Phylogenetics support an ancient common origin of two scientific icons: Devils Hole and Devils Hole pupfish

The Devils Hole pupfish (Cyprinodon diabolis; DHP) is an icon of conservation biology. Isolated in a 50 m² pool (Devils Hole), DHP is one of the rarest vertebrate species known and an evolutionary anomaly, having survived in complete isolation for thousands of years. However, recent findings suggest DHP might be younger than commonly thought, potentially introduced to Devils Hole by humans in the past thousand years. As a result, the significance of DHP from an evolutionary and conservation perspective has been questioned. Here we present a high‐resolution genomic analysis of DHP and two closely related species, with the goal of thoroughly examining the temporal divergence of DHP. To this end, we inferred the evolutionary history of DHP from multiple random genomic subsets and evaluated four historical scenarios using the multispecies coalescent. Our results provide substantial information regarding the evolutionary history of DHP. Genomic patterns of secondary contact present strong evidence that DHP were isolated in Devils Hole prior to 20–10 ka and the model best supported by geological history and known mutation rates predicts DHP diverged around 60 ka, approximately the same time Devils Hole opened to the surface. We make the novel prediction that DHP colonized and have survived in Devils Hole since the cavern opened, and the two events (colonization and collapse of the cavern's roof) were caused by a common geologic event. Our results emphasize the power of evolutionary theory as a predictive framework and reaffirm DHP as an important evolutionary novelty, worthy of continued conservation and exploration.     

MITOCHONDRIAL DNA VARIATION AND EVOLUTION OF THE DEATH VALLEY PUPFISHES (CYPRINODON,CYPRINODONTIDAE)

A phylogenetic analysis of mitochondrial DNA (mtDNA) restriction sites was used to examine the geographic history of the Cyprinodon nevadensis complex of pupfishes, a group of four species (seven extant subspp.) in two endorheic (closed) basins of the Death Valley System in California and Nevada (Owens River Valley and Ash Meadows-Death Valley). The mtDNA results suggest that the group contains mtDNAs from two divergent clades. One such clade is represented by the mtDNAs of the Owens Valley pupfish (C. radiosus) and the existing species in the Colorado River (C. macularius), while the other includes the mtDNAs of the Ash Meadows-Death Valley species (C. nevadensis, C. salinus, and C. diabolis) and a species located much farther to the east (C. fontinalis from the Guzman Basin, Chihuahua, Mexico). These results, together with evidence from other studies, suggest two separate invasions of the Death Valley System by pupfishes carrying phylogenetically divergent mtDNAs. The C. nevadensis complex apparently is either an artificial group or else it is monophyletic and its genetic history includes loss of the original mtDNA in either Owens Valley or Ash Meadows-Death Valley following genetic introgression after an invasion by a pupfish carrying a divergent mtDNA.     

The devil's in the details: genetic and phenotypic divergence between artificial and native populations of the endangered pupfish (Cyprinodon diabolis)

Propagation of threatened or endangered species in artificial habitats is a common strategy for reducing the probability of extinction by demographic or stochastic forces. Differential selection, founder effects and genetic drift can conspire to cause artificial populations to differ irreversibly from native populations for characters important for fitness, thereby compromising conservation efforts. Here we show that artificial propagation of the endangered Devil's Hole pupfish Cyprinodon diabolis resulted in rapid divergence for phenotypic and genetic characteristics despite attempts to replicate key characteristics of the species' native habitat when designing the artificial environments. Although differences in behavior and morphology between the native pool population and the two artificial pools may reflect phenotypic plasticity, the results underscore the need to monitor and control (to the extent possible) closely the evolutionary process when propagating native species in artificial pools for multiple generations.     

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.     

Isolation and characterization of eight polymorphic microsatellite loci for the critically endangered Arenaria nevadensis (Caryophyllaceae)

We report on the isolation and characterization of eight microsatellite markers from enriched libraries for Arenaria nevadensis, one of the most critically endangered plant species in the Iberian Peninsula. These are the first microsatellite loci reported for Arenaria species. The number of alleles ranged from two to eight, and the expected heterozygosity from 0.067 to 0.873. These markers will be useful for characterizing the genetic diversity in A. nevadensis and understanding its population structure, and will provide important genetic data for the conservation and recovery of this species.     

New evidence for the recent divergence of Devil's Hole pupfish and the plausibility of elevated mutation rates in endangered taxa

Sa?lam et al. recently argued that the Devil's Hole pupfish (Cyprinodon diabolis), a conservation icon with the smallest known species range, was isolated 60 kya based on a new genomic data set. If true, this would be a radically long timescale for any species to persist at population sizes <500 individuals, in contrast to conservation genetics theory. However, here we argue that their analyses and interpretation are inappropriate. They placed highly restrictive prior distributions on divergence times, which do not appropriately model the large uncertainty and result in removing nearly all uncertainty from their analyses, and chose among models by assuming that pupfishes exhibit human mutation rates. We reanalysed their data with their same methods, only using an informative prior for the plausible range of mutation rates observed across vertebrates, including an estimate of the genomewide mutation rate from a pedigree analysis of cichlid fishes. In fact, Saglam et al.'s phylogenetic data support much younger median divergence times for C. diabolis, ranging from 6.2 to 19.9 kya, overlapping with our previous phylogenetic divergence time estimates of 2.5–6.5 kya. There are many reasons to suspect an even younger age and higher mutation rate in C. diabolis, as we previously estimated, due to their high metabolism, small adult size, small population size and severe environmental stressors. In conclusion, our results highlight the need for measuring mutation rate in this fascinating species and suggest that the ages of endangered taxa present in small, isolated populations may frequently be overestimated.     

Preliminary analysis of a hybrid zone between two subspecies of Zootermopsis nevadensis

For species largely allopatric in distribution, zones of contact provide an opportunity for hybridization, testing grounds for species boundaries, and may result in the formation of a new species. Thus, hybrid zones have the potential to provide important insights into speciation. In this study, we performed a preliminary analysis of a hybrid zone between two subspecies of the dampwood termite, Zootermopsis nevadensis (Z. n. nuttingi and Z. n. nevadensis) near Bartle, CA, using 12 microsatellite loci and a mitochondrial gene. Fifty-seven colonies collected in 36 locations were analyzed. The analysis of genetic markers revealed a large hybrid zone approximately 104 km in width. Although stepped clines best explained the data, we are unable to rule out the existence of a mosaic hybrid zone. We inferred weak selection (s* < 0.05%) against hybrids, but the data also suggested the existence of a barrier to gene flow from Z. n. nevadensis to Z. n. nuttingi, but not in the other direction. Given the large zone of contact, extensive sampling is needed to obtain a more complete characterization of the hybrid zone. However, the results of this study suggest that despite the accumulation of phenotypic and genetic differences and time since divergence (~2 million years), Z. n. nuttingi and Z. n. nevadensis are capable of extensive hybridization.     

Genomic replacement of native Cobitis lutheri with introduced C. tetralineata through a hybrid swarm following the artificial connection of river systems

River connections via artificial canals will bring about secondary contacts between previously isolated fish species. Here, we present a genetic consequence of such a secondary contact between Cobitis fish species, C. lutheri in the Dongjin River, and C. tetralineata in the Seomjin River in Korea. The construction of water canals about 80 years ago has unidirectionally introduced C. tetralineata into the native habitat of C. lutheri, and then these species have hybridized in the main stream section of the Dongjin River. According to the divergence population genetic analyses of DNA sequence data, the two species diverged about 3.3 million years ago, which is interestingly coincident with the unprecedented paleoceanographic change that caused isolations of the paleo‐river systems in northeast Asia due to sea‐level changes around the late Pliocene. Multilocus genotypic data of nine microsatellites and three nuclear loci revealed an extensively admixed structure in the hybrid zone with a high proportion of various post‐F1 hybrids. Surprisingly, pure native C. lutheri was absent in the hybrid zone in contrast to the 7% of pure C. tetralineata. Such a biased proportion must have resulted from the dominant influence of continually introducing C. tetralineata on the native C. lutheri which has no supply of natives from other tributaries to the hybrid zone due to numerous low‐head dams. In addition, mating experiments indicated that there is no discernible reproductive isolation between them. All the results suggest that the gene pool of native C. lutheri is being rapidly replaced by that of continually introducing C. tetralineata through a hybrid swarm for the last 80 years, which will ultimately lead to the genomic extinction of natives in this hybrid zone.     

A simulation model of the Devils Hole pupfish population using monthly length-frequency distributions

The Devils Hole pupfish, Cyprinodon diabolis, is a federally-endangered fish that is endemic to Devils Hole, a discontiguous part of Death Valley National Park in Nye County, Nevada. Due to its status, Devils Hole pupfish monitoring must be non-obtrusive and thereby exclude techniques that require handling fish. Due to a recent decline in pupfish abundance, Devils Hole pupfish managers have expressed a need for a model that describes population dynamics. This population model would be used to identify vulnerable life history stage(s) and inform management actions. We constructed a set of individual-based simulation models designed to explore effects of population processes and evaluate assumptions. We developed a baseline model, whose output best resembled both observed length-frequency data and predicted intra-annual abundance patterns. We then ran simulations with 5 % increases in egg-larval, juvenile, and adult survival rates to better understand Devils Hole pupfish life history, thereby helping identify vulnerable life history stages that should become the target of management actions. Simulation models with temporally constant adult, juvenile, and egg-larval survival rates were able to reproduce observed length-frequency distributions and predicted intra-annual population patterns. In particular, models with monthly adult and juvenile survival rates of 80 % and an egg-larval survival rate of 4.7 % replicated patterns in observed data. Population growth was most affected by 5 % increases in egg-larval survival, whereas adult and juvenile survival rates had similar but lesser effects on population growth. Outputs from the model were used to assess factors suspected of influencing Devils Hole pupfish population decline.     

Disease swamps molecular signatures of genetic-environmental associations to abiotic factors in Tasmanian devil (Sarcophilus harrisii) populations

Landscape genomics studies focus on identifying candidate genes under selection via spatial variation in abiotic environmental variables, but rarely by biotic factors (i.e., disease). The Tasmanian devil (Sarcophilus harrisii) is found only on the environmentally heterogeneous island of Tasmania and is threatened with extinction by a transmissible cancer, devil facial tumor disease (DFTD). Devils persist in regions of long-term infection despite epidemiological model predictions of species’ extinction, suggesting possible adaptation to DFTD. Here, we test the extent to which spatial variation and genetic diversity are associated with the abiotic environment (i.e., climatic variables, elevation, vegetation cover) and/or DFTD. We employ genetic-environment association analyses using 6886 SNPs from 3287 individuals sampled pre- and post-disease arrival across the devil's geographic range. Pre-disease, we find significant correlations of allele frequencies with environmental variables, including 365 unique loci linked to 71 genes, suggesting local adaptation to abiotic environment. The majority of candidate loci detected pre-DFTD are not detected post-DFTD arrival. Several post-DFTD candidate loci are associated with disease prevalence and were in linkage disequilibrium with genes involved in tumor suppression and immune response. Loss of apparent signal of abiotic local adaptation post-disease suggests swamping by strong selection resulting from the rapid onset of DFTD.     

Population genetics of Galápagos land iguana (genus Conolophus) remnant populations

The Galápagos land iguanas (genus Conolophus) have faced significant anthropogenic disturbances since the 17th century, leading to severe reduction of some populations and the extinction of others. Conservation activities, including the repatriation of captive‐bred animals to depleted areas, have been ongoing since the late 1970s, but genetic information has not been extensively incorporated. Here we use nine species‐specific microsatellite loci of 703 land iguanas from the six islands where the species occur today to characterize the genetic diversity within, and the levels of genetic differentiation among, current populations as well as test previous hypotheses about accidental translocations associated with early conservation efforts. Our analyses indicate that (i) five populations of iguanas represent distinct conservation units (one of them being the recently discovered rosada form) and could warrant species status, (ii) some individuals from North Seymour previously assumed to be from the natural Baltra population appear related to both Isabela and Santa Cruz populations, and (iii) the five different management units exhibit considerably different levels of intrapopulation genetic diversity, with the Plaza Sur and Santa Fe populations particularly low. Although the initial captive breeding programmes, coupled with intensive efforts to eradicate introduced species, saved several land iguana populations from extinction, our molecular results provide objective data for improving continuing in situ species survival plans and population management for this spectacular and emblematic reptile.     

Identification ability of tetraploid microsatellite loci in parentage analysis

An anadromous fish, the Chinese sturgeon (Acipenser sinensis Gray) is included in the 1996 IUCN Red List of Threatened Species. To save populations from extinction, artificial propagation had been sought since the 1980s and was achieved in 2009. Additional genetic information and methods will become crucial components in genetic management of the residual A. sinensis population. Microsatellite markers can provide information on the family structure, to avoid inbreeding or founder effects. However, the polyploidy derivative nature of the A. sinensis genome has added difficulties in settling these problems. Given the costs associated with genetic data collection, the trade‐off between the amount of molecular data (how many loci are needed and which loci should be used) and the number of individuals sampled warrants consideration in order to maximize both efficiency and parentage analysis. Formulas to calculate the resolving power for tetraploid microsatellite loci in a given population are provided; these formulas are adapted from those fitted to the diploid loci in Neff et al. (Mol. Ecol., 9, 2000a, 515) and require genetic data only from the parent or parents in question, and an estimate of population allele frequencies. These formulas will help in addressing practical problems, such as the choice of genetic loci in A. sinensis conservation aquaculture programs.     

Microsatellite markers for two species of dampwood termites in the genus Zootermopsis (Isoptera: Termopsidae)

Dampwood termites in the genus Zootermopsis inhabit forested areas in western North America. To better understand the colony composition and breeding structure of Zootermopsis, we identified polymorphic microsatellite loci to use in population analysis. Microsatellite loci were isolated from Zootermopsis nevadensis nevadensis (Hagen); however, all primers amplified homologous loci in Zootermopsis angusticollis (Hagen) and Zootermopsis nevadensis nuttingi (Hagen). Twelve loci were polymorphic in one or more of the above subspecies and species. The number of alleles per locus ranged from one to six, with some allelic differences among subspecies and species. We are currently utilizing the microsatellite markers to investigate the population genetics of Zootermopsis.     

Allozyme diversity in the federally threatened golden paintbrush, <Emphasis Type="Italic">Castilleja levisecta</Emphasis> (Scrophulariaceae)

Castilleja levisecta (Scrophulariaceae), the golden paintbrush, is an insect-pollinated herbaceaous perennial found in the Pacific Northwest. Currently restricted to two island populations off British Columbia and nine populations (eight on islands) in Washington, C. levisecta is a rare species threatened with extinction. Allozymes were used to describe genetic diversity and structure in these eleven populations. Despite its threatened status and small geographic range, exceptionally high levels of genetic diversity are maintained within C. levisecta. All sixteen of the loci resolved were polymorphic within the species (P_s=100%), while the mean percentage of loci polymorphic within populations (P_p) was 65.7%. The mean number of alleles per polymorphic locus (AP_s) was 2.94 within the species and averaged 2.38 within populations (AP_p). Genetic diversity (H_es) was 0.285 for the species, whereas mean population genetic diversity (H_ep) was 0.213. Smaller populations had, on average, fewer observed alleles and less genetic diversity. A significant negative correlation (r = –0.72) was found between genetic identity and geographic distance, indicating reduced gene flow between distant populations. The most geographically isolated population was one of the larger populations, one of the most genetically diverse and the most genetically divergent. A wide range of pairwise population genetic identities (I = 0.771 – 0.992) was found, indicating considerable genetic divergence between some populations. Overall, 19% of the total genetic diversity was distributed among populations. Results of this survey indicate that genetic augmentation of existing populations is unnecessary. The high allelic diversity found for the species and within its populations holds promise for conservation and restoration efforts to save this rare and threatened plant species.     

Taxonomic status of Cuscuta nevadensis and C. veatchii (Convolvulaceae) in North America

The taxonomy ofCuscuta nevadensis andC. veatchii is investigated.Cuscuta nevadensis is more closely related toC. veatchii andC. denticulata than toC. salina, and the former two taxa are accepted as species. A summary of relevant taxonomic and biological information is provided, including synonymy, distribution and ecology, keys, and a comparison of the morphology of flowers and seeds are examined. The morphological basis of vivipary inC. nevadensis is discussed. The status ofCuscuta vivipara, an invalid name in recent use, is clarified.     

Biodiversity conservation and conservation biotechnology tools

This special issue is dedicated to the in vitro tools and methods used to conserve the genetic diversity of rare and threatened plant species from around the world. Species that are on the brink of extinction because of the rapid loss of genetic diversity and habitat come mainly from resource-poor areas of the world and from global biodiversity hotspots and island countries. These species are unique because they are endemic, and only a few small populations or sometimes only a few individuals remain in the wild. Therefore, the challenges to support conservation by in vitro measures are many and varied. The editors of this invited issue solicited papers from experts from Asia, Africa, Europe, Australia, and North and South America. This compilation of articles describes the efforts in these diverse regions toward saving plants from extinction, and details the direct application of in vitro and cryopreservation methods. In addition, these contributions provide guidance on propagation of rare plants, including techniques for large-scale propagation, storage, and reintroduction. The in vitro techniques for conserving plant biodiversity include shoot apical or axillary-meristem-based micropropagation, somatic embryogenesis, cell culture technologies and embryo rescue techniques, as well as a range of in vitro cold storage and cryopreservation protocols, and they are discussed in depth in this issue.     

EXTINCTION AND RECOLONIZATION: THEIR EFFECTS ON THE GENETIC DIFFERENTIATION OF LOCAL POPULATIONS

In this paper, we use a model by Slatkin (1977) to investigate the genetic effects of extinction and recolonization for a species whose population structure consists of an array of local demes with some migration among them. In particular, we consider the conditions under which extinction and recolonization might enhance or diminish gene flow and increase or decrease the rate of genetic differentiation relative to the static case with no extinctions. We explicitly take into account the age-structure that is established within the array of populations by the extinction and colonization process. We also consider two different models of the colonization process, the so-called “migrant pool” and “propagule pool” models. Our theoretical studies indicate that the genetic effects of extinction and colonization depend upon the relative magnitudes of K, the number of individuals founding new colonies, and 2Nm, twice the number of migrants moving into extant populations. We find that these genetic effects are surprisingly insensitive to the extinction rate. We conclude that, in order to assess the genetic effects of the population dynamics, we must first answer an important empirical question that is essentially ecological: is colonization a behavior distinct from migration?     

Iron reduction in the metal-rich guts of wood-feeding termites

Termites play important roles in lignocellulose and humus turnover in diverse terrestrial ecosystems, and are significant sources of global atmospheric methane and carbon dioxide. All known termite species engage in obligate, complex nutritional symbioses with their gut microbes to carry out such processes. Several hundred microbial species, representing a broad phylogenetic and physiological diversity, are found within the well‐bounded, microliter‐in‐scale gut ecosystem of a given termite. However, most of these species have never been obtained in laboratory culture, and little can be said about their functional roles in the gut community or symbiosis. Herein, an unappreciated facet of the gut chemistry and microbiology of wood‐feeding termites is revealed: the redox metabolism of iron. Gut fluids from field‐collected termites contained millimolar amounts of ferrous iron and other heavy metals. When iron(III) hydroxides were amended to a filter paper diet of Zootermopsis nevadensis, a dampwood termite collected in the San Gabriel Mountains of Southern California, the specimens accumulated high levels of iron(II) in their guts. Additionally, iron was reduced at rapid initial rates in anoxic gut homogenates prepared from field‐collected Z. nevadensis specimens. A Clostridium sp. and a Desulfovibrio sp. were isolated from dilution‐to‐extinction enrichments of Z. nevadensis gut contents and were found to reduce iron(III), as did the termite gut spirochete Treponema primitia. The iron in the guts of wood‐feeding termites may influence the pathways of carbon‐ and electron‐flow, as well as microbial community composition in these tiny ecosystems of global importance.     

Haplotype Diversity in “Source-Sink” Dynamics of <Emphasis Type="Italic">Escherichia coli</Emphasis> Urovirulence

FimH, the mannose-specific, type 1 fimbrial adhesin of Escherichia coli, acquires amino acid replacements adaptive in extraintestinal niches (the genitourinary tract) but detrimental in the main habitat (the large intestine). This microevolutionary dynamics is reminiscent of an ecological “source-sink” model of continuous species spread from a stable primary habitat (source) into transient secondary niches (sink), with eventual extinction of the sink-evolved populations. Here, we have adapted two ecological analytical tools—diversity indexes D _S and α—to compare size and frequency distributions of fimH haplotypes between evolutionarily conserved FimH variants (“source” haplotypes) and FimH variants with adaptive mutations (putative “sink” haplotypes). Both indexes show two- to threefold increased diversity of the sink fimH haplotypes relative to the source haplotypes, a pattern that ran opposite to those seen with nonstructural fimbrial genes (fimC and fimI) and housekeeping loci (adk and fumC) but similar to that seen with another fimbrial adhesin of E. coli, papG-II, also implicated in extraintestinal infections. The increased diversity of the sink pool of adhesin genes is due to the increased richness of the haplotypes (the number of unique haplotypes), rather than their evenness (the extent of similarity in relative abundances). Taken together, this pattern supports a continuous emergence and extinction of the gene alleles adaptive to virulence sink habitats of E. coli, rather than a one-time change in the habitat conditions. Thus, ecological methods of species diversity analysis can be successfully adapted to characterize the emergence of microbial virulence in bacterial pathogens subject to source-sink dynamics. [Reviewing Editor: Dr. Margaret Riley]