GVL members Andrea Schreier, Mandi Finger, and Daphne Gille (in collaboration with Cramer Fish Sciences) were awarded a grant by California Department of Water Resources to bring stakeholders to the table to develop a reintroduction plan for Delta Smelt. The is the first step in coming to terms with the fact that we may need to use hatchery fish to supplement/reinforce the wild population while restoration projects are underway.
One of my favorite projects over the years is the very first project I worked on – the reintroduction of the Paiute cutthroat trout (PCT) to its native habitat in Lower Silver King Creek (SKC), California.
The PCT has the narrowest range of any native trout subspecies in the world. It is endemic to a ~10 mile stretch of stream above a fish barrier in SKC. Lower SKC’s upstream barrier is a waterfall called Llewellyn Falls. SKC is a headwater tributary to the East Fork Carson River. PCT are notable for their near complete lack of spots:
I decided it would be a good time to provide an update about the project, as I just visited SKC to help out with electrofishing sampling.
First some history: if we go back to 1912, some PCT were moved above a fish barrier (Llewellyn Falls) by a Basque sheepherder. This is fortunate, because subsequently multiple stockings of various trout species occurred in Lower SKC during what can be considered the fish stocking heydey of the 20th century. Being an isolated subspecies with no natural competitors, the PCT was quickly outcompeted in Lower SKC, leaving a few refuge populations of genetically pure PCT above Llewellyn Falls. Without this inadvertent “rescue” the PCT might be extinct.
I’m going to leave out a lot of history of chemical treatments and restocking efforts here. Please see this page for some sources of more information.
Fast forward to the early 2000s. Having long recognized that the PCT was threatened yet has high potential for recovery and restoration, the Department of Fish and Wildlife, US Forest Service, and US Fish and Wildlife Service planned on chemically treating Lower SKC to remove nonnative fish. After a number of legal battles that fought to stop the chemical treatment, in 2005 everybody was all set to go and begin the treatment the following morning. However at the very last minute a judge blocked the treatment, sending hikers out with headlamps in the middle of the night to ensure that agency biologists knew that they could not proceed. This was a bit rude given that the biologists had already received word of the judge’s orders via radio. It was not a happy night.
2006 is when I began working on the project. First designing assays to detect hybridization so that we could assess the Lower SKC population. Second I wrote a paper in collaboration with Eric Anderson at NOAA that examined this population further, and showed variability in hybridization amongst the various markers. In 2013 I wrote a Genetic Management Plan for the reintroduction efforts. In the meantime CDFW and the rest of the project team had finally gotten the green light to begin the chemical treatment in 2013. Treatment was conducted in 2013, 2014, and 2015. Lower SKC is now fishless, save PCT getting washed downstream.
At present we are waiting on eDNA results, snorkel surveys, and the lower fish barrier assessment to make 100% sure that the lower fish barrier is intact and there are no rainbow trout in Lower SKC. This is critically important – if any rainbow trout are left behind in Lower SKC, the monumental efforts to chemically treat are likely to have been wasted. We would be back at square one.
Enter the historic California drought from 2012-present, and the latest surveys of the extant refuge populations. The refuge populations in Upper SKC have been regularly surveyed every few years for decades using electrofishing. The first year I worked on the project (2006) I visited SKC and watch the Wild Trout Crew shock Upper and Lower SKC. It was incredible how many fish we found. The populations were healthy and thriving.
Over the past few years coinciding with the drought, the census numbers have dropped precipitously in Upper Silver King, Four Mile Canyon Creek, and Corral Valley Creek. As there is plenty of water in the basin (it’s largely decomposed granite, which acts as a sponge), the prevailing theory is that the drought has resulted in an absence of insulating snowpack, causing the formation of anchor ice, further resulting in winter fish kills. Whatever the cause, the outlook is not good, and we are now left with two known healthy populations in-basin (Coyote Creek and Fly Valley Creek), four populations known to be struggling (Upper SKC, Four Mile Canyon Creek, North Fork Cottonwood Creek (an out of basin population in the White Mountains), and Corral Valley Creek), and at least three whose the status is unknown due to access difficulties.
The whole situation is sadly ironic; it was environmental groups that fought the chemical treatment back in 2005 (pushing back against the use of rotenone, the use of gas-powered equipment in a wilderness area, and what they thought was an insufficient EIS), and now we are worried about the extinction of an iconic trout subspecies. If the PCT had been reintroduced in 2006, their habitat are would have been significantly expanded, and we might not be so worried. At this point we are concerned about whether or not there are enough fish to take from the refuge populations to introduce in lower SKC. It would be a mistake to take so many fish from depleted refuge populations.
What we can do now is wait for the final confirmation that lower SKC no longer has rainbow trout. PCT will be washing downstream into Lower SKC either way. Ideally we would reintroduce a number of fish from the various refuge populations to ensure maximum genetic diversity. Conservation is rarely so straightforward. We must work with what we’ve got, and remember that doing nothing is also a choice.
This is a unique project. Very few areas are able to utilize chemical treatments safely and successfully to remove fish. The PCT is native to a small area, we still have genetically pure individuals, and the reason for their decline is removed when the nonnative fish are removed. We still have hope that the restoration will be successful.
Below are some movies from the latest trip.
Amanda Coen and Shannon Kieran were recently awarded a Jastro Shields grant and Natural Reserve System grant, respectively, to cover a portion of their research expenses. Amanda has also received an NSF Graduate Research Fellowship Program Honorable Mention which is a high honor coming from such a prestigious funding program. Way to go, Amanda and Shannon!
by Andrea Schreier
Sometimes scientific discoveries occur by pure chance – a scientist stumbles upon a new phenomenon without even trying. The discovery of spontaneous autopolyploidy, or abnormally increased genome size, in white sturgeon provides a perfect example.
When I began my PhD work in 2006, I was tasked with helping a senior graduate student develop new microsatellite markers for green and white sturgeon. Once polymorphic markers were identified, my first dissertation chapter focused on how these markers were inherited in white sturgeon. This may sound like a very pedestrian study but white sturgeon are ancient octoploids, meaning each individual possesses eight genome copies. It was unknown at the time how their genome was structured and I needed to confirm that microsatellite markers were transmitted in a Mendelian fashion before I could use them to answer genetic questions in that species. To accomplish this, I examined transmission of microsatellite alleles from parents to offspring in 15 half-sibling families produced at a local caviar farm. When examining the genotype data, I noticed right away that one of the female parents (Y192) had more alleles than she should have (>8) at several microsatellite loci. Interestingly, several of her offspring also had >8 alleles at one or more loci. While a normal octoploid should transmit only four gene copies to its offspring, Y192 transmitted >4 gene copies per locus. What could be going on?
The first thought that came to mind was potential contamination. Perhaps my DNA extracts or PCR reactions for Y192 contained DNA from more than one individual. The fact that the progeny of Y192 had >8 alleles suggested that I was observing a legitimate biological phenomenon rather than an error made at the lab bench. To be certain, I re-extracted DNA from Y192 and re-genotyped her at eight microsatellite loci using new reagents. As expected, I got the same results: >8 alleles at several loci. The only possible explanation for this result was that Y192 wasn’t octoploid but had a higher ploidy level. But how could I test this hypothesis?
It was around that time that I first met Daphne Gille, a PhD student from another lab who was looking to collaborate with the GVL. It just so happened that Daphne was an expert in flow cytometry, a technique that uses fluorescent dye to measure the DNA content of living cells. Now all we needed to test the hypothesis that Y192 had abnormal ploidy was a sample of her blood and access to a flow cytometer. We learned that UC Davis had a flow cytometry core facility. We were ready to go!
It wasn’t possible initially to collect blood from Y192 but Daphne and I decided to randomly sample a small number of white sturgeon at the farm just to validate our flow cytometry protocol and collect some preliminary data. Out of the six individuals we happened to sample, two had elevated ploidy! These abnormal individuals had a genome size 1.25x the normal octoploid (8n) genome size suggesting they where decaploid (10n). Subsequent sampling events at the caviar farm identified individuals that were dodecaploid (12n).
We inferred that Y192 was 12n and when crossed with an 8n male, produced 10n offspring that naturally possessed >8 alleles at each locus. Because the increase in genome size from 8n to 12n occurs spontaneously (no human intervention), we refer to this phenomenon as spontaneous autopolyploidy. The spurious discovery spontaneous autopolyploidy lead to a whole new line of research for me that continues to this day. Stay tuned for future posts about our investigations into the labile genome of sturgeon…
by Shannon Kieran
The GVL works on several environmental DNA projects. Environmental DNA is an emerging field in biology and ecology. Generally, eDNA is any genetic material found in the environment, rather than harvested from tissue, including poop on the trail, tufts of fur caught on a bush, the water a fish swam through, or the twigs a bird nested in. It’s used in ecology to monitor the presence and absence of species of concern.
Recently, GVL members have teamed up with the California Department of Fish and Wildlife to develop eDNA protocols to monitor a number of vernal pool organisms. Vernal pools are temporary, seasonal ponds and contain specially-adapted plants and animals, many of which can live in no other environment. The GVL protocol involves filtering water from these pools and extracting DNA from that water to determine if vulnerable vernal pool species are present in that body of water.
Among the species GVL is monitoring is the Vernal Pool Tadpole Shrimp, Lepidurus packardi:
The vernal pool tadpole shrimp is an endangered branchiopod in the order notostraca. Despite living in a variable, rapidly-changing environment, is has remained noticeably unchanged by evolution for the last 250 million years (which is about 200 million years older than Tyrannosaurus rex).
GVL is also monitoring three species of fairy shrimp which are endemic to California vernal pools (meaning they live nowhere else in the world): The Midvalley Fairy Shrimp, the Conservatory Fairy Shrimp and the Vernal Pool Fairy Shrimp, Branchinecta mesovallensis, conservatio and lynchi respectively:
These endangered fairy shrimp are, like all fairy shrimp, in the order anostraca. They are important food sources for migratory birds
Finally, GVL is monitoring the presence of the California Tiger Salamander, Ambystoma californiensis, which is facing habitat fragmentation and is threatened by climate change:
These are California Tiger Salamander larvae at various stagse of development, all found in the same pool near Merced, CA.
To validate our protocol, California Department of Fish and Wildlife biologists come with us and we dipnet each pool, which is the current standard protocol for monitoring. Here is the team hard at work:
Dipnetting is a process of moving a large, fine mesh net through a pool in order to visually count and identify the creatures found in it. Along with fairy shrimp and salamanders, we often find the water flea daphnia, the tiny, bright red copepods in the vernal pools.
by Bernie May, Research Professor, Department of Animal Science
The lineage that gave rise to current day salmonid fishes (salmon, trout, whitefish, grayling, and char) went through a polyploid event 30-50 million years ago that doubled their number of chromosomes. This phenomenon meant there were four copies of each chromosome (tetraploid) instead of the normal two copies that most organisms, including humans, have (diploid). Since that polyploidy event occurred, the salmonid genome has continued to evolve such that there are now only two copies of each chromosome, with some having residual tetrasomic regions on the ends of the chromosomes (telomeric regions) reflective of their polyploid origin. These residual tetrasomic regions allow normally disomic chromosomes to pair multivalently in salmonid males that upon gametic segregation give rise to residual tetrasomy for these telomeric loci and in some cases to pseudolinkage where loci on different chromosomes appear to be linked, although the segregation shows an excess of recombinant types over parental types. This change from a tetrasomic to a primarily disomic genome has provided the salmonids with more loci that have been able to evolve more specialized functions in metabolic pathways. The unusual meiotic pairing and gametic segregation of these telomeric regions in male salmonids is reviewed and explained in a recent paper by Bernie May (GVL founder) and Mary Delany (CAES Executive Associate Dean), a topic they studied together over 35 years ago when they first met at The Pennsylvania State University.
By Alyssa Benjamin
The Arroyo chub (Gila orcuttii) is a small omnivorous fish native to Southern California coastal streams whose existence has been severely impacted by human development. Because their range overlaps with the densely populated greater Los Angeles area, many streams and rivers once rife with habitat have now been paved over, fragmented, or eliminated completely. The recent decline of Arroyo chub has prompted an urgent need to better understand the remaining populations.
In our study, newly published in Transactions of the American Fisheries Society, we used genetic information as a way to understand the Arroyo chub populations and determine appropriate conservation strategies. Genetic data is especially valuable because it can provide important clues as to the current and historical relationships and status of populations that may not be apparent by simple outward inspection of fish.
For this study, we analyzed over 200 Arroyo chub collected at different sites from six native watersheds and genotyped them using microsatellites. Microsatellites are regions in the genome, generally in non-coding areas, that have a repeating sequence of nucleotides (e.g. ACTGACTGACTG…). The number of repeated units varies greatly between individuals which makes microsatellites useful for looking at population level differentiation.
Our analysis of the genetic data revealed two main points. First, we found a high degree of fragmentation between populations. Essentially, every sampled watershed had a unique Arroyo chub population and some watersheds even had multiple unique populations within the same river. This genetic differentiation is consistent with the overall isolation of fish as a result of human development, habitat loss, the current drought, and flood control measures.
The second finding was that most of the Arroyo chub populations, despite fragmentation and isolation, have managed to retain a lot of genetic diversity thus far. When populations show higher levels of genetic diversity, they are generally better able to adapt to environmental changes and are considered “healthier” populations. However, the fact that most of the Arroyo chub populations are small and fragmented means they have a higher risk of losing genetic diversity in the future. While we did recommend some possible strategies for conservation in the article, habitat restoration will ultimately be most essential to securing the future of the Arroyo chub.
Reference: Alyssa Benjamin, Bernie May, John O’Brien & Amanda J. Finger (2016) “Conservation Genetics of an Urban Desert Fish, the Arroyo Chub.” Transactions of the American Fisheries Society, 145:2, 277-286. DOI: 10.1080/00028487.2015.1121925
by Molly Stephens
The molecular mechanisms underlying an animal’s “decision” to migrate are poorly understood, yet essential to unraveling the mystery of migration in many diverse species. Our new study, published as part of an upcoming Molecular Ecology special issue, ‘Epigenetic Studies in Ecology and Evolution,” offers a potential mechanism: epigenetics. Epigenetic modification, in this case DNA methylation, can alter the expression of genes without modifying the underlying DNA sequence. This type of regulation may provide an important link between external environmental migratory cues and the outward physical and behavioral appearance of a fish, producing rainbow trout (Oncorhynchus mykiss) individuals with one of two very different alternative life history strategies from the same population: smolts or residents (see figure at right).
To explore this potential link, we quantitatively measured genome-scale DNA methylation in fin tissue using a reduced representation bisulfite sequencing (mRRBS) technique on fish produced from crosses of non-migratory steelhead and migratory resident rainbow trout clonal lines. We found 57 differentially methylated regions between smolt and resident juveniles and identified several molecular categories of genes, including some with clear associations with migration, such as the circadian rhythm pathways.
This study provides the first evidence of a relationship between epigenetic variation and life history divergence associated with migration-related traits in any species and demonstrates the power of large-scale epigenotyping in ecological studies. For conservation purposes, resident and migratory rainbow trout are often managed separately based on their environmental and genetic variation. Careful consideration should also be given to the mechanisms by which these factors may interact to produce observable traits in this and other species.
Contact: Melinda Baerwald email@example.com
Reference: Melinda R. Baerwald, Mariah H. Meek, Molly R. Stephens, Raman P. Nagarajan, Alisha M. Goodbla, Katharine M.H. Tomalty, Gary H. Thorgaard, Bernie May, and Krista M. Nichols. 2015. “Migration-related phenotypic divergence is associated with epigenetic modifications in rainbow trout.” Molecular Ecology. doi:10.1111/mec.13231