Americamysis bahia with experimentally manipulated levels of genetic diversity. We created replicate cultures with five distinct levels of genetic diversity and monitored them for 16 weeks in both permissive (ambient seawater) and stressful conditions (diluted seawater). The relationship between molecular genetic diversity at presumptive neutral loci and population vulnerability was assessed by AFLP analysis.When a large number of alleles are lost from a population, increases in individual homozygosity may reduce individual fitness through inbreeding depression. Modest losses of allelic diversity may also negatively impact long-term population viability by reducing the capacity of populations to adapt to altered environments. However, it is not clear how much genetic diversity within populations may be lost before populations are put at significant risk. Development of tools to evaluate this relationship would be a valuable contribution to conservation biology. To address these issues, we have created an experimental system that uses laboratory populations of an estuarine crustacean, Populations with very low genetic diversity demonstrated reduced fitness relative to high diversity populations even under permissive conditions. Population performance decreased in the stressful environment for all levels of genetic diversity relative to performance in the permissive environment. Twenty percent of the lowest diversity populations went extinct before the end of the study in permissive conditions, whereas 73% of the low diversity lines went extinct in the stressful environment. All high genetic diversity populations persisted for the duration of the study, although population sizes and reproduction were reduced under stressful environmental conditions. Levels of fitness varied more among replicate low diversity populations than among replicate populations with high genetic diversity. There was a significant correlation between AFLP diversity and population fitness overall; however, AFLP markers performed poorly at detecting modest but consequential losses of genetic diversity. High diversity lines in the stressful environment showed some evidence of relative improvement as the experiment progressed while the low diversity lines did not.The combined effects of reduced average fitness and increased variability contributed to increased extinction rates for very low diversity populations. More modest losses of genetic diversity resulted in measurable decreases in population fitness; AFLP markers did not always detect these losses. However when AFLP markers indicated lost genetic diversity, these losses were associated with reduced population fitness. The impividuals -9. The eividuals -12, althividuals -16. In aividuals ,18 whileividuals ,20. Becae Nwhile holding the census size constant over three generations in the annual plant Clarkia pulchella, Newman and Pilson [e Nwere more than twice as likely to go extinct as larger populations. Similarly, in a multi-generation experiment using houseflies, Bryant et al. [Many laboratory models have demonstrated the large role of genetic diversity in increasing population fitness mediated through heterosis, particularly when inbreeding levels are high. In one classic example, Leberg  found thd Pilson  were ablt et al.  detectedDrosophila populations. In this study, both mildly bottlenecked and highly inbred populations showed a reduced ability to evolve tolerance to an environmental stressor relative to outbred populations.Frankham et al.  developeDrosophila or Tribolium  reductions in genetic diversity are less well characterized. Further, many laboratory studies of evolutionary processes have relied on m e.g., ,25,26). ,26. Drosm  a small crustacean native to estuaries along the US East coast [A. bahia a reproductive profile more similar to many birds and mammals than to other more fecund invertebrates. Owing to their estuarine habitat, A. bahia tolerate a wide range of salinities. In laboratory settings at 25°C, A. bahia cultures reproduce well in natural seawater with a salinity of 31 ppt NaCl, although they are reproductively viable in as little as 10 ppt NaCl [A. bahia have been collected in waters with salinity as low as 3 ppt, although some field surveys suggest they are uncommon below 9 ppt [Here we present data from laboratory populations of the mysid shrimp  and Navarre Beach, FL USA  during April 2005. Live animals were keyed out under dissecting microscopes at the US-EPA's Gulf Ecology Division in Gulf Breeze, FL USA. Approximately 50 individuals from each collection site were then transported to the US-EPA's Atlantic Ecology Division facilities in Narragansett, RI USA. Populations derived from each of the two collection sites were housed separately in four 80 L tanks with flow-through seawater maintained at 25°C and an ambient salinity of approximately 30 ppt. Animals were fed Selco enriched Artemia ad lib [Americamysis bahia cultures grew quickly to more than 2000 individuals from each source.a ad lib . America1 generation), the founding-females were removed, and their offspring were allowed to grow to maturity and breed for a period of three weeks. After this time, two gravid F1 females were selected from each line and remaining individuals were discarded. These F1 founders were removed after they had released their broods, producing the F2 generation. After the broods matured and became reproductively active, a single gravid F2 female was selected to found F3 and subsequent generations within each line. If the initial founding female was fertilized by a single male (a reasonable assumption given mysid reproductive biology), then pedigree based estimates suggest an average 31% decrease in heterozygosity . In late June 2006, 32 gravid females were selected from each of the two source populations and placed in separate tanks to become founders for 64 low diversity lines . Following the release of young (Fulations . StartinDihybrid (2x) Lines - The viable low diversity lines generated through bottlenecks (above) were designated '1x', and represented the lowest level of genetic diversity in our study. Randomly chosen sets of 1x lines were crossed to generate higher diversity levels. Briefly, to generate 2x lines three gravid females were selected from one of the randomly selected low diversity lines. Their offspring were discarded once they were released, and these mature, now non-gravid females were randomly paired with two males from another low diversity line. Crosses were performed randomly yielding four Navarre × Navarre lines, three Biloxi × Biloxi lines and eight Navarre × Biloxi lines. As the 1x parents of the 2x lines were independently created from the base populations, the inbreeding coefficent for 2x lines was zero . We created these higher diversity levels by combining individuals from different dihybrid lines. The founding number for each population was 12 individuals, so 6x lines were founded by randomly choosing four individuals from three 2x lines. Similarly, 8x lines were founded by choosing three individuals from each of four unique 2x lines. The founding 2x lines were chosen randomly, with the constraint that any ancestral 1x population could be used only one time within a 6x or 8x population . When the salinity was reduced to 7 ppt, reproduction ceased. Based on this preliminary data, together with published findings [A pilot study demonstrated that reproductive rates for findings , and ourad libidum with Selco-enriched [Artemia .Experimental populations were housed in 9.4 L tanks with precisely controlled salinities, light cycles, and temperatures. Both ambient seawater and seawater diluted with dechlorinated tap water were available via a flow through system, and we ran water through the tanks each day for one hour in the morning and one hour in the evening to ensure precise control of salinities. At the observed flow rate, this was sufficient for more than one complete exchange daily. Tanks were kept in two water tables to ensure uniform temperatures between tables and replicates. Tanks were moved within and between tables weekly to further reduce the potential for position effects. Lights were on a 12:12 light:dark cycle with gradual transitions to simulate natural conditions. Salinity was measured using a Hach meter Model 60 d. Salinity and temperature were measured daily in a randomly selected 10% of tanks. The mean temperature for all measured tanks was 25.3°C (± 0.03 S.E.). Low salinity tanks were maintained at a mean of 9.4 (± 0.07) ppt. Normal seawater tanks had an average of 29.4 (± 0.50) ppt. Animals were fed enriched Artemia  and one subjected to low salinity (stressful environment). Experimental populations were founded with 12 individuals (see above) and these were allowed to breed and expand for three weeks in a permissive environment .After this initial census, designated experimental populations were subjected to a stressful environment by gradually reducing the salinity to 9 ppt over the course of four days. Salinity was maintained at this level thereafter. The remaining control tanks were maintained with normal seawater. During the experimental period, a weekly census was conducted in which all individuals were counted and the presence of neonates  was noted.Fifteen pairs of low diversity 1x) lines were established. We intended to establish these cultures from 15 independently bottlenecked lines, however one of the designated lines went extinct before the start of the experiment, so one of the surviving lines was used twice. Fifteen independent pairs of 2x cultures were also established. Higher diversity levels  were replicated 10 times. The entire experiment contained 120 tanks. A summary of the experimental design is shown in Table x lines wAt the end of the 14-week survey period, surviving individuals were preserved in 100% ethanol from each tank for molecular analysis.To estimate the response to selection of each nominal genetic diversity level over the course of the experiment, population sizes in the stressful and permissive environments were compared three weeks (~1 full reproductive cycle) after the environmental stress was introduced and at the end of the experiment (~3 reproductive cycles later).e Nwas estimated to be 110.6 individuals, consistent with a decline in neutral locus heterozygosity of less than 1% per mysid generation. Some lines were excluded from the molecular analysis because fewer than 10 individuals were available.AFLP genotypes generated from surviving control populations at the end of the experiment were used as a measure of starting genomic diversity for each diversity level. It was not possible to genotype the founding populations at the beginning of the experiment because the low diversity stock lines had only a modest number of individuals, and most of these were required to found the experimental populations. For the lowest diversity lines, the harmonic mean population size was 33.8 individuals, which suggests that the populations would have lost about 2% of their heterozygosity due to genetic drift each generation. In the highest diversity populations, A. bahia using DNeasy® Blood and Tissue kit . The manufacture's instructions were followed except that we heated the elution Buffer AE to 70°C for 10 minutes and incubated the sample with Buffer AE for five minutes at room temperature before eluting each DNA sample. Genomic DNA was quantified using Quant-iT™ PicoGreen® dsDNA Assay Kit  with a Synergy™ HT Multi-Mode Microplate Reader .Ten individuals were randomly chosen from each line to estimate genetic diversity. DNA was extracted from whole AFLP analysis followed the procedure of Vos et al. , modifie2. PCR conditions were 2 min at 74°C; 24 cycles of 94°C for 30 sec, 56°C for 30 sec; 72°C for 1 min; followed by 30 sec at 72°C. The pre-amplification product was then diluted ten-fold with 10 mM Tris pH 7.6, 0.1 mM EDTA buffer.Initial PCR enrichment of a subset of fragments (pre-amplification) used 5 μl of the diluted digestion-ligation product as template: 0.5 μM of the EcoRI + A/PstI + C primers  and 0.25 U Taq DNA polymerase (Invitrogen) in 20 μl of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.2 mM each dNTP, and 1.5 mM MgClSelective amplification reactions were similar to pre-amplifications, with 3 μl of diluted pre-amplification product used as template and substituting 50 pM of the appropriate FAM--labeled EcoRI + 3/250 pM PstI + 2 selective AFLP primers. Three selective primer combinations were used on all samples: EcoRI + ACT--PstI + CT; EcoRI + AGG--PstI + CA; and EcoRI + ATG--PstI + CT. PCR conditions were 2 min at 94°C, 12 cycles of 20 sec at 94°C, 30 sec at 66°C dropping 1°C per cycle, 1 min at 72°C; then 20 cycles of 20 sec at 94°C, 30 sec at 56°C, 1 min at 72°C; followed by 30 min at 72°C. AFLP genotypes were electrophoresed and visualized with an ABI 3730 DNA analyzer.® version 1.6 . We manually checked the quality of each AFLP fingerprint and bin using the method described by Whitlock et al. [http://www.R-project.org/ script to convert the raw peak intensity data output from GeneMarker to a format compatible for AFLPScore version 1.3 [1.0 error rate), and maximized the number of loci retained. We included all pairwise comparisons for the samples that had greater than two replicates in our mismatch analysis. We generated AFLP genotypes for each restriction enzyme pair with the optimized locus selection and genotype thresholds using AFLPScore.Bins within the range of 100 to 500 bp  were genk et al.  with slik et al. ,40. The 1.0 error rate was 19.484 ± 2.3992. Following the intensive screening and quality control process, 59 loci (bins) were available to estimate genetic diversity. AFLP based estimates of genetic diversity were calculated using AFLP-Surv v1.0 [j) [The locus selection threshold was 1000 bp and the genotype threshold was 10% for each restriction enzyme pair. The average mismatch error rate for the three restriction enzyme pairs was 8.3504 ± 1.7367 (S.D.) and the average εurv v1.0 . AFLP bav1.0 [j) .Last Census (LC), 2) the Median Population Size (MPS) using data from all 13 censuses for each experimental tank and 3) the Reproductive Index (RI), which was calculated as the number of weeks in which reproduction was observed divided by the total number of weeks that the population survived for each population.Three different indices of population fitness were evaluated: 1) the number of individuals in the Statistical relationships among fitness, genetic diversity , and environmental stress were evaluated using general linear models. All calculations were performed using either JMP 7.0 or SAS 8.0 .The genetic load of the inbred 1x was estimated relative to the outcrossed AMX lines using the methods of Morton and Crow . GeneticA substantial proportion of the bottlenecked lines either failed to thrive or did not survive long enough to be used in the main experiment. Of the 64 lines initiated, only 14 achieved a population size of at least 26 individuals, the predetermined threshold deemed sufficient to generate dihybrid lines and maintain the 1x lines.Genetic load within the main experiment was estimated to be higher in the stressful environment for all three fitness indices. Using LC, the number of lethal equivalent loci in the permissive environment was 2.87 compared to 10.97 in the stressful environment. Lethal equivalents for MPS were 3.45 in the permissive environment and 6.71 in the stressful environment. For RI, in a permissive environment we estimate that there were 1.06 lethal equivalents compared to 3.19 in the stressful environment.j was 0.14 ± 0.05. In comparison, AMX lines had an average PLP of 52.1 ± 4.3 and an average Hj of 0.19 ± 0.02. Nominal genetic diversity explained a moderate amount of variation in AFLP diversity estimates . In post-hoc tests, neither estimator was effective at differentiating among the three highest genetic diversity treatments; however the 1x, 2x and higher diversity lines were distinguishable from each other when PLP was used to estimate genetic diversity  and average HAbundance after three weeks of culture under permissive conditions Phase 1) was significantly correlated with nominal genetic diversity level . AFLP diversity estimated as Hj explained less but still significant abundance variation .AFLP diversity estimated as PLP explained a modest percentage of the variation in abundance after three weeks in permissive conditions . Both factors contributed strongly to the relationship . Treatment means ranged from 9.7 individuals  to 123.2 individuals . There was no significant interaction between salinity stress and genetic diversity level   . Abundance at initiation of experimental treatments was a significant covariate . In this more complex model, there was a significant interaction between initial abundance and genetic diversity level , but no interaction between environment and genetic diversity . Both nominal diversity level  and environment  were significant individually.An additional model that included the results of the first census  as a covariate also explained much of the variation in MPS , and the covariate was marginally insignificant .The last census sizes ranged from a mean of 2.5 individuals  to 84.4 individuals . A model including nominal genetic diversity and environmental stress explained 53% of the observed variation in LC (p < 0.0001). Genetic diversity  and environmental stress  were both significant, but the interaction term was not . An expanded model for LC that included the results of the first census  as a covariate explained no additional variation in LC . Both factors were statistically significant  with a marginally insignificant interaction between these two factors .Environmental stress and genetic diversity explained much of the variation in RI  and environmental stress were evaluated for three different fitness indices: MPS, LC, and RI.The effects of AFLP diversity . Both variables were statistically significant , and there was no significant interaction between the two terms . Similar results were obtained when Hj was substituted for PLP .A significant portion of the variation in MPS is explained by a model incorporating AFLP diversity measured as PLP and environmental stress . There was no interaction between genetic diversity and stress in either model using PLP  or Hj .Models evaluating the effect of AFLP diversity and environmental stress on LC were also significant overall , . Neither genetic diversity estimator had a significant interaction with environmental stress.Similarly, both PLP and HPopulation extinctions were rare during the course of the study, and were confined to the low diversity populations Table . Three oj in extinct lines was 0.11 and 0.18 in surviving lines, and the difference was statistically significant (p = 0.014) . HAfter three weeks exposure to low salinity (Week 6 of the experiment), the average census size for 1x populations in this stressful environment was 57% smaller than those in the high salinity control environment, while the high diversity AMX lines were, on average, 24% smaller in the stressful environment relative to their controls. At the end of the experiment (Week 16), census sizes for 1x stressed populations were 94% smaller than their controls on average, whereas average AMX populations reared in low salinity was only 7% smaller than their controls. The relative decline in census size of the salinity stressed 1x populations was partly driven by the extinct lines; however, when these were excluded the net decline relative to the control population was still 83%  the relative performance of high and low diversity populations in both good and bad environments; 2) the power of AFLP markers to detect meaningful losses of genetic diversity; 3) the magnitude of genetic load in both good and bad environments; 4) the potential utility of genetic rescue and heterosis; and 5) the relative potential for adaptation to novel environments.The experimental results presented here indicate that the In this simplified laboratory environment, lower population genetic diversity was associated with lower population fitness, although this decrease was not always statistically significant in all post-hoc tests. As expected, average population fitness in the stressful environment was always lower than fitness in the permissive environment for a given level of genetic diversity.Interestingly, none of the interaction terms between nominal genetic diversity and environmental stress were significant for any of our three fitness indices although the interaction was significant for RI. This may indicate that factors interact less in our laboratory setting than might be expected in a more complex natural environment, but we cannot rule out the possibility of insufficient statistical power. Power analysis could potentially address this issue, however we lack a non-arbitrary estimate of the magnitude of a meaningful effect . Similarj based on our final set of 59 screened AFLP markers do not reliably detect differences between the three highest nominal diversity levels. Similarly, both estimators explain only a modest amount of the variation in the three fitness indices. Despite the lack of a detectable molecular genetic difference, the observed mean fitness was always lower in 8x populations than in AMX populations in the stressful environment and for two of the three fitness proxies in the permissive environment. Post-hoc tests showed these differences were statistically significant for two of the three proxies in the stressful environment  was explained by nominal diversity level. The overall relationship was in the expected direction; however, post hoc tests Table  reveal tt Figure .In this simplified experimental environment, AFLP markers detected large decreases in genetic diversity but missed more modest but ecologically meaningful losses. This may have important implications for the application of AFLP genotypes. While AFLP markers lack the power to detect all meaningful losses of genetic diversity, these markers are unlikely to cause false positives; when detectable losses in AFLP diversity occur, our data suggest they signal a serious decline in population viability.A. bahia populations was obtained before the formal experiment started. In order to generate the 1x lines used in this study, we started with 64 founding lines. Fully three quarters of these lines failed to generate the 26 individuals that were required to found the experimental lines after several months in culture. Some early losses may also be due to demographic stochasticity--initial brood sizes are small in young mysid females. However many lines that survived failed to thrive during more than four months under permissive conditions. Thus, inbreeding effects were a major determinant of the number and types of lines available for our main experiment making it necessary to construct experimental populations using only the modest number of lines that were most resistant to inbreeding depression. This result is typical of animals with large, panmictic populations [The clearest evidence for the effects of inbreeding on ulations ,45.From a conservation genetics perspective, it is important to understand the population level consequences of individual inbreeding depression . It has been repeatedly noted that the impact of individual inbreeding depression varies with environment ,47, and Because we constructed our higher diversity populations by combining different numbers of low diversity lines, our study may be viewed as a series of replicated 'genetic rescue' experimeWe did not detect a statistically significant difference between the 6x and 8x populations in any of the fitness assays or by using molecular markers. We note that the best performing 8x were superior to the best performing 6x populations, however the worst performing 8x populations were inferior to the worst performing 6x populations. Because the 8X lines were founded with only three individuals from each of four founding 2x lines, it is possible that some of the founding lines did not establish themselves in some 8x populations. In any case, genetic diversity levels in these two classes are expected to be quite similar. Even for a locus that is fixed for alternate alleles in the 1x populations, expected heterozygosity of 6x and 8x populations would only differ by 4% on average . The acMany studies have focused on the individual fitness consequences of inbreeding in benign and stressful environments due to inbreeding depression effects  but thisTo assess the strength of selection in the stressful environment, we calculated the ratio of populations in the stressful environment to those in the permissive environment three weeks (~1 mysid generation) after the stressful environment was introduced. We hypothesized that the relative proportions should be similar at both time points if inbreeding and heterosis are influencing the relationship, but that when adaptation has occurred, population sizes in the stressful and permissive environments will grow more similar over time. We found that after three weeks of selection the 1x population sizes in the stressful environment were 57% smaller than those in the permissive environment, while the AMX population sizes were only 24% smaller in the stressful environment. These declines represent the selection pressure imposed by the stressful environment. After 10 more weeks of selection, the AMX population sizes in the stressful environment were only 7% lower than those in the permissive environment while the 1x population sizes were 94% lower  was also notably lower in low genetic diversity populations, so a much higher fraction of low diversity lines are likely to fall below the minimum number of individuals required to maintain population viability . In geneUsing the mysid experimental system, we found that: 1) reduced population genetic diversity reduces population fitness in both permissive and stressful environments; 2) even some modest reductions in genetic diversity can reduce the value of some fitness measures, especially in stressful environments; 3) environmental stress and genetic diversity appear to independently influence population fitness; and 4) AFLP genotypes detected large reductions in population genetic diversity, but did not reliably detect modest reductions in genetic diversity that may influence population fitness. Therefore, many more AFLP loci than are commonly used would be necessary to detect these losses. However when genetic diversity losses are detected using a moderate number of AFLP loci, they are likely to be ecologically important. We also found that: 5) low diversity populations show more inter-population variability than high diversity populations for most estimates of population fitness; and 6) high diversity populations showed some capacity to adapt to the stressful environment, but low diversity populations did not.In natural populations the relationship between population fitness and genetic diversity will depend on specifics of the environment and the organism. Genetic diversity may not always enable populations to persist, but a lack of diversity essentially guarantees that adaptation to altered environments will not occur. Despite the importance of diversity for population survival, our understanding of the relationship between diversity and long-term population viability is limited. Studies in simplified laboratory environments, such as the one described here, can be used to determine a baseline for the relationship between diversity and population risk under the best possible conditions  and provide an important way to assess molecular tools that are potentially useful in conservation biology.j : Heterozygosity estimate derived from dominant molecular markers; LC: The Last Census; the number of individuals in an aquarium at the end of the experiment; MPS: Median Population Size for a single line over the course of the experiment; NI: Net Increase in population size after three weeks in permissive conditions; PLP: Proportion of Loci Polymorphic; the fraction of AFLP bands that vary within an experimental populations; RI: Reproductive Index; the fraction of census weeks in which neonates were observed; TTE: Time To Extinction.AMX: Admixed lines; HJAM, DEN and MJB took the lead in designing overall experiment, while AK, JSG and DMC provided critical insights into key sections of the design. DMC, AK and JAM also conducted an extensive series of pilot studies that made this project possible. RG-G designed methods that allowed us to precisely maintain the experimental environment. TJM-optimized objective AFLP scoring parameters, produced AFLP genetic diversity estimates, and wrote the AFLP scoring section. AR managed the collection of molecular data and wrote the molecular section of the AFLP methods. All Narragansett based authors participated in weekly censuses and daily culture activities. All authors contributed to the writing of the paper and have read and approved the final manuscript.


Salamandra salamandra), as found in Middle Europe (Germany), can be framed in an explicit phylogeographic framework that allows for the evolution of habitat adaptation between distinct populations to be traced. Typically, females of S. salamandra only deposit their larvae in small permanent streams. However, some populations of the western post-glacial recolonization lineage use small temporary ponds as larval habitats. Pond larvae display several habitat-specific adaptations that are absent in stream-adapted larvae. We conducted mate preference tests with females from three distinct German populations in order to determine the influence of habitat adaptation versus neutral genetic distance on female mate choice. Two populations that we tested belong to the western post-glacial recolonization group, but are adapted to either stream or pond habitats. The third population is adapted to streams but represents the eastern recolonization lineage.Although some mechanisms of habitat adaptation of conspecific populations have been recently elucidated, the evolution of female preference has rarely been addressed as a force driving habitat adaptation in natural settings. Habitat adaptation of fire salamanders (ST values around 0.5, the stream-adapted females preferred males from the same habitat type regardless of genetic distance. Conversely, pond-adapted females did not prefer males from their own population when compared to stream-adapted individuals of either lineage.Despite large genetic distances with FA comparative analysis of our data showed that habitat adaptation rather than neutral genetic distance correlates with female preference in these salamanders, and that habitat-dependent female preference of a specific pond-reproducing population may have been lost during adaptation to the novel environmental conditions of ponds. A crucial step in ecologically driven population differentiation and potential speciation is the adaptation of recently diverged sub-populations to new habitats ,2. AlthoSalamandra salamandra, Linnaeus 1758). We have studied this species in detail to describe the processes and effects of recent habitat differentiation in a vertebrate population. In the current paper, we integrate behavioral data of S. salamandra within an explicit phylogeographic framework in order to describe the extent to which habitat adaptation and genetic distance between populations influences female mate preference.By integrating molecular, ecological and demographic approaches -19, we hS. salamandra deposit their larvae in small permanent streams. However, as a habitat-dependent adaptation, small temporary ponds are also used as larval habitats by the western recolonization lineage [S. salamandra in Middle Europe, whereas adaptation to pond reproduction is a post-glacially derived adaptation of certain populations of the western recolonization lineage [[Fire salamanders recolonized Germany through two independent lineages, one eastern and one western, after the end of the last European glaciation event, roughly 8,000 years ago ,16,20, s, s20, se lineage ,22. Larvlineage [, and matS. salamandra, such mate recognition signals may be communicated by olfactory cues, as is found in other salamanders [S. atra and S. lanzai [Lyciasalamandra [S. salamandra is involved in mate recognition, and in this study we used odor preference tests to investigate the preference of female salamanders for males.It is still generally thought that geographic distance results in greater neutral genetic divergence, which in turn increases the diversification of mate recognition mechanisms and associated signals -27, but , but 27,amanders -31. Inde. lanzai ,33 and Llamandra ) use odoThe major aim of this study was to test whether the preferences that females displayed in the odor tests correlated with reproductive adaptation to either stream or pond environments, or to the neutral genetic distance measured by divergence of nuclear microsatellite loci.5,68 = 3.304, p = 0.046), indicating that female mate choice has been influenced either by habitat or by genetic distance. When paired with males from the pond habitat . However, the opposite trend  was observed if they were given access to stream-adapted males from the eastern post-glacial recolonization lineage  compared to males from pond habitats  between populations of the different post-glacial recolonization lineages based on putative neutrally evolving microsatellite loci ranged between 0.522 (s-E/s-W) and 0.590 (s-E/p-W), whereas the FST distance between populations s-W and p-W of the western recolonization lineage was nearly 10-fold lower (FST = 0.071).When tested for significant deviations from Hardy-Weinberg equilibrium, only locus Sal 29 in population s-E (Nasenbach) showed a significant deviation (p = 0.001), whereas all other comparisons were in Hardy-Weinberg equilibrium. All pairwise population comparisons based on nine microsatellite loci were significant on a 5% level. As expected from the post-glacial population history of For the tested combinations in which the habitat component was constant and the genetic distance component was different  have adapted to different larval habitats , where larvae develop until metamorphosis. It has previously been ambiguous whether female mate preference in S. salamandra is correlated with the habitat type as mating in S. salamandra is strictly terrestrial and not associated with the habitat where females will deposit larvae. Since olfactory cues are known to be involved in intraspecific communication in terrestrial salamanders [It is widely accepted that adaptation to new habitats can drive population differentiation and speciation through reproductive isolation. In this context, female preference for males from the same habitat type is interpreted as habitat dependent assortative mating. In three-spine sticklebacks, it has been demonstrated that differences in body size are coupled with ecological differentiation , and that females use this characteristic to recognize and mate assortatively according to habitat ,14. Germamanders -34, we samanders ,28. The ST = 0.071). The neutral genetic divergence between populations of the different recolonization lineages was nearly 10-fold higher (FST(s-E/s-W) = 0.523 and FST(s-E/p-W) = 0.590). Interestingly, adaptation to reproduction in ponds seems to be specific for the western recolonization lineage, and is thought to be a post-glacial adaptation [Fire salamanders recolonized Germany through both eastern and western recolonization lineages after the last glaciation, and different lineages are associated with diagnostic mitochondrial haplotypes . Our stuaptation . The facWe found a clear trend to indicate that stream-adapted females of both lineages discriminated against pond-adapted males (combination A (p < 0.01) and D (p = 0.076), Fig. Although our data support the hypothesis that habitat adaptation rather than genetic distance influences female mate preference, our results also show that adaptation to reproduction in ponds did not induce specific mating preferences in salamanders from this specific population. From our phylogeographical-ecological analysis, we can infer that pond reproduction evolved as a post-glacial adaptation to new environmental conditions, a situation that has thus far only been described in the western recolonization lineage. Reproduction in streams, however, must have been the ancestral condition . As streversus metamorphic) has been described for the Alpine newt , as in pestris; .S. salamandra is coupled with female preferences for males of the same habitat type from our present study. However, such mating preferences must be present when pond and stream adapted salamanders occur under sympatric conditions; otherwise, the observed genetic differentiation in such situations would not be maintained  of the eastern post-glacial recolonization lineage  and one stream-adapted  population of the western post-glacial recolonization lineage of S. salamandra in a very similar habitat ranged between 78 – 2266 m2 [S. salamandra under natural conditions occurs after the deposition of larvae [Fire salamanders in Middle Europe usually deposit larvae that were fertilized in the preceding reproductive period in the oviduct of the female  after each trial and let it dry before performing the next trial.In total, 136 individuals  were used in the female preference tests. All preference tests simulated possible contact situations in which a female could choose between a male from the same population , and a male from a different population differing in one or both respects  or with the shared habitat adaptation ? In order to determine which component had a stronger impact on female mate preference, we tested specific combinations of choice trials so that either the genetic distance component or the habitat component was constant. In each of these four tests  Do females prefer males from their own population, i.e., did they spend significantly more time with the male from their own population than with the other male? We analyzed six different choice trial combinations  - transformed before performing all analyses in order to achieve consistent variances.After using one-factorial ANOVA to reject the null hypothesis that the females spend equal amounts of time with males of their own population in choice trial combinations A-F, (a) for definitions) predominantly influenced female preference between populations, we determined the neutral genetic distance between these populations based on differentiation of nine microsatellite loci . Amplification of loci, scoring of alleles and loci characteristics are described in [ST values (Reynolds' FST) were inferred and pairwise differences were tested for significance (α = 0.05). All analyses were done by using the Arlequin program (version 2.0 [Genomic DNA was extracted from tissue samples (toe clips) of the same individuals that were used in the preference tests following published procedures . Accordiribed in . Each posion 2.0 ).The authors declare that they have no competing interests.Conceived and designed the experiments: SS, MW. Performed the experiments: BC, CJ. Analyzed the data: BC, MW, CJ, SS. Designed figures: CJ, BC. Wrote the paper: SS, BC, MW. All authors read and approved the final manuscript.


Ctenomys australis (the sand dune tuco-tuco) is a territorial subterranean rodent that inhabits a relatively secure, permanently sealed burrow system, occurring in sand dune habitats on the coastal landscape in the south-east of Buenos Aires province, Argentina. Currently, this habitat is threatened by urban development and forestry and, therefore, the survival of this endemic species is at risk. Here, we assess population genetic structure and patterns of dispersal among individuals of this species at different spatial scales using 8 polymorphic microsatellite loci. Furthermore, we evaluate the relative importance of sex and habitat configuration in modulating the dispersal patterns at these geographical scales.The population genetic structure of subterranean rodent species is strongly affected by demographic  and stochastic factors (e.g. random genetic drift among subpopulations and habitat fragmentation). In particular, gene flow estimates at different spatial scales are essential to understand genetic differentiation among populations of a species living in a highly fragmented landscape. C. australis is not restricted at regional spatial scales (~ 4 km). Assignment tests revealed significant population substructure within the study area, providing support for the presence of two subpopulations from three original sampling sites. Finally, male-biased dispersal was found in the Western side of our study area, but in the Eastern side no apparent philopatric pattern was found, suggesting that in a more continuous habitat males might move longer distances than females.Our results show that dispersal in Overall, the assignment-based approaches were able to detect population substructure at fine geographical scales. Additionally, the maintenance of a significant genetic structure at regional (~ 4 km) and small (less than 1 km) spatial scales despite apparently moderate to high levels of gene flow between local sampling sites could not be explained simply by the linear distance among them. On the whole, our results support the hypothesis that males disperse more frequently than females; however they do not provide support for strict philopatry within females. The characterization of population genetic structure and dispersal patterns offers fundamental information on the ecology and evolution of species and provides vital knowledge for the conservation of threatened species . In suchCtenomys (tuco-tucos), which present restricted mobility and are usually distributed in patches with low local effective population numbers [Fully characterising dispersal patterns is particularly difficult in the case of solitary species with territorial behaviour living in complex landscapes in which spatial structure can be described as a more or less continuous habitat, irregularly interrupted by discontinuities ,5. Many  numbers ,7.Ctenomys australis) is solitary, highly territorial, and most likely polygynous [C. australis is one of the largest species within the genus (250-500 g) and the extreme energetic costs associated with digging restrict the availability of suitable habitat for this species. The capacity of this species to remove soil decreases dramatically in harder substrates [Ctenomys species [C. australis is currently restricted to a much reduced range of approximately 100 km2 [Like most species of tuco-tucos, the sand-dune tuco-tuco , only interrupted by small towns and streams, and a large river, the Quequén Salado River [The evolutionary history of province . Along tdo River ,11. Thisdo River .However, at a smaller spatial scale (less than 5 km), the habitat is recurrently interrupted by low inter-dune grasslands with harder soils ,10. ThusC. australis.In a previous study, Zenuto & Busch  implemenC. australis represent important information for guiding conservation plans [In particular, natal dispersal, which occurs in most subterranean rodents, tends to be sex biased and has important effects on social structure, population demography and genetic composition ,12, parton plans .C. australis is currently being lost to urban development, forestry and the progressive advance of grasslands, so survival of this endemic species is also linked to the preservation of the whole sand-dune ecosystem [The sand-dune habitat of cosystem . In ordeC. australis. Our objective is to determine to what extent the sex of individuals and landscape features influence dispersal patterns.In general, little is known about dispersal in subterranean rodents because of the difficulties associated with direct quantification in species that live underground ,7. HowevThe mean fragmentation index was greater in the Eastern and Central sampling sites  than in the Western site 0.021). Both the Eastern and Central sites showed highly significant differences in the degree of habitat fragmentation in comparison to the Western site  are presented in Table FST was observed between the two most distant sampling sites (E and W) but the central one was more similar to the more distant Western site than to the Eastern .The global Mantel test based on between-individual geographic and genetic distances did not uncover a relationship between genetic and geographic distances . These results are in accordance with the pair wise The Bayesian analysis using STRUCTURE clearly identified the presence of substructure among the sampling sites; the model that best fit the data was K = 2. All runs at K = 2 produced identical clustering solutions with similar values of cluster membership (Q) for all individuals Figure . One of There were a total of 24 individuals that could not be assigned to either genetic cluster using the threshold value of Q > 0.7; 14 of these were from site E, 6 from site C and 4 from site W. Six of them were identified as individuals that did not belong to any of the two clusters using the GeneClass exclusion test. The remaining 18 individuals could neither be assigned to a genetic cluster nor excluded from both of them; therefore, they were not taken into consideration for the inference of migration patterns.The six individuals that were excluded from both clusters were considered as immigrants that came from unsampled populations. One of them was sampled in site W and the other five in site E. The average genetic distance among these six individuals (GD = 50.13) was not significantly different from that obtained for the western genetic cluster  or from that obtained for the Eastern cluster . These results suggest that they probably are immigrants from the same external source population.In order to make inferences about the direction of migration, we divided the sampling area into two halves that are separated by a strip of vegetation . Although we observed an influence on the proportion of each individual genotype that is assigned to each inferred cluster, the inferred number of clusters itself and the cluster to which each individual was assigned remained unaffected. We also carried out an analysis for studying genetic structuring at a local level within the Eastern and Western sampling sites using TESS. These analyses indicated that there is a lack of spatial substructuring within both sampling sites.We also carried out separate Mantel tests for each sampling site. The test was significant for the Western sampling site , suggesting a restricted geographical dispersal at this geographical scale. In contrast, this test was non-significant for the Eastern sampling site .Q) estimated by STRUCTURE between males and females [Q were observed between sexes  at regional scale.We used two approaches to test for sex biased dispersal at regional scale. First, we looked at differences in the percentage of ancestry . Also, both FIS and FST were not significantly different between sexes at the regional scale . In sum, these four estimates are in agreement with the absence of a sex-biased dispersal pattern at the regional level; and are in concordance with the results of the STRUCTURE analysis.We also investigated sex-biased dispersal using the approach of Favre et al.  implemenet al. . NeitherIn order to test for sex biased dispersal at a small geographic scale (at the level of sampling sites), we calculated multilocus autocorrelation coefficients among males and among females within the sampling sites E and W  and an Eastern (E) clusters. Both Eastern and Western sampling sites are clearly two different subpopulations, while the Central sampling site represents a more or less even genetic mixture of genetic clusters E and W. Overall, the results indicate that dispersal within distances of less than 4 km is frequent. However, the rates of dispersal are not sufficient to avoid substructuring. These moderate dispersal capabilities suggest that small and seemingly minor disturbances may fragment habitat and subpopulations. Additionally, an asymmetric migration is detected among sampling sites, being predominantly in the West-East direction. More specifically, 14.38% of individuals from the Eastern sample site were originated in the Western genetic cluster. On the contrary, only 2.05% of individuals that were sampled in the Western site were identified as immigrants from the Eastern genetic cluster. In addition, the Eastern subpopulation presented a greater percentage of individuals whose origin is ambiguous. The assignment coefficients of these individuals suggest that they may be descendants from parents coming from different genetic clusters. On the other hand, the Western subpopulation is genetically much more structured as evidenced by its local FST, which is higher than those of the Central and Eastern sampling sites.The genetic structure and demography of local populations is closely linked to the scale and rate of dispersal . In thisMantel tests provided no evidence for distance dependence of genetic structure at regional level, although it must be kept in mind that the statistical power of this test is relatively low here.Migration at a small spatial scale (less than 1 km) seems to be enough to preclude spatial substructuring within sample sites. However, in the Western site there is an isolation by distance pattern and there is also evidence for sex biased dispersal, with males being more likely to disperse than females. The Eastern site, on the other hand, shows no signs of isolation by distance or sex biased dispersal. These results suggest that dispersal is more limited within the Western site, indicating a restriction in dispersal among individuals due to geographic distance.The difference in dispersal patterns between Eastern and Western sampling sites seems to be explained by a combination of factors such as variation in landscape configuration (and the distribution of resources associated with this variation), density dependent factors , and differences in social structure and/or sex ratio.In terms of landscape configuration we note that the sampling area is located near the Eastern limit of the species distribution . SuitablC. australis strongly depends on the availability of suitable habitat [et al., unpublished data). Possibly, these differences, in conjunction with social factors  would limit the availability of vacant habitat, making the establishment of immigrants more difficult in the Western site.The population size in  habitat ,11, whicAs we said previously, the high local turnover of individuals in the Eastern site of the study area could be reflected, more probably, by the lack of a correlation between genetic and geographical distances at this scale. However, we must also consider that small and unstable populations that are recurrently experiencing extinctions and recolonizations are unlikely to have reached equilibrium .et al. [In this context, there are several studies in rodents showing the genetic consequences of dispersal among patchy populations at very low spatial scales ,13. Partet al. .Ctenomys rionegrensis is one of the most impacted species within the genus, and like C. australis, is strictly limited to sandy soils. Particularly, this species occur on a highly fragmented habitat in a strongly restricted geographic range in Uruguay. Populations of this species are separated by very few kilometers (from 5 to 40 km among them), conforming a classic metapopulation system along the coast of the Uruguay River. Interestingly, the microsatellite genotype data showed that C. rionegrensis is strongly structured geographically, with subpopulations constituting distinct genetic units and, like C. australis, showing an absence of an isolation-by-distance pattern [C. rionegrensis than linear distances among subpopulations. A reanalysis of these data [Among tuco-tucos,  pattern . At thisese data  suggestsC. australis along a coastal portion of its distributional range  showed that patterns of occupancy in this species were highly associated to the occurrence of the larger and more continuous suitable patches at shorter distances to the coastline. In contrast, the highest proportion of unoccupied suitable patches was observed in the most fragmented habitat. In sum, these results showed the great importance of the patch size and the landscape discontinuities over the patterns of habitat occupancy at medium and low spatial scales (between 1 and 10 km) in this species.A metapopulation study carried out by Mora  in C. auCtenomys in the field [Ctenomys is known as a low vagility group of species, due to their morphology, energetic consumption associated with excavating new burrows and their exposition to predators aboveground. For an ecologically closer species - C. flamarioni (adult body size between 300 and 400 g) - Stolz [C. australis showed that males were able to move larger distances than 160 meters on a continuous habitat in a period of 10 days . However, on the basis of this last study, we cannot affirm that dispersal is male-biased or that the movement capacity in this rodent is larger than the distances reported in its closer species C. flamarioni using direct estimates of dispersal .C. australis subpopulations at fine scale than the linear distance among them. Similar conclusions have been suggested for other mammalian taxa that occupy fragmented landscapes [Overall, the results suggest that stochastic events like genetic drift, in conjunction with differences in habitat configuration among sampling sites and the availability of propitious habitats, could probably be more important in structuring the ndscapes ,26. HoweLife-history traits , and ecological features  could be viewed as a complex set of variables that interact to influence philopatry in rodents -31.FST, FIS, mAIc and vAIc between sexes provided evidence for a significant male-biased dispersal rates at the regional level.If dispersal is male-biased, we expected that the level of genetic structure over the study area will be lower among males than among females. However, neither the immigrant proportions nor additional statistical tests based on et al. [C. australis is not restricted at a regional spatial scale.In this sense, the absence of different patterns of migration between sexes at regional level could be an artefact of the moderate number of migrants detected, or maybe in part due to different behaviours according to the spatial scale of analysis ,33. As wet al. , additioet al. ,34. In tet al. ,22,29,30Ctenomys report patterns of philopatry at local level considering small spatial scales [et al. [et al. [Ctenomys talarum, Ctenomys sociabilis and Ctenomys flamarioni, respectively.On the other hand, at the local scale in the Western site, the multilocus spatial autocorrelation analyses showed that males seem more likely to disperse than females. This pattern of dispersal is in agreement with the most common pattern reported in subterranean rodents ,12, and l scales ,12,29. C [et al.  report tC. australis at the local level. The home range sizes in this polygynous species are notoriously larger in males than those of the females ; therefore, we expected that males should move longer distances than females.Two major hypotheses intend to explain sex biased dispersal in mammals and birds . As was On the other hand, sex-biased dispersal could be explained by the inbreeding avoidance hypothesis, which is based on the principle that the sex that incurs the maximum cost from inbreeding will be more likely to disperse . FurtherC. australis are most likely a polygynous rodents that do not associate frequently with the opposite sex except for mating; they live alone in burrow systems and are very aggressive toward conspecifics [FIS was not significant and near to zero (= 0.001), supporting the idea of a random mating system (most likely in accordance to the inbreeding avoidance hypothesis).According to Solomon , male-bipecifics ,11. EvenCtenomys  these dC. australis. In this study we found both mature as well as subadults individuals among the migrants, and because mature migrants could have moved during any stage of their life, differences in migration related to age classes cannot be easily inferred. According to Zenuto & Busch [Ctenomys [On the other hand, no previous study describes the patterns of movements for different age classes in  & Busch , we obseCtenomys ,24. ThisCtenomys ,7.Several species have discontinuous or patchy distributions even at a local scale , and indC. australis within this sand dune habitat. Future analysis over different landscape configurations and at different spatial scales will allow us to increase our understanding of the factors that model the complex processes of population differentiation and microevolution in subterranean rodents.Due to the progressive urbanization, forestry and the impact of human activities during the last decades the habitat of coastal sand dunes is rapidly being lost ,11. The C. australis habitat is covered by natural grassland [Sampling was conducted in a sand dune habitat along the south-eastern Atlantic coast, localized at 15 km to the south-west of Necochea city , Province of Buenos Aires, Argentina  of 4 ha, an Eastern site (E) of 7 ha, and a Western site (W) of 8 ha. Sample sizes at each location are presented in Table Panicum racemosum, Hydrocotyle bonariensis and Achyrocline satureioidis, [A total of 112 individuals of eioidis, ,11), theC. australis in these two sites is surrounded by harder, deeper and more developed soils . Previous studies showed strong differences in the Normalized Difference Vegetation Index  and the habitat heterogeneity  between the Eastern/Central area, regarding to the area including the Western sampling site . In spite of these differences, we carried out an independent analysis of habitat fragmentation. In order to quantify the differences in habitat configuration among sampling sites we employed an additional study of habitat fragmentation using a high resolution satellite images (Landsat ETM+) and a package of analysis available in the IDRISI software [At this geographical scale  the habitat is patchy, being interrupted by low inter-dune grasslands that constitute potential barriers to dispersal. The Eastern and Central sampling sites have a smaller extension of suitable habitat and are characterized by a more patchy configuration than the Western sampling site Figure . The suispecies, ). In consoftware . Firstlysoftware ). The hasoftware ; where nWe obtained tissue samples  from the captured individuals, who were live trapped with Oneida Victor N°0 snap traps , with a rubber cover to avoid injuring animals . After collection of tissue samples for genetic analyses, animals were immediately released back within the same burrow system where they had been originally captured.Position of captures was recorded with GPS to determine their location. To determine different age classes among individuals we obtained detailed information about the reproductive status and body size of collected individuals. An open or plugged vagina and signs of pregnancy or postpartum pregnancy was indicative of sexual maturity in females (following ). The aget al. [Tissue samples for DNA extraction were preserved in 95% ethanol and transferred to freezers at -70°C. Tissues were prepared and deposited in the collection of Laboratorio de Evolución, Facultad de Ciencias, Montevideo, Uruguay. Genomic DNA was isolated following a protocol modified from Miller et al. , involviCtenomys haigi , 3 μl of a total DNA solution 1/100, 2 mM MgCl2, 0.2 μM each primer, and 0.8 μM dNTPs (0.2 μM of each), using a "Rapidcycler" . The thermocycling profile included an initial denaturing at 94°C for 10 min (polymerase activation), followed by 30-35 cycles of denaturing at 94°C for 30 s, annealing at 48-56°C for 30 s, extension at 72°C for 45 s, and a final extension at 72°C for 5 min. Relative concentrations of primers were adjusted by trial and error to create multiplexes. Final PCR fluorescently labelled product were analyzed with a capillary sequencer ABI3100 . Considering the relative sizes of fragments, and the number of dye labels compatible with the capillary electrophoresis system we used the following four loci combination of multiplexing: Soc7/Soc8/Hai9; Soc2/Soc6; Hai3/Soc1; and Soc5/Hai4. Fragments were scored with the programs GENESCAN 3.0, GENOTYPER 2.5 (Applied-Biosystems).Primers of nine dinucleotide microsatellite loci developed from the Argentinean species , Hai11; ) and Cte6, Soc8; ) that prGenetic diversity within each sampling site was measured as the number of alleles per locus (At) and per sampling site (A), the percentage of loci that were polymorphic (%P), observed (Ho) and expected (He) heterozygosity , using AAnalysis of linkage disequilibrium between pairs of loci and deviations from Hardy-Weinberg equilibrium were tested using ARLEQUIN 3.0 . We usedFST between sampling sites were computed using Weir & Cockerham [FSTs. This method estimates individual FST values for each local population using the approach first proposed by Balding & Nichols [FSTs values mean greater genetic structure of this particular population compared with the other ones. We used 10 pilot runs of 2000 iterations to obtain the parameters of the proposal distributions used by the MCMC. We also used an additional burn in of 5 × 105 iterations and a thinning interval of 50. All posterior probability estimates were obtained using a sample size of 20000.We used the software Cervus 3.0  to evaluockerham  estimatoockerham . These aockerham . Additioockerham , as impl Nichols . Greaterr statistic, using the software POPTOOLS 2.6.2 [In order to determine if there was a pattern of isolation by distance we carried out a correlation analyses between pair-wise genetic distances and linear geographical distances considering each pair of individuals ,50. We dLS 2.6.2 .6 iterations. We thus used this as burn-in and based the estimations on 3 × 106 additional iterations. We used Q > 0.7 as threshold for assigning individuals to populations. Individuals that were below this level were subjected to the exclusion test implemented in GeneClass [et al. [et al. [We used the method of Pritchard et al.  implemeneneClass  to detereneClass  and the  [et al. , both of [et al. , we usedet al. [et al. [et al. [We used GENALEX 6  to calcuet al.  to deteret al.  among thet al. . Rough eet al. , Paetkau [et al.  and Mane [et al. . Individet al. [6 followed by 106 additional iterations. The interaction parameter (ψ) of the Hidden Markov Random Field was set to 0.6. The spatial Bayesian clustering algorithm implemented by TESS is supposed to perform better than STRUCTURE when genetic differentiation is low and when the genetic units are closely distributed in a continuous or quasi-continuous habitat [In order to detect regional and local substructuring within the sampling sites we also used the software TESS  that impet al. . We cons habitat .FIS and FST [Genetic inferences of sex-biased dispersal patterns were done using four statistical indices calculated by Fstat 2.9.3.2 . This an and FST ; the two and FST ). Assign and FST  was usedr with those obtained from 999 spatial permutations of individuals belonging to the site of interest. Then, the observed data were added as the 1000th permutation, on the null hypothesis that our data were genetically random with respect to geographic position. The 95% confidence intervals over the correlograms were obtained as described by Peakall & Smouse [In order to determine if there were differences in dispersal between sexes at a fine scale (within the sampling sites), we carried out a spatial autocorrelation analysis (SAA) on the genetic multilocus data using GENALEX 6 . Due to & Smouse . This anMSM designed and conceived this study. MSM and FJM collected the genetic data, and with OEG, MJK and EPL performed all the genetic analysis and drafted the manuscript. All authors read and approved the final manuscript.


Zalophus wollebaeki) are top predators in this unique setting and have an essentially unlimited dispersal capacity across the entire species range. In theory, this should oppose any genetic differentiation.Oceans are high gene flow environments that are traditionally believed to hamper the build-up of genetic divergence. Despite this, divergence appears to occur occasionally at surprisingly small scales. The Galápagos archipelago provides an ideal opportunity to examine the evolutionary processes of local divergence in an isolated marine environment. Galápagos sea lions  that exclusively dwells in the west. Stable isotope patterns correlate with significant differences in foraging-related skull morphology. Analyses of mitochondrial sequences as well as microsatellites reveal signs of initial genetic differentiation.We find significant ecological, morphological and genetic divergence between the western colonies and colonies from the central region of the archipelago that are exposed to different ecological conditions. Stable isotope analyses indicate that western animals use different food sources than those from the central area. This is likely due to niche partitioning with the second Galápagos eared seal species, the Galápagos fur seal (Our results suggest a key role of intra- as well as inter-specific niche segregation in the evolution of genetic structure among populations of a highly mobile species under conditions of free movement. Given the monophyletic arrival of the sea lions on the archipelago, our study challenges the view that geographical barriers are strictly needed for the build-up of genetic divergence. The study further raises the interesting prospect that in social, colonially breeding mammals additional forces, such as social structure or feeding traditions, might bear on the genetic partitioning of populations. The relative role of ecologically mediated divergence in speciation processes is still under debate . Theory Marine environments provide excellent study cases, as they typically allow broad dispersal in mobile taxa and, compared to terrestrial habitats, offer low travel costs . Still, Zalophus wollebaeki) is endemic to the archipelago and genetically distinct from its nearest relatives . SAT 1.5d  and usedSAT 1.5d .4 permutations was used to test for the statistical significance of this relationship ['ecodist package' in R [Stepping stone models on a two-dimensional space predict a linear relationship between Fst/(1-Fst) and the logarithm of geographic distance . Becausege' in R ]. In migge' in R . We therJBWW conceived of the study, did the field work together with FT, conducted the genetic analyses and wrote the manuscript together with DT. CH was responsible for the stable isotope analysis, SB for the morphometric part of the study. SS helped to collect samples. DT hosted the project in his lab and together with FT provided significant input concerning the interpretation of the results. All authors read and approved of the final manuscript.Condylobasal lengths for skulls from western and central habitats. Raw data of skull measurementsClick here for fileMembership coefficients of colonies to genetic clusters from STRUCTURE analysis. The data provided describes the proportions of individuals assigned to one of four population clusters given for each of the sampled rookeries. Clusters where the majority of individuals were assigned are highlighted. In addition, the mean of the greatest membership coefficients of each individual is reported for each of the sampled rookeries.Click here for fileSpecimens used for morphometrics. Specification of specimens used for morphometric analysis and the according Institutions that provided them.Click here for file


Philornis downsi, parasitises nestlings of endemic birds on all major islands of the Galápagos archipelago. The fly's high mortality and fitness impacts are of conservation concern for vulnerable and declining species of Darwin's finches. Using microsatellite data in Bayesian clustering and landscape genetic analyses, we examine gene flow and dispersal in P. downsi between three islands and across habitats  and examine for the presence of population bottlenecks. We also examine variation at the mitochondrial gene CO1 across islands to establish if cryptic species were present.Understanding the dispersal and genetic structure of invasive insects across islands is important for designing management plans that are appropriate at spatial and temporal scales. For invasive parasites, population dynamics are largely determined by the distribution and density of their host species. The introduced parasitic fly, P. downsi population was found to have undergone a population bottleneck.Both the mitochondrial and microsatellite data were consistent with there being a single species across islands. We found low genetic differentiation between islands and strong evidence for inter-island gene flow, or shared recent ancestry among individuals. Landscape genetic analysis identified two genetic clusters: one encompassing Santa Cruz and Isabela, and one on Floreana Island. There was no evidence of genetic differentiation between habitats and molecular variance was mainly attributable to within individuals. The combined Philornis downsi populations have high connectivity within and between islands, with low levels of genetic differentiation between Floreana and the other two islands examined. The genetic bottleneck found across islands suggests there was a small founding population or few introduction events of P. downsi. The high dispersal capacity and wide habitat use of P. downsi highlights the significant threat that this parasite poses to the Galápagos avifauna. Our findings are relevant for assessing the viability of methods to control P. downsi on Galápagos, such as the sterile insect technique. Biological invasions threaten biodiversity and ecosystem function, with pronounced negative effects on islands in particular -3. GenetFor invasive arthropod parasites, these factors are inextricably linked with the distribution, genetics, and behaviour of host species -10. The Philornis downsi, is an avian ectoparasite that is considered to be a serious threat to the persistence of endemic finch populations on the Galápagos Islands [P. downsi was given the highest risk ranking affecting endemic fauna in the Galápagos archipelago [P. downsi colonised the islands at least 40 years ago, as the fly was identified recently from collections made in 1964 [P. downsi are associated with 62–100% nestling mortality in Darwin's finches [Philornis flies and the dispersal behaviour and population genetics of the genus Philornis or of any other myiasis-causing parasite of birds [reviewed in [The introduced fly,  Islands -14. Recehipelago . Other phipelago  and intehipelago  are of lhipelago ,13. Howe finches ,14,17, a finches  and reduP. downsi is the sterile insect technique (SIT), which is renowned for its effectiveness at eradicating or suppressing fruit fly and screw-worm fly populations across the globe [One potential control method to eradicate he globe ,20. SIT he globe ,21,22. She globe . Thus, tP. downsi.The Galápagos archipelago offers a unique system to examine the population genetics of an introduced avian parasite that causes severe fitness costs and that is still within a relatively early phase of invasion. We collected parasites in 2004, 2005 and 2006 from three islands of the Galápagos. Using mitochondrial data, we firstly determine whether the three island populations from which we sampled are of the one fly species. We then use microsatellite data to examine gene flow within and among islands to: (1) determine whether dispersal and genetic divergence are occurring among islands and between habitats within islands , (2) determine the presence of population bottlenecks resulting from the invasion process, and (3) determine whether inter-island genetic differentiation may be of concern to the potential success of an archipelago-wide SIT program for controlling Philornis downsi  is a semi-haematophagous obligate avian parasite in its three larval stages, whereas adult flies are non-parasitic and feed on organic matter [P. downsi appear to reach their third instar phase at the time of host fledging. The larvae pupariate at the base of the nesting material and remain for approximately two weeks before emerging as adult flies [c matter . Adults c matter ,24. Secoc matter . Most lalt flies ,25.Philornis downsi were collected from three islands of the Galápagos: Santa Cruz , Floreana , and Isabela . Fly samples were collected from nests during the January to March finch breeding season in 2004, 2005 and 2006 from two contrasting habitats, the arid lowlands (0–100 m asl) and the humid highlands (300–600 m asl)  on Isabela Island.m fagara . In contm fagara , abundan2, except for the highland site on Isabela, where our sample site was only 100 m2 because habitat fragmentation has reduced the Scalesia forest to small remnant patches. The distance between highland and lowland sites was much shorter on Floreana (3–5 km) than on Santa Cruz and Isabela (both 15–25 km), while on Santa Cruz, the distance between all four sites  varied between 15 and 27 km. Data were obtained from all three islands in 2004, from just Floreana in 2005, and from Santa Cruz and Floreana in 2006  , while one nest was opportunistically sampled from each of the Galápagos mockingbird (Nesomimus parvulus) and the yellow warbler (Dendroica petechia aureola). Fourteen recently fledged nests were sampled for P. downsi where the finch species was unknown. GPS coordinates were recorded at each nest location. Inactive nests were collected and sealed in individual plastic bags and later dismantled for counting of P. downsi individuals. All flies were immediately preserved in 95% ethanol.For the purpose of our study, larvae, puparia and puparia cases were sampled from 64 bird nests of five Darwin finch species  were homogenised and washed three times in 10 mm TRIS prior to digestion with Proteinase K to remove traces of ethanol, excess lipids, and other potential contaminants. Across all three islands, 1012 P. downsi individuals (larvae and pupae) were genotyped . To minimise and estimate genotyping error, each run of the DNA analyser contained eight repeated samples and a control sample run each time. In total, this resulted in 70 individual samples  being re-amplified and genotyped at least once.DNA extraction was carried out using the salting out procedure described in  with thec et al. . SamplesCO1 gene was amplified in five P. downsi individuals collected from Santa Cruz (1 highlands), Floreana , and Isabela . Samples were amplified using primers M202  according to the manufacturer's instructions. Products were sequenced on ABI 3700 (version 3.7) automated DNA sequencers. SeqEd (version 1.0.3) (Applied Biosystems) was used to edit chromatogram files to determine bi-directional consensus sequences and to manually align sequences across samples.An 822-bp region of the 3' end of the 1-J-1751 ) and M701-J-1751 ). AmplifRELATEDNESS 5.0.8 [P. downsi individuals collected from a single bird's nest). Exact tests were performed for each microsatellite locus to test deviation from Hardy-Weinberg equilibrium using GENEPOP[P. downsi offspring within nests of Darwin's finches is low, and the individuals found within each nest are produced by up to approximately five ovipositing females that have each mated with between one and five males . To eliminate the effect of sibs in the data, we selected unrelated individuals that were identified using the sib-ship reconstruction method implemented in the program COLONY 1.2 [P. downsi individuals taken from an independent bird nest was run in COLONY 1.2, which uses a maximum likelihood method that partitions individuals into pure full-sib families , or full-sib families nested within half-sib families  using progeny genotypes without known parental genotypes [We calculated allele frequencies using SS 5.0.8  by randog GENEPOP. All locg GENEPOP and thesLONY 1.2 . Each saenotypes ,38. ThreCOLONY 1.2) [We selected one individual per reconstructed maternal family . In nested-half sib families , individuals were only selected from full sib families with the largest number of members that had the highest posterior probability. Only individuals genotyped at all eight loci were included in the analysis and individuals were not sampled from families that contained Class I or Class II typing errors (identified by ONY 1.2) . These cGIMLET[To examine the probability that two randomly selected individuals from the same population will have the same multi-locus genotype, a Probability of Identity (PI) analysis was performed using GIMLET. The outGIMLET.Fst [MICROSATELLITE ANALYSER (MSA) 4.05 [GENEPOP[GENALEX version 6 [Heterozygosity, and pairwise Fst  was calcSA) 4.05 . Genotyp [GENEPOP. P-value [GENEPOP. The AMO [GENEPOP was condersion 6  to partiBOTTLENECK version 1.2.02 [P. downsi by analysing within-population heterozygosity and allele frequency using the constructed dataset of individuals sampled from all islands. Both the stepwise mutation model (SMM) and two-phase model of mutation (TPM) were used, with the latter model being considered the most appropriate for microsatellites. The variance for the TPM was set at 5% and the proportion of SMM in TPM was set at 95% [Recently colonised species may experience a population bottleneck, resulting in a reduction in the number of alleles and expected heterozygosity at polymorphic loci. However, alleles may be lost at a faster rate than the loss of heterozygosity, so observed heterozygosity is higher than the expected heterozygosity at equilibrium . The pron 1.2.02  was usedt at 95% . To detet at 95% . We alsot at 95% .STRUCTURE 2.1 [GENELAND[a priori knowledge of population units and limits. Both software packages implement a Bayesian clustering method that uses a MCMC technique to define the number of populations in a sample that are at Hardy Weinberg Equilibrium. The methods implemented in these two programs differ in that GENELAND determines the optimal number of populations or 'clusters' and then allocates individuals  to these clusters using geographic coordinates, whereas STRUCTURE carries out the allocation sequentially for different numbers of clusters, and then flags the number of clusters with the highest likelihood [STRUCTURE, the following run parameters were used: admixture without population information used, correlated allele frequency model, a burn-in period of 100 000 simulations followed by a run length of 1 million Markov Chain Monte Carlo (MCMC) simulations and three iterations for each number of potential clusters (defined as k = 1–5) to check for consistency of results. Estimation of k was taken to be the values of k with the highest Pr (X|k).For inferring genetic structure among the three sampled islands, we conducted two complementary individual-based Bayesian clustering analyses using TURE 2.1  and the [GENELAND without kelihood . In STRUSTRUCTURE, the algorithm implemented in GENELAND[k) in our data set, we used the Dirichlet model, which assumes independent allele frequencies with the following parameters: 1000 000 MCMC iterations, uncertainty attached to spatial coordinates = 0, variable number of populations = TRUE, minimum k = 1, maximum k = 5, and spatial information included in the model = TRUE. This procedure was performed three times to establish consistency of k across runs. The established k was then run five times to check the consistency of individual assignment to the inferred populations across runs. The same parameters were used but k was fixed at the modal number found in the first analyses. These five runs were post-processed (with a burn-in of 1000 × 100 iterations) to obtain posterior probabilities of population membership for each individual. Consistency of results across the five runs was checked visually.In contrast to  GENELAND is consi GENELAND,52. The  GENELAND,53. All o) and expected (He) heterozygosity, inbreeding coefficients (Fis), and genetic differentiation (estimated using Fst) using FSTAT v. 2.9.3 [Inferred populations were further examined for heterozygosity, allelic richness (corrected for sample size), observed  having an identical single nucleotide substitution (T-G). This supports the existence of one sampled species across the three islands.Sequences of the -06, and the PI for sibs was 2.610-3. This equates to one individual in approximately 751 880 having a non-unique genotype where individuals are unrelated (unbiased), and one individual in approximately 383 individuals having a non-unique genotype if all individuals are siblings.Probability of identity (PI) analyses showed that the microsatellite loci had sufficient power and resolution for the analyses. The unbiased PI value was 1.333X2 = 72.75; df = 16; P < 0.001). Mean allelic richness across loci was almost identical on each island  and the range of observed heterozygosity across loci was also similar . The number and size of alleles from each island population were the same at each locus with two exceptions: there was a unique allele at locus Pd6 on Isabela , and at locus Pd7 on Floreana , which were each detected only in a single individual. Pairwise Fst analysis showed low, but significant levels of genetic differentiation between Santa Cruz and Floreana  Isabela and Floreana , but not between Santa Cruz and Isabela . The low genetic differentiation found between islands was reflected in an AMOVA, which showed that just 2% of the molecular variance was attributable to variation among islands (sum of squares (SS) = 14.23; df = 2; variance components (V) = 0.052), 4% among individuals , and 94% within individuals .The total number of alleles observed at each locus was as follows; Pd1 = 4; Pd2 = 3; Pd4 = 4; Pd6 = 5; Pd7 = 3; Pd8 = 4; Pd9 = 3; Pd10 = 3 (Table eq) was observed, indicative of a population bottleneck under the TPM model  and under the SMM model (P < 0.01). A mode-shift distortion in the distribution of allele frequencies was evident , a clear excess of heterozygosity (He) relative to the equilibrium heterozygosity  between them demonstrated the low divergence between individuals on Floreana Island and those on Santa Cruz and Isabela  = 0.014 – 0.034; P < 0.05). Tests of departure from HW equilibrium showed no significant deviation in either of the two clusters across all loci.The two clusters identified by i Tables  and 3. APhilornis on the islands from which we sampled. A population bottleneck was detected in the entire sample of individuals from the three islands, which is consistent with the pattern expected from an invasive, recently colonised species [P. downsi on the Galápagos archipelago. Fly populations on Santa Cruz, Floreana, and Isabela showed strong evidence for high inter-island gene flow. However, low levels of divergence were detected between individuals from Floreana Island and those from Santa Cruz and Isabela when incorporating geographic sampling information. The molecular variance was mainly explained at the level of individuals, and not by island, which further demonstrates the low genetic differentiation between islands. Bayesian clustering analysis with geographic data assigned individuals to two genetic clusters, one comprising individuals from Santa Cruz and Isabela, and the second comprising all individuals from Floreana Island  between populations is found to be a useful predictor of the performance of assignment methods [STRUCTURE to confidently assign individuals to any cluster with certainty may reflect the lack of power to do so due to the low genetic differentiation (i.e. Fst) between sampling locations. Thus, we conclude there was an insufficient signal in the data to confidently assign individuals under the model of Pritchard et al. [STRUCTURE to assign individuals correctly where genetic differentiation is low and dispersal is common requires further study using empirical field data [The Bayesian clustering method implemented in  defined . Further defined ,55. The d et al. , despited et al. . Verifyield data ,55.P. downsi populations and data from all islands of the Galápagos where P. downsi occurs, which will be necessary for a detailed examination of founder effects, bottlenecks, introduction events and colonisation pathways. Thus, without knowing where P. downsi populations originally came from, or where they most recently arrived on the Galápagos archipelago, a comprehensive invasion history can not be constructed on a demographic or evolutionary scale [P. downsi invasion on the Galápagos archipelago. It is possible that P. downsi arrived on Ecuadorian cargo ships that were transporting fruit to the islands for human consumption [P. downsi.The current study lacks genetic data from mainland ry scale ,56. Howesumption ,58, whilsumption ). Strongsumption , while tsumption . Such paP. downsi across the three islands examined, which could be due to a small founding population, low immigration rates, or few introduction events [P. downsi is further evidence for a small effective population size upon initial colonisation. However, the occurrence of multiple introductions can not be excluded, particularly in the absence of comparisons with potential source populations  [P. downsi, the fly has clearly succeeded at establishing and spreading itself across the archipelago in high numbers.Recently colonised invaders are often subject to a reduction in genetic variation and population bottlenecks because populations are not in genetic equilibrium -6. We prn events . The low Brazil) . DespiteP. downsi on Galápagos because the fly was not detected in finch nests and identified until 1997 [Recently established species may persist at low and possibly undetectable numbers before becoming noticeably abundant and invasive years or decades later , which mtil 1997 , despitetil 1997 ,17. The til 1997 , indicattil 1997 ).P. downsi has spread successfully in at least 12 avian host species on the Galápagos Islands [P. downsi population on Floreana Island has detectable levels of genetic differentiation when compared with two other island populations, which might be the result of a separate introduction event(s) or colonisation pattern. A wider geographic sample of locations across habitats and islands is needed to examine this more definitively in combination with a larger number of highly polymorphic genetic markers. However, it is clear that P. downsi populations generally have high connectivity between islands or high shared ancestry, although variation in population processes  between particular islands may allow for low levels of inter-island genetic differentiation.Ecological ,28,59 fi Islands . In the P. downsi on the Galápagos archipelago may reflect the estimated short time period since the flies' introduction (~40 years ago) [Local populations are expected to evolve adaptive differences in response to differing environmental conditions . The lacars ago)  such thaDrosophila subobscura, just two decades after its introduction into North America. The evidence we present for high gene flow between habitats implies that morphological variation in P. downsi is unlikely, though other insect species on Galápagos show morphological variation and genetic differentiation between habitats and islands of the archipelago [Bulimilus land snails on Galápagos [Fly populations may show rapid evolution with geographic cline, as shown by Huey et al.  who founhipelago ,66,67. Calápagos  and Darwalápagos .P. downsi on the Galápagos Islands is perhaps the most appropriate method for eradicating an invasive fly within this ecologically fragile island ecosystem. SIT is a non-disruptive method as it does not introduce toxic or foreign chemicals into the environment, it is species specific, and does not introduce new genetic material into populations because the released organisms are not self-replicating [The use of SIT to control licating ,69.P. downsi within and between three islands of the Galápagos is high, and unlikely to result in reproductive isolation. Thus, release of a single sterile strain of P. downsi could effectively suppress and eradicate the fly across the archipelago. Captive breeding experiments of adult P. downsi from multiple island populations are necessary to determine this with high confidence.The effectiveness of SIT is affected by population genetic differentiation within the target species because the occurrence of undetected sub-species or strain differentiation across geographic populations can be detrimental to widespread sterile male release . ReinfesP. downsi highlights the significant threat that this parasite poses to the Galápagos endemic avifauna. Our findings are concordant with the prediction that parasites with low host specificity [P. downsi, this window of opportunity appears to have passed, prompting the need for a long-term eradication program combined with sustained quarantine and monitoring practices.The wide habitat range and high dispersal capacity of cificity , good dicificity . Ideallycificity . For P. RYD and MGG performed all molecular laboratory work. RYD performed all genotyping, data analysis and drafted the manuscript. MGG and SD participated in data analysis and interpretation. SK participated in the design and coordination of the study and provided funding. SK and RYD conducted field work and collection of samples. All authors read and approved the final manuscript.