat. It is poachers seeking the horns, which are ground up for various applications in Chinese and Vietnamese medicine and used for supposed aphrodisiac qualities; for Yemeni knife handles; and merely for the bizarre thrill of disfiguring a large mammal.
So what, exactly, is the chemical composition of a rhino horn? Nothing special. It consists primarily of keratins, the same family of proteins that form nails, hairs, bills, beaks, and hooves. A horn’s interior includes calcium for strength and melanin for protection against ultraviolet radiation. Cut off a horn and it’ll grow back, if the animal doesn’t die. The keratin of a rhino’s horn isn’t the same as the proteins of tooth enamel or the collagen-based ivory of an elephant, hippo, or walrus tusk or a whale’s baleen.
The northern white rhinoceros once lived in south Sudan, the Democratic Republic of the Congo, Chad, the Central African Republic, and Uganda. The 1970s and 1980s brought poaching and war. By the 1990s and into the new millennium the remaining population of 30 rhinos, in Garamba National Park in the Democratic Republic of Congo, dwindled as human violence escalated. The last northern white rhino born into captivity was in 2000.
By 2008, experts considered the northern subspecies extinct in the wild.
Only four individuals were left in the world, living at a zoo in the Czech Republic. Wildlife biologists moved them to the Ol Pejeta conservancy in Kenya, hoping the more natural habitat might spur romance among the two females and two males. They mated, but no progeny resulted. Nor did introducing a southern white rhino stud help – Najin and Fatu were infertile. And even if they could have given birth, their genetic diversity would likely not have been enough to reconstitute a founding population, let alone save the subspecies.
Human history doesn’t go back very far, and so the authors of the new paper sequenced the genomes of nine northern rhinos (the first done) and four southern rhinos from fibroblasts (connective tissue cells) frozen in time at the San Diego Zoo, stored over the past three decades.
Analyzing genome sequences enables a look back farther in time than human memories or historical records by applying the rate of mutation of known genes to how different those genes are in cells from extant (or frozen) individuals. Mutation rate serves as a molecular clock of sorts, enabling mathematical models to reconstruct an approximate timing of ancient events. The mutation rate for rhinos (and humans) is approximately 2.5 DNA base changes per 100 million bases per generation.
Reconstructing Deep History
According to one type of statistical analysis of molecular clock data, the white rhino populations in the north and the south began to decline about 800,000 years ago, but started to rebuild about 100,000 years ago. But then about 80,000 years ago, the rhinos separated into northern and southern populations.
After the split, the northern population grew quickly and then declined, while the southern population initially declined but later resurged to surpass the numbers to the north. This opposite pattern may help to explain the greater diversity in the northern white rhino genome today, despite the animals’ near-disappearance.
A second type of statistical analysis based on mutation rate placed the divergence of the north and south population to 10,000-20,000 years ago. So the researchers estimate the split somewhere between 80,000 and 10,000 years ago, after which DNA swapping was minimal. Other ungulates (hoofed mammals) split into northern and southern groups during this time, in sync to the ebb and flow of lakes and forests with the glacial cycle.
Whenever the parting of the ways occurred, the ancestral group of about 16,000 rhinos split and must have encountered some stress in the journey, because just 1,300 ended up in the north and 2,800 in the south.
A Robust Genome
According to the genome sequences, the north and south rhino populations haven’t mixed much, and weren’t very different when they diverged. Although their thawed genome sequences differ by only about .1%, the researchers teased out some intriguing findings. And the news is promising for the northern rhinos.
The researchers assessed the 13 rhino genomes for evidence of genetic variation, inbreeding, and positive natural selection (persistence of new adaptive traits – in Darwinian parlance, those that enhance survival to reproduce). Here’s a closer look:
- Genetic Variation. The northern rhino genomes revealed 4,065,345 unique sites (single nucleotide polymorphisms, or SNPs), compared to 2,511,658 for the southerners. The greater genetic variation in the north makes sense because the southern group plummeted in the early 20th century to 20 to 50 individuals. Such a population bottleneck strangles the gene pool into genetic uniformity once mating resumes.
- Runs of homozygosity – stretches in the genome where the sequence is the same on both DNA strands – are a hallmark of inbreeding. These signposts take up more than 3 percent of the southern white rhino genome, but a bit less for the northerners. Plus, the stretches of sameness are up 32 million base pairs for the southern rhinos but max out at 23 million base pairs for the north. Put another way, the southerners are more inbred.
- Positive selection. One hundred SNPs, in 28 genes, are under positive selection, in either northern or southern rhinos. A mutation that alters the amino acid and persists indicates positive selection – a helpful genetic change that confers a reproductive advantage. Eleven of these genes encode olfactory receptors, reflecting the importance of the sense and perception of smell.
Four of the frozen cell lines from northern white rhinos are the top candidates for reconstructing the subspecies because they are the most genetically diverse – and that’s what brings disease resistance and fertility, an effect opposite that of inbreeding.
The Next Step: Reproduction
Going from analyzing genomes to creating embryos could happen in different ways, from low tech to high tech.
Scenario 1: Conventional breeding. Mating the 2 surviving northern white rhinos to southern males hasn’t worked, and the only known hybrid, Nasi, was born in 1977 and died in 2007, unhealthy and childless. The approach is similar to breeding the Przewalski horse to modern horses to “bring back” the wild ass, which supposedly went extinct in the 1960s and is known mostly from cave paintings in Europe dating back 20,000 years. (I remember seeing a half-breed at the Catskill Game Farm years ago and read the plaque about the efforts to restore them, a key moment in my drive to become a biologist. Oliver Ryder, who is the Kleberg Endowed Director of Conservation Genetics at the San Diego Zoo and co-author of the new rhino paper, is a Przewalski expert.)
Scenario 2: IVF. A second way to create embryos would use frozen sperm from northern white rhinos to fertilize an egg from Najin or Fatu in vitro and gestate the result, if viable, in a southern surrogate. But IVF is iffy, and the southern genomes not as diverse as those in the frozen fibroblasts from the top four animals. And those four are the basis of two more options that make more genetic sense.
Scenario 3: Stem cells. Expose the fibroblasts to cocktails of transcription factors to create induced pluripotent stem (iPS) cells, then coax them to differentiate as sperm and ova. Next, follow the IVF-surrogate route.
Scenario 4: Cloning. Isolate a nucleus from a northern fibroblast and transfer it to a southern rhino’s egg that has had its nucleus removed. This is somatic cell nuclear transfer (SCNT), aka cloning. Continue development in a surrogate.
Genetic Modification No, Epigenetics Yes?
As long as we’re talking iPS cells and SCNT, should we, could we, genetically manipulate the new rhinos to alter instructions for horn development, rendering the appendages stunted, absent, or otherwise unattractive to poachers? This occurred to me while reading about the tuskless elephants of Addo Elephant National Park in last Sunday’s New York Times.
But tusks aren’t horns. Nor are toothless mammals models for genetically engineering a hornless rhino, although it would be convenient if they were. Baleen whales lost their teeth when a jumping gene harpooned an enamel gene, enamelysin. And toothless anteaters, pangolins, sloths, armadillos, and aardvarks have a different gene, enamelin, altered into silent pseudogenes. Toothless turtles, crocodiles, and chickens harbor mutations in a gene that controls bone development, not keratin. Oh well.
A better bet may be to approach horn development via gene expression (epigenetics), not searching for an absent or otherwise mutant gene needed to craft a horn. Candidate genes? Those that control how keratin is distributed, including thyroid hormone, growth factors, and retinoic acid and other transcription factors.
Perhaps a cocktail of existing drugs that alter epigenetics targeted to keratin genes or the genes that control them would work. (The three categories of epigenetic intervention are methylation of DNA, modification of acetyl groups bound to the histone proteins that DNA loops around, and use of microRNAs to suppress activity of certain genes.)
The cocktail might be delivered in a capture-and-release program, or even in feed, to southern rhinos gestating northern embryos created via stem cells or cloning. And while the new population grows, geneticists could continue hunting for a more permanent solution. Meanwhile, the new generation of northern white rhinos could be introduced into the abundant habitat in the north, along with their epigenetic elixir.
What makes the most sense, of course, is to stop the poaching.