Acquired predator recognition via epidermal alarm cues but not dietary alarm cues by isolated pupfish.

We tested whether Shoshone pupfish Cyprinodon nevadensis shoshone and Amargosa River pupfish C. n. amargosae respond behaviourally to conspecific chemical alarm cues released when epidermal tissue is damaged by a predator. We found that both subspecies reduced activity and vertical position in the water column in response to alarm cues. We then tested if pupfish can use alarm cue to acquire recognition of a novel predator. We trained pupfish with (1) water + odour of largemouth bass fed a diet of earthworms, (2) alarm cues from skin extract (epidermal alarm cues) + odour of bass fed a diet of earthworms, or (3) water + odour of bass fed a diet of pupfish (dietary alarm cues). Pupfish responded to epidermal alarm cues but not to dietary alarm cues. Pupfish were retested with the odour of bass that were fed an earthworm diet. Pupfish that had previously received epidermal alarm cues reduced vertical position and activity relative to the other two treatments. This is the first demonstration of acquired recognition of a novel predator by a pupfish, the first report of partial predator naiveté, and opens the possibility of predator-recognition training as a tool for management and conservation of endangered desert fishes.

Ignorance is not bliss: evolutionary naiveté in an endangered desert fish and implications for conservation.

Predator naiveté has been invoked to explain the impacts of non-native predators on isolated populations that evolved with limited predation. Such impacts have been repeatedly observed for the endangered Pahrump poolfish, Empetrichthys latos, a desert fish species that evolved in isolation since the end of the Pleistocene. We tested Pahrump poolfish anti-predator responses to conspecific chemical alarm cues released from damaged epidermal tissue in terms of fish activity and water column position. Pahrump poolfish behavioural responses to conspecific alarm cues did not differ from responses to a dechlorinated tap water control. As a positive control, the well-studied fathead minnow, Pimephales promelas, showed significant alarm cue responses in terms of reduced activity and lowered water column position. The density of epidermal club cells, the presumptive source of alarm cues, was significantly lower in Pahrump poolfish relative to fathead minnows. Therefore, anti-predator competence mediated by conspecific alarm cues does not seem to be a component of the ecology of Pahrump poolfish. These findings provide a proximate mechanism for the vulnerability of Pahrump poolfish to non-native predators, with implications for the conservation and management of insular species.

Epidermal Club Cells in Fishes: A Case for Ecoimmunological Analysis.

Epidermal club cells (ECCs), along with mucus cells, are present in the skin of many fishes, particularly in the well-studied Ostariophysan family Cyprinidae. Most ECC-associated literature has focused on the potential role of ECCs as a component of chemical alarm cues released passively when a predator damages the skin of its prey, alerting nearby prey to the presence of an active predator. Because this warning system is maintained by receiver-side selection (senders are eaten), there is want of a mechanism to confer fitness benefits to the individual that invests in ECCs to explain their evolutionary origin and maintenance in this speciose group of fishes. In an attempt to understand the fitness benefits that accrue from investment in ECCs, we reviewed the phylogenetic distribution of ECCs and their histochemical properties. ECCs are found in various forms in all teleost superorders and in the chondrostei inferring either early or multiple independent origins over evolutionary time. We noted that ECCs respond to several environmental stressors/immunomodulators including parasites and pathogens, are suppressed by immunomodulators such as testosterone and cortisol, and their density covaries with food ration, demonstrating a dynamic metabolic cost to maintaining these cells. ECC density varies widely among and within fish populations, suggesting that ECCs may be a convenient tool with which to assay ecoimmunological tradeoffs between immune stress and foraging activity, reproductive state, and predator-prey interactions. Here, we review the case for ECC immune function, immune functions in fishes generally, and encourage future work describing the precise role of ECCs in the immune system and life history evolution in fishes.

Landscape genetics reveal broad and fine-scale population structure due to landscape features and climate history in the northern leopard frog (Rana pipiens) in North Dakota.

Prehistoric climate and landscape features play large roles structuring wildlife populations. The amphibians of the northern Great Plains of North America present an opportunity to investigate how these factors affect colonization, migration, and current population genetic structure. This study used 11 microsatellite loci to genotype 1,230 northern leopard frogs (Rana pipiens) from 41 wetlands (30 samples/wetland) across North Dakota. Genetic structure of the sampled frogs was evaluated using Bayesian and multivariate clustering methods. All analyses produced concordant results, identifying a major east-west split between two R. pipiens population clusters separated by the Missouri River. Substructuring within the two major identified population clusters was also found. Spatial principal component analysis (sPCA) and variance partitioning analysis identified distance, river basins, and the Missouri River as the most important landscape factors differentiating R. pipiens populations across the state. Bayesian reconstruction of coalescence times suggested the major east-west split occurred ~13-18 kya during a period of glacial retreat in the northern Great Plains and substructuring largely occurred ~5-11 kya during a period of extreme drought cycles. A range-wide species distribution model (SDM) for R. pipiens was developed and applied to prehistoric climate conditions during the Last Glacial Maximum (21 kya) and the mid-Holocene (6 kya) from the CCSM4 climate model to identify potential refugia. The SDM indicated potential refugia existed in South Dakota or further south in Nebraska. The ancestral populations of R. pipiens in North Dakota may have inhabited these refugia, but more sampling outside the state is needed to reconstruct the route of colonization. Using microsatellite genotype data, this study determined that colonization from glacial refugia, drought dynamics in the northern Great Plains, and major rivers acting as barriers to gene flow were the defining forces shaping the regional population structure of R. pipiens in North Dakota.

Characterization and phylogenetic analysis of complete mitochondrial genomes for two desert cyprinodontoid fishes, Empetrichthys latos and Crenichthys baileyi.

The Pahrump poolfish (Empetrichthys latos) and White River springfish (Crenichthys baileyi) are small-bodied teleost fishes (order Cyprinodontiformes) endemic to the arid Great Basin and Mojave Desert regions of western North America. These taxa survive as small, isolated populations in remote streams and springs and evolved to tolerate extreme conditions of high temperature and low dissolved oxygen. Both species have experienced severe population declines over the last 50-60years that led to some subspecies being categorized with protected status under the U.S. Endangered Species Act. Here we report the first sequencing of the complete mitochondrial DNA genomes for both E. l. latos and the moapae subspecies of C. baileyi. Complete mitogenomes of 16,546bp nucleotides were obtained from two E. l. latos individuals collected from introduced populations at Spring Mountain Ranch State Park and Shoshone Ponds Natural Area, Nevada, USA, while a single mitogenome of 16,537bp was sequenced for C. b. moapae. The mitogenomes of both species contain 13 protein-encoding genes, twenty-two tRNAs, and two rRNAs (12S and 18S) following the syntenic arrangement typical of Actinopterygiian fish mitogenomes, as well as D-loop control regions of 858bp for E. latos and 842bp for C. baileyi moapae. The two E. latos individuals exhibited only 0.0181% nucleotide sequence divergence across the entire mitogenome, implying little intraspecific mtDNA genetic variation. Comparative phylogenetic analysis of the poolfish and springfish mitochondrial genomes to available mitogenomes of other Cyprinodontoid fishes confirmed the close relationship of these oviparous Empetrichthys and Crenichthys genera to the viviparous goodeid fishes of central Mexico, and showed the combined clade of these fishes to be a sister group to the Profundulidae killifishes. Despite several significant life history and morphological differences between the Empetrichthyinae and Goodienae, estimates of evolutionary genetic distances using two partial regions of mtDNA point to inclusion of the Empetrichthys and Crenichthys genera within the family Goodeidae along with the goodeid fishes of central Mexico.

Complete mitochondrion genome of the endangered Mohave tui chub Siphateles bicolor mohavensis (Cypriniformes: Cyprinidae).

The Mohave tui chub (Siphateles bicolor mohavensis) is an endangered cyprinid fish endemic to the Mojave Desert region of southeastern California, USA. Here, we describe the complete 16,607 base pair (bp) mitochondrial genome of S. b. mohavensis. The mitogenome has a nucleotide base composition of A (29.08%), T (26.91%), G (17.58%), and C (26.43%), and encodes 13 protein subunits, 22 tRNAs, a 12S rRNA of 956 bp and 16S rRNA of 1691 bp, and a 929 bp D-loop control region, each located in the conserved mtDNA structure typical for cyprinid fishes. All protein-coding genes have initiation codons of ATG or GTG, and only the ND1, CO1, ATPase8, NDL4, ND4, ND5, and ND6 genes have complete stop codons. Phylogenetic analyses confirmed the relationship of S. b. mohavensis to several genera of cyprinids (e.g. Gila, Acrocheilus) also endemic to western North America. This characterized mitogenome may help inform management practices for S. b. mohavensis by facilitating future studies on how allopatric populations of this imperiled species are evolving across refuge habitats.

Evaluating an icon of population persistence: the Devil's Hole pupfish.

The Devil's Hole pupfish Cyprinodon diabolis has iconic status among conservation biologists because it is one of the World's most vulnerable species. Furthermore, C. diabolis is the most widely cited example of a persistent, small, isolated vertebrate population; a chronic exception to the rule that small populations do not persist long in isolation. It is widely asserted that this species has persisted in small numbers (less than 400 adults) for 10 000-20 000 years, but this assertion has never been evaluated. Here, we analyse the time series of count data for this species, and we estimate time to coalescence from microsatellite data to evaluate this hypothesis. We conclude that mean time to extinction is approximately 360-2900 years (median 410-1800), with less than a 2.1% probability of persisting 10 000 years. Median times to coalescence varied from 217 to 2530 years, but all five approximations had wide credible intervals. Our analyses suggest that Devil's Hole pupfish colonized this pool well after the Pleistocene Lakes receded, probably within the last few hundred to few thousand years; this could have occurred through human intervention.

Contemporary evolutionary divergence for a protected species following assisted colonization.

BACKGROUND: Contemporary evolution following assisted colonization may increase the probability of persistence for refuge populations established as a bet-hedge for protected species. Such refuge populations are considered "genetic replicates" that might be used for future re-colonization in the event of a catastrophe in the native site. Although maladaptive evolutionary divergence of captive populations is well recognized, evolutionary divergence of wild refuge populations may also occur on contemporary time scales. Thus, refuge populations may lose their "value" as true genetic replicates of the native population. Here, we show contemporary evolutionary divergence in body shape in an approximately 30-year old refuge population of the protected White Sands pupfish (Cyprinodon tularosa) resulting in a body-shape mismatch with its native environment. METHODOLOGY/PRINCIPAL FINDINGS: Geometric morphometic data were collected from C. tularosa cultures raised in experimental mesocosms. Cultures were initiated with fish from the two native populations, plus hybrids, in high or low salinity treatments representing the salinities of the two native habitats. We found that body shape was heritable and that shape variation due to phenotypic plasticity was small compared to shape variation due to population source. C. tularosa from the high salinity population retained slender body shapes and fish from the low salinity population retained deep body shapes, irrespective of mesocosm salinity. These data suggest that the observed divergence of a recently established pupfish population was not explained by plasticity. An analysis of microsatellite variation indicated that no significant genetic drift occurred in the refuge population, further supporting the adaptive nature of changes in body shape. These lines of evidence suggest that body shape divergence of the refuge population reflects a case of contemporary evolution (over a 30-year period). CONCLUSIONS/SIGNIFICANCE: These results suggest assisted colonization can introduce novel, and/or relaxed selection, and lead to unintended evolutionary divergence.

Parasites and salinity: costly tradeoffs in a threatened species.

Parasites and environmental conditions can have direct and indirect effects on individuals. We explore the relationship between salinity and parasites in an endemic New Mexico State threatened fish, the White Sands pupfish (Cyprinodon tularosa). Spatial variation in salinity limits the distribution of the endemic springsnail (Juturnia tularosae) within Salt Creek, a small desert stream. The springsnail is the presumed intermediate host for trematodes that infect the White Sands pupfish, and trematode prevalence and intensity in pupfish are positively associated with the springsnail. Salinity and parasites both have negative impacts on pupfish, but in areas of high salinity, pupfish can effectively escape parasites. Pupfish trematodes were absent from sites lacking snails. At the upstream site, the absence of parasites and lower variance in salinity were correlated with larger pupfish that were in better condition than pupfish at either the middle or lower sites. Springsnails were present in the middle section, an area with moderate salinity, and all pupfish had trematodes (median abundance 847 trematodes/fish). Lipid levels and condition were lowest in fish from the middle site. Additionally, fewer older fish indicated an increased mortality rate. At the lower site, springsnails were absent due to high salinity; pupfish trematode abundance was much lower (six trematodes/fish), and fish condition was intermediate. An additional experiment revealed that snail activity and survival were significantly reduced at high salinities commonly present at the lower site. Although both high salinity and parasites significantly affect pupfish, parasites might be more detrimental.

PHOSPHOGLUCONATE DEHYDROGENASE POLYMORPHISM AND SALINITY IN THE WHITE SANDS PUPFISH.

The phosphogluconate dehydrogenase (Pgdh) locus is the only polymorphic allozyme locus observed among 37 loci examined in all four populations of a New Mexico state Endangered species, the White Sands pupfish (Cyprinodon tularosa). We report evidence suggesting that this polymorphism may be associated with salinity. Salinity levels vary widely within and between habitats occupied by White Sands pupfish. The frequency of the Pgdh100 allozyme was correlated with salinity but not with temperature. Frequency of Pgdh100 differed between low (3.76 parts per thousand (ppt)) and high (9.23 ppt) salinity sites at Malpais Spring despite no obvious barriers to fish movement. Frequencies of Pgdh100 in two introduced populations differed from that of the presumptive founding stock and correlated with salinity in the current habitats.