Monday, January 12, 2015

Of Salamanders and Men

A red-bellied newt in hand in the field  
"In addition to being a delightful place to live, northern California offers an asset that is perhaps less well known to the millions who have been flocking there in recent years: In my somewhat prejudiced opinion, its newts are unrivaled anywhere in the world!"- Victor Chandler Twitty, Of Scientists and Salamanders, 1966
While they were sorting through the genetic code, the Berkeley team asked us to keep quiet about the unusual sightings of red-bellied newts in Santa Clara County until they had published their results. As a child of two university professors, I was not surprised by this request. So we did what we were best at, tromping around in the forest to learn more about this population especially where they lived.

I dove into the literature to tease out tips on the behavior of red-bellied newts which might help us focus our surveys. Many of the red-bellied newt publications were by Dr. Victor C. Twitty and students in his Stanford laboratory.

Dr. Twitty first described red-bellied newts as a separate species in 1935 and he continued to study their behavior and biology for 32 years. Indeed, throughout this time, his academic home was Stanford University located in Palo Alto, California at the foot of the Santa Cruz Mountains, the very same mountain range that shelters the Stevens Creek watershed. Did our improbable discovery of a disjunct population of red-bellied newts in Stevens Creek have anything to with Twitty's groundbreaking salamander research decades earlier, especially his approach, unusual in academia at that time, of combining laboratory experiments and field research? Or were Twitty's and our discoveries random and unrelated?

Dr. Twitty attested to the mix of random and methodical events that shape a scientific career and led him to invest so much of his seasoned years into this small amphibian in his popular book, Of Scientists and Salamanders.
"The role of chance in research and discovery is greater than is generally recognized, and this will be well exemplified from microsurgery to natural history and back again, and from the study of cell populations in tissue culture to the study of animal populations in the streams and hills of northern California."
Walking the Stevens Creek watershed   
Although microsurgery was not my bailiwick, wandering streams and hills of California was, so I bought a copy of the out-of-print book and journeyed back decades in its pages searching for clues. As the park team hiked in and out of Stevens Creek gradually adding more dates and more locations where the red-bellied newts were found, and narrowing down the period of the year when they make their above-ground breeding appearance, I shared anecdotes about Twitty and his remarkable research findings. We kept comparing our sightings under the oaks, madrones, maples, and Douglas firs of Stevens Creek to his published words. Most of his words came from a special place called Pepperwood Creek in Sonoma County, named after a common tree with spice-scented leaves, a tree most of us now call California bay. It was a twisty road that took him to Pepperwood Creek 140 miles from the Stanford campus, and his time there with the newts unquestionably changed his life.
Victor C. Twitty as young embryologist at Yale University.
Image from Embryo Project Encyclopedia
ISSN: 1940-5030     
Victor Twitty was born in Indiana in 1901. When he was a graduate student and then instructor at Yale University from 1929 to 1931, he first studied salamanders as experimental subjects in the laboratories of early embryologist Ross Granville Harrison. Salamanders were popular subjects for embryology since their embryos more readily accept foreign grafts than other types of vertebrates. Much of the early embryology experiments consisted of taking tissue from one area of the embryo and implanting it into another location on the same embryo or even into another type of salamander to see how the host and donor cells responded and thus detect the stimuli that guide the differentiation of embryonic cells.

When he originally came to Stanford in fall of 1932, after a one-year stint as a National Research Council Fellow in Germany at the Kaiser Wilhelm Institute in Berlin, Twitty was relieved to find an academic position during the Great Depression. He continued to cut up salamander embryos grown from the familiar tiger salamander eggs which his East Coast associates shipped to him, but he also ventured into the Santa Cruz Mountains to find the abundant eggs of the California newt. On his first foray into the hills above Stanford, he described collecting on a "pond floor almost solidly carpeted by the spherical jelly masses in which the eggs are deposited" - a bounty of study subjects for the young professor.

Warning colors - this newt contains tetrodotoxin, a powerful neurotoxin.   
One of his earliest findings at his California dissecting microscope was at first a frustration and then an important clue to the physiology of his new research subjects. Whenever he grafted the embryonic eyes and limbs from western newts into eastern salamander embryos, all growth would freeze in the eastern salamander larvae for awhile. This led to another set of experiments in which the ground-up tissues of western newt embryos were injected into well-developed eastern salamander larvae. Even though the later were at an active stage, they always became paralyzed for an hour or two after the injection. Frogs, toads, turtles and mice were likewise paralyzed after similar injections, fatally so for the mice. Twitty and other Stanford professors were on the trail to discovering a potent neurotoxin that is present in all four of the western Taricha newts - initially called tarichatoxin and eventually determined to be the same toxin as in Japanese puffer fish, tetrodotoxin. The Stanford labs were expanding their exciting salamander research and that required more study subjects.

Red-bellied newt on left, rough-skinned newt on right.
Hard to imagine that these two newts were not recognized as separate species until 1935, however, maybe this is due to the limited season that the red-bellied newt is above ground and the remoteness of much of its streams.   
Twitty's need for more newt embryos and his love for fishing California streams took him farther afield to collect newt eggs. In June 1933, Twitty traveled 150 miles north to Ukiah, a location he heard that salamander eggs could be found late in the California breeding season. Successful in this venture, he returned again and earlier in 1934 and 1935 for more collecting. Through observations in the field and subsequently in the lab, he noticed that the Ukiah salamander eggs, larvae and adults looked different than those he collected along the central California coast. Eventually, Dr. Twitty determined that he had been collecting three* species of Taricha newts in California with somewhat distinct breeding seasons and habitats and partially overlapping ranges. The Ukiah collecting trips provided Twitty with the opportunity to distinguish and describe a new species, the red-bellied newt (Taricha rivularis), and a known but commonly confused species, the rough-skinned newt (Taricha granulosa initially called T. similans by Twitty) from the more commonly recognized California newt (Taricha torosus).

California newt larva showing two dark dorsal bands.   
Without the genetic tools we have today, the Twitty lab plunged into hybridization experiments between the three similar-looking Taricha species by removing eggs from oviducts of spawning females and treating them with sperm from males of the other two species. The hybrid embryos developed normally through the larval stage but as they transformed into juveniles, the researchers found it difficult to meet their new diet requirements in the lab and were initially unable to keep the hybrids alive.

Red-bellied newt larva with black pigmentation spread evenly across back and sides. As dwellers of the fast-moving portions of streams, red-bellied newt larvae have a dorsal fin that ends before the head and other modifications for life in rough water. Photo by Gary Nafis at   
This was not about to stop the curious minds of the Twitty lab. To answer questions as to how closely these three species are related, they needed to raise the hybrid larvae to maturity to determine if they were able to successfully breed. As Twitty explained, "One cannot become so deeply involved as I had in the systematics of a genus of organisms without wondering about such things as the nature and extent of the evolutionary divergence that the species have undergone."

Rough-skinned newt larvae with black pigmentation somewhat banded in a pattern intermediate between  the California  newt and the red-bellied newt.  Photo by Gary Nafis at   
If the Stanford-produced hybrids could mature and reproduce, then this indicated that the parent species had probably diverged from each other in relatively recent time on an evolutionary scale.

The lengthy descriptions of embryological processes and lab experiments in Of Scientists and Salamanders started to overwhelm me. The Twitty lab seemed far away from our wanderings at Stevens Creek.

But as I slowly found the time to read on, I was surprised to see how Dr. Twitty's academic focus suddenly shifted in 1953. Unexpected circumstances brought him to Pepperwood Creek and over the next thirteen years, he and his students conducted a flurry of research experiments on red-bellied newts in their natural habitat. In 1966 he published Of Scientists and Salamanders, partially to share this remarkable change in career, and then within six months he committed suicide. Our little Stevens Creek team couldn't help but wonder if this sad event contained any clues to how our and Twitty's paths crossed.

To be continued as The Pepperwood Creek Affair.
This post is the eighth in a series on the discovery of red-bellied newts in the Santa Cruz Mountains of California. To start at the beginning of the series go to Mystery of the Red-Bellied Newt.

*Eventually, a fourth species, the Sierra Newt of the Sierran foothills, Taricha sierrae, was recognized as a separate species from the similar looking California or coast range newt.

Rough-skinned newt, Taricha granulosa
California newt, Taricha torosa
Red-bellied newt, Taricha rivularis
Eastern tiger salamander Ambystoma tigrinum formerly Amblystoma t.
California bay, Umbellularia californica

See also:

Victor Chandler Twitty, Stanford University, 1966, Of Scientists and Salamanders, W.H. Freeman and Co., San Francisco. All quotes in this post are from this book.

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