Progress 09/01/19 to 01/30/21

Outputs
Target Audience:There are four target audiences of the product(s) we seek to generate: 1) California State mussel farmers, 2) the West Coast aquaculture industry, 3) seafood consumers, and 4) student communities. For California State mussel farmers, more options for aquaculture will allow for a more competitive sector. For the entire West Coast aquaculture industry, a new crop variety may help alleviate issues with seasonal disease, regional die-offs, OA, and natural mortality (predation). Utilizing a native mussel species may lower barriers of entry for new Southern California based seed producers, adding stability to the West Coast industry, as currently a vast majority of bivalve seed products are sourced from only a very small handful of operations in the PNW. For consumers, more variety is always better, and more-so if said options represent a localized 'seed to table' model. Finally, the future of sustainable aquaculture must be bolstered by informative public education on the topic. We delve further into each target audience next, with related "Efforts" achieve during Phase I of this project: California State mussel farmers: The market opportunity in California alone presents a lucrative incentive to deliver the California Mussel as a new aquaculture species. Currently operating aquaculture in the state is ~6,000 acres, distributed across 14 farms, with potential and incentive to expand. Two examples of local mussel farms currently invested in producing and selling mussels include Santa Barbara Mariculture's 25-acre plot and the Pacific-6/Pacific Mariculture's (previously Catalina Sea Ranch) 100 acres in federal waters off of Southern California. Both sites have historically and/or currently purchase seed from Whiskey Creek Hatchery in Washington, primarily because a source of local seed simply does not exist. Other PSW mussel farms are also expected in the next five years. Growing non-native Pacific Northwest derived mussels presents a major disadvantage for SBM, Pacific6, and any other Southern California based grower- the cost of production is higher, animals underperform compared to PNW counterparts, and the mussels are more vulnerable to disease and other factors that lead to loss. Clearly, there is a niche that the California Mussel may be destined to fill. Holdfast will help promote market expansion of CM in California through direct marketing with their extensive network of seafood distributors and end-users (mostly chefs). They will also leverage their network of academic and non-profit partners that specifically work on changing public perception of aquaculture, including California Sea Grant, the Wrigley Institute, Aquarium of the Pacific, and Port of San Diego to advocate for CM. Marketing campaigns to the general public will highlight the hyper-local 'seed to table' pipeline that a CM market will establish in California, adding to the list of environmental benefits already delivered by shellfish aquaculture. Phase I Efforts: Because Santa Barbara Mariculture and Holdfast Aquaculture are in the commericalization stage of this project, no efforts to educate the West Coast commercial industry have yet been made. Once the project completes (after Phases II and III) documents concerning the rearing, grow-out, and economics of the California Mussel are expected after appropriately protecting intellectual property generated. The US West Coast Aquaculture Industry: After becoming fully established in California, commercialization efforts will shift to the West Coast shellfish industry, valued at $103M (USDA 2017 State Agriculture Overview). Like other California agricultural products, we anticipate the West Coast and US to embrace the CM as another high-quality product of California, not only responsibly grown but extremely nutritious. The name 'California Mussel' or similar will be trademarked as a unique product of California, and national and global marketing strategy goals will aim to obtain the same status as the trademarked New Zealand Green Lipped Mussels. However, the so-called "California mussel" has a range that extends from Baja through Alaska, and so regional production might be developed along this entire range. One can envision something similar to the oyster industry, whereby individual farms and sites market their specific niche varieties. This project therefore serves the entire West Coast industry, as more available species create resilience in the market, can provide more economic opportunities, and can serve as a model for bringing other under-utilized species into aquaculture production markets. Phase I Efforts: None. Seafood Consumers: Santa Barbara Mariculture and Holdfast have a common goal to produce sustainable seafood in close proximity to the consumer. This project supports these priorities by adding a new sustainable option which, critically, might outperform other non-PSW derived options such as the oyster or Mediterranean mussel. Consumers benefit further from knowing that California mussel seed does not have an added greenhouse gas footprint associated with shipping costs from out-of-state seed or seeded line purchases. Finally, our group's collective mission is to deliver food to under-resourced communities that have not historically had access to these healthy and nutritious foods. Phase I Efforts: Santa Barbara Mariculture and Holdfast Aquaculture have begun an outreach effort (the 'Aquaculture Hub') which seeks to study the perception of bivalves in and under-resourced community food program, both before and after an educational cooking video is watched by consumers. This project, in association with colleagues at USC and Community Services Unlimited, will inform future best-practices and -approaches for introducing new seafood species into markets that historically do not have sustainable local seafood options. We anticipate that the California mussel, once produced at a commercial scale (from Phase II efforts), can be used in this and similar programs as both an informal education tool for the general public and to continue to support and/or subsidize under-resourced community food programs. Student Communities: In order to ensure that the future reaps the potential benefits of sustainable aquaculture, the subject needs to be taught at all educational levels, with programs tailored to appropriate student levels. We therefore consider student communities as Target Audiences for this work. Co-PI Dr. Sergey Nuzhdin and collaborators at Holdfast Aquaculture (Dr.s Diane Kim and Nathan Churches) hold professorial positions that allow for dissemination of information in both formal and informal learning environments at the college level. Additionally, educators at Holdfast Aquaculture host high school interns and have voluntarily lectured at various local schools and events. Phase I Efforts: An informal educational project is being undertaken at Holdfast, in collaboration with AltaSea and SnapChat, to produce a 'virtual dive' educational program for high school students. Participants will use SnapChat's Lens Studio to create an underwater farm, participate in a 'virtual SCUBA dive', and access educational information built into the 3-D world, similar to a video game. The primary learning objectives are for students to be able to describe what sustainable aquaculture is, label and identify several local species, and discuss the benefits of a multi-trophic aquaculture system. While currently informal, this program could be adapted to existing curricula and/or museum and aquarium audiences after Beta testing. This program could help reach many young student communities via during a Phase II efforts. Changes/Problems:The Coronavirus significantly altered the work schedule for Phase I, and directly prohibited the possibility of working at the Wrigley Institute for Environmental Science (Goal 1), resulting in a setback due to the necessity of generating a new hatchery site, and subsequently stalling efforts to produce 'significant CM stocks' (Goal 2 and 3) which were necessary to enable both a full economic assessment of CM (Goal 2) and genetic improvement approaches (Goal 3). One other major factor contributing to the failure to produce an on-farm run of the California mussel was an outbreak of vibriosis at the Holdfast Aquaculture site, which ultimately led to high mortalities of line-settled mussels. These types of bacterial infections are unfortunately somewhat common in the industry and have resulted in multiple years of halted production in some cases. The reason behind the consistent larval mortalities (bacterial contamination) was not deduced until the end of the Phase I period. Vibrio is suspected as the key factor for failure to grow settled CM to juvenile stages because the team at HF was able to settle a suspected vibriosis-challenged batch of CM (see Phase II figures for successfully settled CM) by utilizing an over-the-counter aquatic antibiotic treatment, whereas batches that did not receive the antibiotic treatments did not settle. HF understand the gravity of getting a handle on this key water quality parameter. To this end, HF continues to consult with an industry expert who also successfully dealt with a similar vibrio outbreak at their hatchery, regarding appropriate and effective preventative protocols (Peter Struffenegger, retired, previous production manager at several West Coast aquaculture ventures). The most likely culprit for source infections was deemed to be sodium thiosulfate crystals, which, surprisingly, have been shown to harbor harmful bacteria, rendering chemical water treatments obsolete. It is unfortunate that this outbreak stunted the Phase I effort, but HF has a plan to eliminate the problem during Phase II. Namely, heavier investment in UV lamps in recirculation scrubbing systems, continued consultation with our industry expert, and a rigorous weekly standard operating procedure involving bacteria-specific marine agar plate swabs from all possible water sources and water-interfacing equipment. These precautionary measures will be implemented in Phase II to ensure near-axenic larval cultures. Allowing us the leeway to paraphrase the overarching goal of Phase I as an effort to establish a production hatchery, record mass-spawns, gamete conditioning potential, and settlement onto lines for the California mussel for the first time, and to produce important scientific data for the industrial and academic field of aquaculture, then Phase I was successful despite the hurdles of 2020. What opportunities for training and professional development has the project provided?Throughout Phase I and in the interim between Phase II funding, SBM owner/operator Bernard Friedman has been interacting with regulatory groups in order to verify proper management of the farm in regards to long term permitting of the CM. In this process, SBM has prepared and submitted a formal request to add the CM as a new and permanent species for commercial cultivation on the lease (submitted Jan. 28, 2021). Importantly, the United States Army Corps of Engineers has already signed off and no further regulatory action is need by them, and the process now sits with the CADFW. This represents professional development, if at first apparently only in an oblique manner, because many multiagency and stakeholder workshops are necessary to push authorizations like these forward. Mr. Friedman is also a board member of Commercial Fisherman of Santa Barbara, whose mission is to "provide healthy,high quality seafood to local and global markets, ensure the economic and biological sustainability of fisheries, and maintain California's fishing heritage". In their bi-monthly meetings, the group discusses issues related to commercial fishing, including regulation and advocacy for the commercial fleet. This represents professional development for both SBM and the members of the CFSB, because these conferences allow for smoother implementation of longline aquaculture efforts in the region via continuous stakeholder feedback and engagement on the topic. How have the results been disseminated to communities of interest?As mentioned elsewhere in this Final Report, the collaborative team here consists of several academics who regularly lecture and engage student communities (high school through graduate level). For one example, Dr. Churches has incorporated an entire "Aquaculture" lecture in his environmental science courses at Mount Saint Mary's University, where he includes a background of this project specifically and the overall potential of the California mussel (and other new aquaculture species targets). This is an example of formal education, whereas the "Virtual Dive" project represents an informal educational opportunity for high school students. In this program, students create an underwater multi-trophic aquaculture farm using SnapChat's Lens Studio program, and get to "dive" the site before and after installation, all the while observing and learning the changing dynamics of the waterway as a function of sustainable farmed seafood. Other programs that work to disseminate results from this work include the Aquaculture Hub, a collaboration with Community Services Unlimited and other USC colleagues, which will eventually generate informational and educational cooking videos to encourage consumption of local sustainable bivalves in under-resourced communities. A study measuring the impacts of this kind of information dissemination is forthcoming, and this could be further supported with support in Phase II. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Phase I outcomes: · A new commercial & research hatchery, established in San Pedro. · The California Mussel (CM) can be mass-spawned. · The CM can be induced to spawn (protocols developed). · The CM may be amenable to Gamete Conditioning System ripening. · Larval CM produced in a hatchery will settle onto fuzzy rope. · Juvenile CM will respond to re-socking techniques. · Market metrics: farm-grown CM may outperform PNW-derived crops. · Scientific studies: bivalves have an extreme generational mutation rate, with implications for selective breeding of CM. Goal 1 Major activities completed / experiments conducted: The Wrigley center was significantly impacted by the Covid-19 pandemic and was off limits. We constructed a new ~2500 square-foot aquaculture facility which contained all necessary aquaculture stations and equipment necessary to complete commercial production. Data collected: Site testing, including algae cell densities and quality, water parameters (temperature, contamination, chemistry [nitrates, nitrites, ammonia, pH, salinity, copper], and environmental parameters [room temperature and light]. Summary statistics and discussion of results: A new site was generated that was capable of both research level questioning and commercial-scale production. Key outcomes or other accomplishments realized: In general, Goal 1 was realized, though not at the original site. Changes in knowledge: NA Changes in action: NA Changes in condition: NA Goal 2 Major activities completed / experiments conducted: Activity: Wild monitoring, collections, and spawns. Several spawn and settling attempts. A significant amount of work: tides must be amenable and dearth of literature pertaining to the factors that might predict a gravid wild CM. Experiment: spawn induction protocol using hydrogen peroxide. CM are thought to be "trickle spawners", releasing gametes at low-but-consistent rates. A very important outcome of Phase I is the first reported evidence that mass spawns are both possible and potentially inducible. This increases their potential for commercial adaptability substantially. Key factors: A cohort of relaxed and filtering mussels. A 3% hydrogen peroxide in filtered salt water solution must be used. The solution must be expelled directly into the incurrent siphon with disposable pipette. Further notes: Time from collection did not seem to affect success. Activity: first ever demonstration of multiple mass spawns and settlement in hatchery produced CM larvae. The team was able to produce 8 mass spawns (>1M larvae); two spawns producing at a typical commercial scale (~20-50M larvae). From these latter two commercial scale spawns, the team was further able to demonstrate settlement onto industry-common fuzzy rope. This is the most significant outcome of Phase I. Experiment: settlement substrate preferences. No preference noted in non-traditional materials (rope, mesh, or cultch). Activity & Experiment: Gamete Conditioning Systems for the CM. Production of animals that appeared more ripe than wild-sourced counterparts using GCS systems. This bodes well for the potential to ripen the CM out of season and create a year-round supply of larvae. Activity: Market Metric data collection. Metrics include count per kilogram, weight per dozen, average size measurements, cooked meat-to-shell ratios, raw meat-to-shell ratios, survival time on ice, and shell strength crush tests. We generated this data for wild CM and several commercial Mytilid varieties including a Holdfast & Santa Barbara Mariculture produced set of Mytilus at 5 months post-outplanting and at 2021 harvest. Activity/Experiment: re-socking the CM. HF collected a few hundred wild juvenile (~0.5-2 cm) CM from area beaches, de-clumped and isolated them, and then successfully re-settled them to fuzzy ropes in a tank. This is the first unequivocal demonstration that the CM will reattach to a substrate after being completely removed, which clearly suggests that re-socking will not be a barrier to production for the species. Data collected (in correspondence with the activity/experiment letter designation above): General collection notes. Full notes regarding exact parameters of successful spawn induction protocol. Spreadsheets, phenotypes, photographic evidence. Experiment notes and photographic evidence. Water quality parameters (nitrate, nitrite, ammonia, pH) for GCS, recording of various GCS prototypes, and photographic evidence. Market metrics (Spreadsheets) Photographic evidence. Summary statistics and discussion of results: 1) CM can be mass-spawned, 2) spawning in CM may be inducible, 3) CM seems amenable to gamete conditioning systems, 4) CM larvae produced in a hatchery will settle onto fuzzy ropes, and that 5) juvenile CM will respond to re-socking techniques. In Phase II, considering this new data, we plan to pursue commercial scale production, which may result in the emergence of a new sustainable seafood for the US West Coast. Key outcomes or other accomplishments realized: Changes in knowledge: This work has resulted in a change of knowledge for both academics and industrial personnel on our team in terms of the understanding of the amenability for the CM for hatchery-based production. Changes in action: Santa Barbara Mariculture and Holdfast Aquaculture anticipate pursuing the CM further during Phase II, which qualifies as a change in action, considering that SBM long line acreage is at a premium monetary value Changes in condition: Much new data supporting CM aquaculture. Increased presence of nutritious ocean-sourced proteins in local food markets in under-resourced communities. Goal 3 Major activities completed / experiments conducted: Experiment: Mutation rate analysis. USC colleagues pursued generating data to directly measure mutation rate in the industry's best "model organism", the Pacific oyster. This is a necessary knowledge gap to fill, and guides Phase II efforts concerning the California mussel. The data suggests that the Pacific oyster (and likely many or all bivalves) has the highest generational mutation rate ever observed in eukaryotes. The implications of this study are huge for both industry and evolutionary theory. Data collected: Full genome sequencing of three sets of parental crosses and their adult F1 offspring, for a total of 18 genomic libraries. Many related datasets. Summary statistics and discussion of results: The team found that the mutation rate of the Pacific oyster (Crassostrea gigas) has a mutation rate on the order of 3 times higher than that of any previously directly observed eukaryote (plant, animal, fungi, or protist). Key outcomes or other accomplishments realized: Changes in knowledge: This will represent a change in knowledge to the scientific and industrial communities regarding the fundamental properties of mutation rates in plants and animals. Changes in action: Holdfast Aquaculture has changed its approach to selective breeding programs as a result of this data (see Phase II approach) Changes in condition: NA Discussion of stated goals not yet met: During Phase I our collaborative team was able to establish a new commercial hatchery facility (overarching theme of Goal 1), close many remaining critical gaps and questions regarding hatchery potential of the CM (overarching theme of Goal 2), and produce possibly groundbreaking scientific work that will inform best-practices for bivalve selective breeding approaches (overarching theme of Goal 3). We therefore are inclined to deem Phase I a successful effort in generating a platform for Phase II follow-on goals. The last major goal yet to be met is the establishment of commercial-scale quantities of hatchery-started and subsequently farm-grown CM. This will be the major overarching effort in Phase II.

Publications

• Type: Journal Articles Status: Other Year Published: 2021 Citation: Title: Pacific oysters (Crassostrea gigas) dramatically recalibrate models for the upper limit of the eukaryotic mutation rate. This article was submitted for review and returned requesting another experiment (the explanation of which is beyond the scope of this Final Report). In summary: our findings, which were supported by the Phase I grant funds, suggest that bivalves have dramatically higher mutation rates than previously appreciated. This finding has huge implications for evolutionary theories and for future selective breeding programs of any bivalves, including the California mussel. Follow up experiments and associated data are anticipated within the next few months (summer 2021) and this work will be resubmitted thereafter. NIFA will be appropriately acknowledged in the paper. Working Abstract: Advanced terrestrial selection breeding programs for food crops serve as a template for future domestication of ocean-based crops. One consideration for marine organisms and their suitability for artificial selection is their mutation rate. Bivalves, one of aquacultureâ¿¿s most heavily produced crop, are thought to maintain among the highest observed mutation rates. Predicted rates of de novo mutations have been indirectly calculated from mutation loads or segregation distortions, frequently from allozyme or microsatellite data in the Pacific oyster (Crassostrea gigas). Based on solid theoretical framework, the observed levels of heterozygosity, given effective population size, cannot be easily explained unless the rate of de novo mutations far exceeds other eukaryotes. However, to date, no trios (two parents and their offspring) have been sequenced at sufficient depth to directly evaluate this hypothesis. Using Pacific oysters, this study utilized three single parent pairings, and randomly selected juvenile individuals from each corresponding family, in order to empirically determine mutation rate. Our findings rank among the highest ever recorded for any organism (10-5 per nucleotide), and dramatically recalibrates the upper limit of the eukaryotic mutation rate from previous models. The authors realize the boldness of this claim, and encourage future work to confirm these results. If borne out, the data herein provide the first direct measurement of mutation rate in a broadcast spawning species with considerable commercial importance, thereby informing best breeding practices.

Progress 09/01/19 to 08/31/20

Outputs
Target Audience:The target audience for this project include industry partners invested in the development of sustainable aquaculture in Southern California, as well end-users of the mussel food product that include restaurants, wholesale/retail partners and individual consumers from direct retail sales. The industry partners include locally owned small businesses Santa Barbara Mariculture (SBM), the only mussel farm in Southern California now in operation for over 10 years, and Holdfast Aquaculture (HFA), the only mussel seed provider in the region and industry leader in developing locally sourced and locally adapted aquaculture species. SBM produces over 100,000 pounds of sustainable mussel products annually, and is the model for Southern California nearshore and offshore aquaculture. Unfortunately, SBM is in the position of purchasing seed from hatcheries that are based in the Pacific Northwest, which impacts product quality and generates an industrial bottleneck. HFA seeks to mitigate this constriction by generating locally produced seeds for SBM and other emerging aquaculture entities in the region. This work will also potentially benefit the Ventura Shellfish Enterprise (VSE) and individual farmers who lease the twenty 100-acre ocean plots designated for mussel farming as they become available in 2-3 years. VSE has hired SBM and other industry leaders in consultation for their project, and as such any products of this SBIR can directly influence or inform VSE output, planning, and species production. ? Changes/Problems: USC boat (Miss Christi) problems beginning Dec 2019 - had to switch experimental hatchery site to Holdfast in San Pedro. Covid beginning March 2020 caused significant delays. Anomalous temperature/weather patterns: One major issue encountered this summer was significantly colder than usual area waters. The California mussel typically ripens as a function of temperature and food availability, and 2020 had only the narrowest window of ideal temperature ranges (65-70F), with only a few weeks in the ideal range. Storm systems also moved through during the summer time-frame and the Southern California region experienced several cold water dips which may have hindered wild broodstock ripening. Indeed, the spawn attempts we were able to successfully generate came at the end of long and stable warm periods, and spawn success typically dropped off after cold water dips. Vibrio infections: One of the major ordeals challenging the success of this project has been massive larval die-off in the hatchery. HFA was able to secure 3-4 high volume spawns of the Calfiornia mussel, with the best batches having a maximum of ~30M larvae produced. This would/should equate to approximately 4000 feet of line per spawn run, if larval survival remained high. However, at approximately 2 weeks and for multiple species, HFA experienced massive mortalities. Water chemistry and all other variables were addressed to the best of our ability and in consultation with other hatcheries, but changes in systems did not lead to better success. The second settling experiment (ongoing and described above) allowed HFA personnel to perhaps finally identify the culprit: vibrio bacterial infections. The bivalve literature says nothing in regards to California mussel vibriosis, but other examples with oysters and scallops indicate very similar phenotypes to those observed at HFA. After noticing this congruence, HFA began implementing low levels of antibiotics and saw a decline in the mortality rate for the latest batch of California mussel larvae. This revelation was only recently discovered, and mitigation is an ongoing discussion with the team. The authors hypothesize that they will be able to generate commercial quantities (many tens of millions) of California larvae if vibriosis is indeed the culprit, via bacterial mitigation techniques based on literature (e.g., Karim et al. 2013). What opportunities for training and professional development has the project provided?This grant provided professtional development and research training for one graduate student. The grant also enabled an online internship program with the Port of LA High School to support two high school students interested in sustainable aquaculture. How have the results been disseminated to communities of interest?Personal communiations with stakeholders, including industry partners and emerging businesses such as the Ventura Shellfish Enterprise, have taken place and information about the project can be found on Holdfast Aquaculture's website. What do you plan to do during the next reporting period to accomplish the goals?Authors will continue working onTO.2 (Using data generated from TO.1, determine CM's potential and commercial feasibility for full-cycle, hatchery based, longline aquaculture, including an economic assessment), as well asTO.3 (Generate significant CM stocks to provide pathway for selective breeding and genetic improvement approaches in Phase II). This grant was extended to Jan 31, 2021.

Impacts
What was accomplished under these goals? Starting with the Phase I Technical Objectives, TO.1 was not fully realized as originally planned because of problems with the Miss Christi (USC's commuter ferry) and inability to reliably access the island's facilities, exacerbated further after the Covid-19 pandemic. This has been documented in other reports regarding this SBIR. Because of this logistical issue, the majority of the work has taken place at Holdfast Aqaculture (HFA) facilities in the Port of Los Angeles, where T.O.1 objectives were fully realized. The change in research hatchery location did not hinder the ability to perform proof-of-concept studies as detailed throughout this report, and in fact streamlined the seed to farm pipeline and strengthened the relationship between the only mussel seed producer and the only mussel farmer in the region. Regarding TO.2, the authors here suggest that we were able to accomplish many of the goals described, falling short of being able to provide a full economic assessment (though value-added product development did get underway). Authors are beginning work on an economic assessment with the remainder of the time on this grant. The one area that has not yet been fully realized as of the time of this report is TO.3, which aims to develop enough broodstocks to perform genomic work for California breeding research. However, as outlined in this report, the academic, computational, and laboratory pipelines were generated and quality checked specifically for bivalves, and are at the ready when Phase II begins. The authors would also like to add a note here that ongoing and immediate future settling experiments may yet produce enough seeded California mussel line to fully realize TO.3 targets. The technical questions posed for this project are answered here using project outcomes: Will CM larvae reliably settle onto longlines, as seen in commercially available Mytilus edulis complex (MEc) species? The answer to this question is likely yes, with some changes to overall settling strata density and increased bubbling. Full production run settling (high density, many hundreds of feet of seeded line) has not yet been realized, but current trials indicate this is achievable. Are specific longline substrata more successful than others? Our project results indicate that fuzzy rope is the preferable substrate for California mussel larvae, and that other materials (polypropylene rope, PVC, cultch) are not as attractive to the larvae. Will CM be amenable to commercial harvest approaches (e.g. re-socking, de-bearding, survival on ice, etc.) and at comparable rates to MEc species? Our results have not indicated any answer to this question at this point. Is environment a major factor influencing the success of CM longline approach? Our results have not indicated any answer to this question at this point. Are market-relevant phenotypes improved with hatchery-based aquaculture of CM, compared to similarly aged/sized wild CM? Though this question remains largely unanswered at this time, It does appear that GCS systems can ripen California mussels outside of season, indicating that improvements of this species compared to wild specimens are quite possible.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Churches, Nathan, P Change, J Chancellor and S Nuzdhin. In review. Pacific oysters (Crassostrea gigas) dramatically recalibrate models for the upper limit of the eukaryotic mutation rate. Molecular Biology and Evolution.