Evolutionary and ecological correlates of thiaminase in fishes

Abstract

Thiamine (vitamin B 1 ) is required by all living organisms in multiple metabolic pathways. It is scarce in natural systems, and deficiency can lead to reproductive failure, neurological issues, and death. One major cause of thiamine deficiency is an overreliance on diet items containing the enzyme thiaminase. Thiaminase activity has been noted in many prey fishes and linked to cohort failure in salmonid predators that eat prey fish with thiaminase activity, yet it is generally unknown whether evolutionary history, fish traits, and/or environmental conditions lead to production of thiaminase. We conducted literature and GenBank BLAST sequence searches to collect thiaminase activity data and sequence homology data in expressed protein sequences for 300 freshwater and marine fishes. We then tested whether presence or absence of thiaminase could be predicted by evolutionary relationships, trophic level, omega-3 fatty acid concentrations, habitat, climate, invasive potential, and body size. There was no evolutionary relationship with thiaminase activity. It first appears in Class Actinoptergyii (bony ray-finned fishes) and is present across the entire Actinoptergyii phylogeny in both primitive and derived fish orders. Instead, ecological factors explained the most variation in thiaminase: fishes were more likely to express thiaminase if they fed closer to the base of the food web, were high in polyunsaturated fatty acids, lived in freshwater, and were from tropical climates. These data provide a foundation for understanding sources of thiaminase leading to thiamine deficiency in fisheries and other organisms, including humans that eat uncooked fish.

Full text

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Evolutionary and ecological
correlates of thiaminase in shes
Freya E. Rowland
*, Catherine A. Richter , Donald E. Tillitt & David M. Walters
Thiamine (vitamin B1) is required by all living organisms in multiple metabolic pathways. It is scarce in
natural systems, and deciency can lead to reproductive failure, neurological issues, and death. One
major cause of thiamine deciency is an overreliance on diet items containing the enzyme thiaminase.
Thiaminase activity has been noted in many prey shes and linked to cohort failure in salmonid
predators that eat prey sh with thiaminase activity, yet it is generally unknown whether evolutionary
history, sh traits, and/or environmental conditions lead to production of thiaminase. We conducted
literature and GenBank BLAST sequence searches to collect thiaminase activity data and sequence
homology data in expressed protein sequences for 300 freshwater and marine shes. We then tested
whether presence or absence of thiaminase could be predicted by evolutionary relationships, trophic
level, omega-3 fatty acid concentrations, habitat, climate, invasive potential, and body size. There
was no evolutionary relationship with thiaminase activity. It rst appears in Class Actinoptergyii
(bony ray-nned shes) and is present across the entire Actinoptergyii phylogeny in both primitive
and derived sh orders. Instead, ecological factors explained the most variation in thiaminase: shes
were more likely to express thiaminase if they fed closer to the base of the food web, were high in
polyunsaturated fatty acids, lived in freshwater, and were from tropical climates. These data provide
a foundation for understanding sources of thiaminase leading to thiamine deciency in sheries and
other organisms, including humans that eat uncooked sh.
iamine (vitamin B1) is an essential cofactor in multiple enzyme complexes required for metabolism of car-
bohydrates and amino acids1. Yet despite being necessary for all life, animals cannot synthesize thiamine de
novo, and so the majority must obtain it through diet or direct uptake in the case of fry2–5. Biological thiamine
synthesis is energetically expensive and complicated6, 7. iamine in aquatic systems is present at extremely low
(picomolar) concentrations; spatially heterogenous; degrades rapidly in the presence of UV8, alkaline conditions9,
and high temperatures10; and tends to be rapidly taken up aer synthesis11–13. Furthermore, having too lit-
tle thiamine leads to a suite of cardiovascular and neurological issues in humans3, foxes14, shes15, and other
wildlife16. Although some eects of thiamine deciency are reversible, early life deciency can cause death3, 17,
and permanent brain damage has been documented in humans18. e long-term eects of temporary or inter-
mittent thiamine deciency in shes17 and the reasons thiamine deciency complex is showing up more in wild
populations19 remain poorly understood, in part because thiamine supplementation is inexpensive and easily
applied in managed populations.
ere are two demonstrated mechanisms for aquatic animals to become thiamine decient: through a diet
that lacks enough thiamine16 (e.g., due to poor absorption or lacking nutrients) or through eating something
that destroys thiamine before it can be absorbed17, 20, 21. Previous research has hypothesized that a diet of lipid-
rich prey can lead to thiamine deciency in sh predators, but these correlative studies have not considered
if lipid-rich forage sh also contain thiaminase22. Early life stage mortality and sublethal eects in salmonids
is linked with low egg thiamine concentrations caused by elevated thiaminolytic enzymes (i.e., thiaminase)
present in the maternal diet21, 23, 24. Fishes dier drastically in their thiaminase activity25–27, but the sources
and reasons for thiaminase production are relatively unknown. Bacteria can use thiaminase as a salvage path-
way for thiamine biosynthesis28, and originally thiaminase activity in forage shes was thought to be linked to
thiaminase-producing bacteria such as Paenibacillus thiaminolyticus that had been isolated from Alewife (Alosa
pseudoharengus)29. However, later research revealed no relationship between thiaminase activity and either the
amount of P. thiaminolyticus thiaminase I protein or the abundance of P. thiaminolyticus cells30. More recently,
Richter etal.31 provided evidence for de novo production of thiaminase I by sh (Zebrash, Danio rerio) and
identied the genetic basis for thiaminase production in shes.
e question remains why shes produce thiaminase if it destroys an essential nutrient that sh cannot syn-
thesize? It is highly unlikely that thiaminase production is a prey response to limit predators for two reasons:
OPEN
U.S. Geological Survey, Columbia Environmental Research Center, 4200 New Haven Rd, Columbia, MO 65201, USA.
*email: frowland@usgs.gov
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(1) predators develop TDC when a large portion of their diet has thiaminase, but it can take years21; and (2) this
feedback loop is too slow to benet prey producing thiaminase who might have two or three generations until
predator populations begin to experience reproductive failure. One possible explanation of thiaminase produc-
tion is the result of strong selective pressure as a method to partially resynthesize thiamine6. Fish that express
thiaminase activity are not themselves decient in thiamine26, suggesting that mechanisms exist to partition
thiaminase activity from thiamine within the tissues of sh that express thiaminase. However, the mechanisms,
eciency, and energetic requirements of thiaminase partitioning have not been elucidated. iaminase activity
has been found to increase with disease challenges32 and with diet quality and stress33. However, not all shes
produce thiaminase, and thiaminase activity levels are highly variable26, 33, 34. e evolutionary history of thiami-
nase production in shes is also unknown. iaminase may be more common in primitive than derived North
American freshwater shes34, 35, but these studies did not consider marine species.
We sought to explore whether evolutionary and ecological characteristics could explain thiaminase presence
in shes. Specically, we evaluated: (1) if phylogenetic relationships would predict thiaminase presence or activity,
consistent with previous work on North American freshwater shes34, 35; and (2) if ecological or physiological
characteristics of trophic level, habitat use during foraging, salinity tolerance, or lipid content predicted thiami-
nase presence or activity in shes. Associations of thiaminase with these ecological or physiological factors would
aid in evaluation of risk for thiaminase-induced TDC in piscivorous shes, wildlife, and humans.
Methods
Data collection
Data on thiaminase I activity of 300 shes were compiled from existing literature25–27, 32–55 and a GenBank search
for protein sequences coded by expressed transcripts with signicant homology to Zebrash (Danio rerio)
thiaminase I (Richter etal. 2023; GenBank accession number NP_001314821.1). e thiaminase I enzyme of
Zebrash is homologous to a candidate thiaminase gene identied in Alewife. e empirically derived mass and
isoelectric point of the thiaminase I activity extracted from Alewife tissues exactly matched that predicted for
the candidate Alewife thiaminase I gene31. We limited the search to sh species with at least 10,000 predicted
protein sequences in GenBank. We conducted a protein BLAST sequence search for each of the species against
the Zebrash thiaminase predicted protein sequence31. Fishes were scored thiaminase positive if they had an
expressed predicted protein sequence with at least 35% sequence identity56 to Zebrash thiaminase and contained
the predicted active site cysteine (C153 in NP_001314821.1).
e BLAST search and literature agreed for 23 shes where both genetic and literature thiaminase data were
available. ere were few disagreements. Sea Lamprey (Petromyzon marinus) were categorized as thiaminase
positive in the literature36 but negative in BLAST. Since Boggs etal. (2019) suggested little to no thiaminase
activity for another lamprey speciesand the BLAST data are more comprehensive, we categorized them as nega-
tive. Ninespine Stickleback (Pungitius pungitius) was listed as thiaminase positive in Riley and Evans35 based on
activity of 85 ± 60pmol/g/min26. However, the BLAST data indicated it was thiaminase negative and the high
standard deviation in measurements suggested many low values. Furthermore, a more recent study showed
very little thiaminase activity57. us, we categorized the Ninespine Stickleback as thiaminase negative. Lake
Whitesh (Coregonus clupeaformis) was categorized as thiaminase positive based on Deutsch and Hasler58 but
was BLAST negative. Since we have lower condence in the sole literature value, we categorized Lake Whitesh
as negative. Bown (Amia calva) was positive in two studies36, 46 but also listed as both positive and negative36.
iaminase analysis in these studies was conducted on whole-body Bown homogenates, and it is possible that
positive results may have resulted from analyzing Bown that contained thiaminase-rich prey in their guts, so
they were eliminated from analysis. European Perch (Perca uviatilis) was positive in one study48 but negative
in BLAST. We put higher trust in the BLAST data since published work48 only reports positive or negative activ-
ity and did not report a range of measured values. All entries were checked closely by two separate people for
completeness and accuracy.
We obtained family, order, and ecological data from shbase.org59 for all species included in our nal thiami-
nase database. Data on maximum total length (cm), trophic level estimate, and median Omega-3 concentration
were treated as continuous variables. A species’ invasive ability (i.e., documented negative ecological impacts
in areas where they are introduced or labeled as potential pest species on Fishbase59), climate range (i.e., polar,
boreal, temperate, subtropical, tropical, deep-water), habitat (benthic, benthopelagic, or pelagic), and whether
a sh spends the majority of its life in marine or freshwater environments were treated as categorical variables.
Data analysis
We analyzed all data in R v4.1.260. We tested for phylogenetic relationships among sh families and presence/
absence of thiaminase using a published sh phylogeny including the Classes Sarcopterygii, Chondrichthyes,
and Actinopterygii61, and among orders using a phylogeny of ray-nned shes of Class Actinopterygii only62.
We used the R package ape63 to prune the tree to sh orders/families where we had data using the ‘drop.tip’ func-
tion. We then used ‘make.simmap’ in the phytools package64 to t continuous-time reversible Markov models to
estimate the evolution of thiaminase at each node for 500 simulations. e models assumed equal (0.5/0.5) root
node prior probabilities of presence or absence of thiaminase conditioned on the published sh phylogenies. We
used these simulations to estimate the probability that an ancestral state/root node had thiaminase, represented
as pie charts at each node.
To explore the ecological determinants of thiaminase activity we used Bayesian binomial models with a
logit-link function in the rstanarm65 R package. We used weakly informative priors with a mean of zero and
standard deviation of 2.5. We ran each Bayesian model for 10,000 iterations and discarded the rst half as a
warm-up to obtain 20,000 simulations for analysis. We conrmed convergence using Gelman–Rubin statistic
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(R < 1.01)66 and by examining trace plots. None of the models had inuential outliers as assessed by leave-one-
out cross-validation (“loo”) in the rstan package67. We report the coecients as the mean and the 95% credible
interval (95% CRI), which is the range of values for posterior samples. We computed Bayesian R2 for the regres-
sion models (i.e., multiple regression, trophic level, and omega-3 models) to explore the proportion of variance
explained by the models68.
Results
Our species pool included 300 shes that were tested for thiaminase activity or were searched for thiaminase
proteins in their expressed sequence libraries. Of these, less than half (n =119) had thiaminase. ese species
represent a broad range of sizes, climates, and habitats (Supplementary TableS1), with representation from
124 families, 56 orders, and 3 classes of shes. Despite having broad evolutionary representation, we found
no evidence of an evolutionary pattern in thiaminase of shes (Fig.1, Supplementary Fig.S1). We found no
evidence for thiaminase production in more primitive shes like Coelacanth (Class Sarcopterygii), lampreys
(Class Hyperoartia), or cartilaginous shes like sharks, rays, and skates (Class Chondrichthyes; Supplemen-
tary Fig.S1). iaminase rst appears in the most primitive ray-nned shes (Class Actinopterygii) such as
the Bichir (Order Polypteriformes), Mississippi Paddlesh (Order Acipenseriformes), and Spotted Gar (Order
Lepisosteiformes). iaminase is distributed across the entire Actinopterygii phylogeny to more derived orders
such as the live-bearing shes (Order Cyrinidontiformes; Fig.1). e Markov model simulations indicated equal
probability of thiaminase across all nodes of the phylogeny (Fig.1, Supplementary Fig.S1). Ecological factors
explained nearly 40% of the variation in thiaminase presence or absence (Bayesian R2 = 0.36, Fig.2). Trophic
level (bTL = − 0.88, 95% CRI [− 1.72, 0.11]; Supplementary TableS2), and association with marine environments
(bmarine = − 2.01, 95% CRI [− 3.07, − 1.12]) were negatively related to thiaminase presence in shes (Fig.2). Habitat
(benthic, benthopelagic, or pelagic), invasive potential, and size (as maximum total length) were not predictors
of thiaminase (Fig.2).
Probability of thiaminase production decreased as trophic level increased, meaning that lower trophic levels
were more likely than top predators to have thiaminase, and the trophic level model alone explained 10% of the
variation in the data (Bayesian R2 = 0.10, Fig.3a). Marine species were less likely to have thiaminase (Fig.3c); only
21.8% of marine shes compared to 59.5% of freshwater shes had thiaminase. Two ecological traits increased
probability of thiaminase. Omega-3 concentration (bomega-3 = 0.94, 95% CRI [0.07, 1.82]; Supplementary TableS3)
and tropical climate (btropical = 1.16, 95% CRI [0.03, 2.23]) were positively related to thiaminase in shes (Fig.2).
Higher omega-3 concentrations resulted in higher probability of thiaminase production, and omega-3 concen-
tration alone explained 5% of the variation in the data (Bayesian R2 = 0.05, Fig.3b). Tropical species had a nearly
equal proportion of thiaminase positive shes as non-tropical species (Fig.3d), so the increased probability of
thiaminase presence in tropical shes only appears aer trophic level, omega-3 concentrations, and marine/
freshwater status are included in the model.
Discussion
ere are two previous studies exploring why thiaminase is present in some shes but not others34, 35, and with
addingthe current study we still do not know why any shes make thiaminase. Yet dietary thiaminase has been
linked to thiamine deciency since the 1940s in taxa as diverse as silver foxes14, reptiles69, 70, shes17, 71, marine
mammals72, and humans3. We found that ecological traits rather than evolutionary patterns explain thiaminase
presence among hundreds of shes. Fishes with lower trophic levels, high polyunsaturated fatty acids, freshwater
habitats, and from tropical climates were more likely to produce thiaminase.
Evolutionary patterns of thiaminase in shes
iaminase is not present in ancient shes. Lampreys, cartilaginous shes, and the Coelacanth (Latimeria cha-
lumnae) all lack the genes to produce the thiaminase protein. For both family- and order-level analyses, there was
no phylogenetic relationship of thiaminase presence/absence. e trait rst appears in the Class Actinopterygii
(ray-nned shes) Order Polyteriformes (e.g., bichirs, reedshes), which evolved 368 million years ago61, but
the presence/absence of thiaminase has no discernable patterns within Class Actinopterygii. Previous work has
reported thiaminase activity and presence were generally higher in basal teleosts (clupeids, cyprinids, and catos-
tomids) than in more derived neoteleosts (e.g., percids and centrarchids)34, 35. However, with a tenfold larger data
set we were unable to discern any phylogenetic patterns. Certain orders have more members with thiaminase
(Clupeiformes and Cypriniformes), suggesting there must be some evolutionary reason for its presence. While
other species can and do produce thiaminase, clupeids in particular are well known to cause thiamine deciency
if they are a large portion of predator diets17, 50, 70, 73–75. A better understanding of how thiaminase in Clupeiformes
such as alewife diers from that in Cypriniformes such as carps and other shes is needed.
Ecological patterns of thiaminase in shes
Ecology appears to be an important determinant of thiaminase presence in shes. iaminase I activity in carp
increased in response to pathogenic bacterium exposure, suggesting that thiaminase may be modulated in
response to disease challenges32. It may be that tropical and freshwater shes are more likely to have thiaminase
because of higher exposure to pathogens. Previous work has found higher disease prevalence for shorebirds
occupying tropical freshwater than marine temperate or arctic regions76, and viral and bacterial loads are higher
in freshwater systems77. Moreover, a large-scale metanalysis across taxa found biotic interactions are stronger
in the tropics78. is suggests that habitat and climate and their inuence on exposure to pathogens may be a
reason for thiaminase presence.
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Trophic level was the strongest predictor of thiaminase among the ecological variables we explored. Very few
top trophic level predators (TL > 4) were thiaminase positive. e reasons for shes producing thiaminase remain
unclear. Lower trophic food items like seston and zooplankton are highly variable in thiamine concentrations79.
Likewise, median thiamine concentrations can dilute with increases in trophic level80, 81. ere is some evidence
that thiaminase production may be related to diet composition33, but there are many remaining questions. More
research is needed to understand how thiaminase-producing shes compartmentalize thiamine and thiaminase
within their tissues and in identifying the ecological advantages of producing thiaminase.
One of the more interesting results was the strong positive relationship between omega-3 concentration
and thiaminase presence, independent from trophic level (omega-3 vs. trophic level: F1, 154 = 0.203, p = 0.653).
iamine-deciency complex in North American sheries is thought to be the result of thiaminase in prey
shes destroying thiamine in predators gut contents as they pass through the gut15, 17, 23, 26. In contrast, thiamine
Figure1. Presence (black) or absence (white) of thiaminase across 42 sh orders within Actinoptergyii
that overlap between our data and Rabosky, etal.62. If at least one species within the order had evidence
ofthiaminaseactivity, we coded it as having thiaminase present. Branch lengths indicate time since evolution
such that longer branches show orders that evolved longer ago. e pie chart at each node is the probability of
the ancestral state having thiaminase based on 500 Monte Carlo simulations. Silhouettes represent common
body forms within each order (downloaded from phylopic.org; all images public domain/creative commons).
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deciency (called M74) in Atlantic Salmon (Salmo salar) from the Baltic Sea has been correlated with consump-
tion of Sprat (Sprattus sprattus) and Atlantic Herring (Clupea harengus)50. In the Baltic, thiamine deciency is
presently thought to be caused by high lipid density leading to low thiamine concentrations per unit energy22, 82, 83.
However, there has been no reported evidence that oxidative stress from highly unsaturated omega-3 fatty acids
results in thiamine deciency in consumers eating diets without thiaminase. Diets containing both high con-
centrations of omega-3 fatty acids and thiaminase confound attempts to uncover drivers of thiamine deciency
observed in M74-aected sh. It is also possible forage sh with high lipids22 and thiaminase presence such as
in Sprat27 have additive negative eects on thiamine concentrations in predators. More eorts to disentangle
whether thiaminase, high lipids (such as omega-3), or both cause thiamine deciency are needed to understand
threats to sheries.
Future directions
A critical step forward in determination of which sh species produce thiaminase will come from our under-
standing of biological function(s) of thiaminases in sh. Why shes produce thiaminase remains unknown, but
the discussions may have been hindered because of the tendency to focus on thiaminase’s thiamine-degrading
properties (and aforementioned impact on predators) rather than its function as a benet to organisms. In bacte-
ria, thiaminase II hydrolyzes the thiamine break-down product of formylaminopyrimidine (N-formyl-4-amino-
5-aminomethyl-2-methylpyrimidine) to 4-amino-5-hydroxymehtyl-2-mehtylpyrimidine (HMP) which is then
recycled in a thiamine biosynthetic pathway7. More recently, Sannino etal.28 demonstrated that the bacterium
Burkholderia thailandensis uses thiaminase I to salvage precursors from environmentally available thiamine
derivatives, and then preferentially uses these precursors for thiamine synthesis. is preference of auxotrophic
B. thailandensis for thiamine precursors over thiamine itself has also been observed in the abundant SAR11 clade
marine bacteria84. ese mechanisms for salvage of thiamine precursors have not been demonstrated in shes
but oer areas of investigation within the sh microbiome.
iaminase I most certainly oers a selective advantage in shes that possess this gene. Some possible
advantages oered by thiaminase production include: (1) aide in thiamine production of commensal bacteria
of the microbiome; (2) ecological advantages through population control of predatory species that forage on
thiaminase-producing shes; and (3) enhanced health of thiaminase-producing species of sh through greater
immune function. iamine is the least metabolically stable B vitamin85 due to an oxidative side-reaction that
readily damages the thiazole moiety86. iamine is also degraded in the presence of UV, sultes, or high pHs,
exacerbating its scarcity in natural environments and oering an advantage to organisms with microbiomes
containing the ability to resynthesize thiamine from its breakdown products or precursors2. It is interesting that
thiaminase production is most common in lower trophic levels, meaning that forage shes are most likely to
produce it. iaminase I as a thiamine salvage pathway in their microbiome would oer a strong advantage to
shes in environments with low and unstable thiamine supplies. However, it is unlikely that thiaminase I in forage
Figure2. Results from the Bayesian multiple regression of ecological variables vs. thiaminase presence/absence.
Trophic level, omega-3, and maximum length (cm) are continuous variables, and the others are categorical
(yes = 1, no = 0). e dotted line shows 0, so posterior histograms that do not overlap zero have good evidence
of being related to thiaminase (e.g., if most of the posterior is positive, this suggests it is related to thiaminase
presence). e 95% credible interval within each histogram is shaded and the median is represented as vertical
solid line. Overall, this model explained 36% of the variation in the data.
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species serves as a selective force to control reproduction and subsequently population size in salmonine preda-
tors. Laboratory experiments demonstrated that it took at least two years to induce TDC in eggs and fry of Lake
Trout fed a thiaminase-rich diet21, a timeline too long for the feedback loop to benet prey producing thiaminase.
Last, there is evidence that thiaminase I in shes may be associated with immune function. iaminase activ-
ity within shes is found to be greatest in tissues known to have immune function, such as head, kidney, gill, and
spleen44, 47. Additionally, thiaminase activity increased in carp injected with a pathogenic bacterium (Aeromonas
salmonicida), suggesting a relationship between thiaminase expression in sh and immune status32. iaminase I
in shes may have antimicrobial activity, which would be a signicant health benet for survival. e subcellular
localization of thiaminase in lysosomes87 is consistent with such an antimicrobial activity.
e physiological function of thiaminase I in shes remains in question at this point. Better understanding
of these functions will ultimately help our predictions of ecological determinants for thiaminase production in
shes, as well as evolutionary signicance of this fascinating enzyme.
Conclusions
e present work shows that de novo thiaminase production in shes is widespread. We found no evolution-
ary relationship with thiaminase activity. iaminase appears in Class Actinoptergyii and is present across the
entire phylogeny in both primitive and derived sh orders within this Class. Computer simulation resulted in
Figure3. Exploration of coecients that had the most support for predicting thiaminase in the multiple
regression (Fig.2). Top panels show the continuous relationships between (a) trophic level and (b) omega-3
concentration and thiaminase activity. Each point represents a sh that either does not have thiaminase (y = 0)
or had evidence of thiaminaseactivity (y = 1) in the literature or BLAST sequence, color coded by a sh’s climate
region. Each regression was t separately with freshwater only (dotted line), marine only (dashed line), or all
shes included (solid line). Bottom panels show the presence (dark bars, labeled as “yes”) or absence (light bars,
labeled as “no”) of thiaminase activity separated by (c) freshwater vs. marine, and (d) non-tropical and tropical.
Percentage of shes with thiaminase within a group is on top of the dark bars.
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the probability of all families in the Actinoptergyii Class having thiaminase, suggesting the genes have been
widely retained. Ecological factors explained the most (40%) variation in thiaminase; shes were more likely to
express thiaminase if they feed closer to the base of the food web, were high in polyunsaturated fatty acids, lived
in freshwater, and were from tropical climates.
Determining sources of thiaminase can help predict spatial and temporal patterns of the risks of thiamine
deciency globally. iamine deciency is considered one of the top emerging issues for wildlife19. As the climate
changes, certain sh communities are shiing their ranges. Species like Northern Anchovy (Engraulis mordax,
a thiaminase positive sh) have reached record abundances in the Pacic Ocean along the southern portion of
the United States88, causing thiamine deciency in Pacic Salmon74. Understanding which prey species produce
thiaminase, why they produce it, and how prey range and population sizes may change with climate is a necessary
foundation for predicting and managing thiamine deciency in sheries.
Data availability
All data and R code are publicly available onZenodo (https:// doi. org/ 10. 5281/ zenodo. 82639 18).
Received: 17 August 2023; Accepted: 11 October 2023
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Acknowledgements
We thank K. Klymus and R. Betancur for help with phylogenies, and the Chinook Salmon thiamine working
group for feedback on the analysis. D. Honeyeld provided excellent insight on thiaminase status of shes in our
database and on the manuscript, and C. Kra helped us reframe the paper. Any use of trade, rm, or product
names is for descriptive purposes only and does not imply endorsement by the U.S. Government.
Author contributions
CRediT(Contributor Roles Taxonomy) roles in the study were as follows: F.E.R., Conceptualization; F.E.R. and
C.A.R., Data curation; F.E.R., Formal analysis; F.E.R., C.A.R., D.M.W., and D.E.T., Methodology; F.E.R., Writ-
ing – original dra; F.E.R., C.A.R., D.M.W., and D.E.T., Writing—review & editing.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
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