M-ratio Test Research Articles

Low Bottleneck Detection in Long-Lived Species Despite Lost Genetic Diversity: A Case Study of Tuatara and Eastern Massasauga Rattlesnakes.

Population bottlenecks can reduce genetic diversity and may lead to inbreeding depression. However, some studies have provided evidence that long lifespans buffer negative genetic effects of bottlenecks. Others have cautioned that longevity might merely mask the effects of genetic drift, which will still affect long-term population viability. We used microsatellite data from actual populations of tuatara (Sphenodon punctatus) and eastern massasaugas (Sistrurus catenatus) as a starting point for simulated population declines to evaluate the performance of bottleneck tests under a range of scenarios. We quantified losses in genetic diversity for each scenario and assessed the power of commonly used tests (i.e., M-ratio, heterozygosity excess, and mode-shift) to detect known bottlenecks in these moderate- to long-lived species. Declines in genetic diversity were greater in bottlenecks simulated for eastern massasaugas, the shorter-lived species, and mode-shift and heterozygosity excess tests were more sensitive to population declines in this species. Conversely, M-ratio tests were more sensitive to bottlenecks simulated in tuatara. Despite dramatic simulated population declines, heterozygosity excess and mode-shift tests often failed to detect bottlenecks in both species, even when large losses in genetic diversity had occurred (both allelic diversity and heterozygosity). While not eliminating type II error, M-ratio tests generally performed best and were most reliable when a critical value (Mc) of 0.68 was used. However, in tuatara simulations, M-ratio tests had high rates of type I error when Mc was calculated assuming θ = 10. Our results suggest that reliance on these tests could lead to misguided species management decisions.

Genetic Diversity and Population Structure of the Forgotten Geum, Geum geniculatum Michx.

Geum geniculatum (Rosaceae), bent avens, is a perennial herb restricted to the high elevations of three mountaintops near the North Carolina/Tennessee border (USA). Although geographically restricted, occurrences on these mountaintops can have up to hundreds and occasionally thousands of individuals. While Geum geniculatum has been censused thoroughly, some sites have not been visited in over a decade, and formal biological studies are lacking. To understand genetic variation within the species and connectivity among populations, individuals were sampled from each of three extant mountain locations and were genotyped using 14 microsatellite markers previously developed for other congeners. Results show that G. geniculatum displays high genetic diversity, and that each mountain acts as a highly structured metapopulation with moderate interpopulation differentiation. Based on the high numbers of private alleles and results of an M-ratio test, genetic drift is likely driving structure and differentiation among metapopulations. Results will inform management of the species, and suggest that transfer of seeds or genetic material among sites should be minimized.

Effects of urbanization on population genetic structure of western gray squirrels

Habitat loss and fragmentation due to urbanization are key contributors to the decline of biodiversity. The consequence of these factors is small, isolated populations that are more susceptible to deterministic and stochastic threats of extinction. There is an increasing trend in population reductions of the western gray squirrel (Sciurus griseus) in urban areas of Southern California, USA. Griffith Park (GP) contains one of the last urban populations of western gray squirrels (WGS) present in Los Angeles. We used hairtubes to collect hair of WGS at 3 sites within GP and at 5 sites outside of GP. Twelve microsatellite loci and a 550 bp segment of the mitochondrial control region were used to examine the genetic diversity within GP and among all sample sites, and to determine gene flow within GP. Results revealed subpopulations within GP have low levels of allelic richness at microsatellite loci (AR = 2.28–2.53) and low mitochondrial haplotype diversity (HD = 0.000–0.271). We found significant genetic differentiation (FST = 0.109–0.156, p < 0.001), high levels of relatedness within each GP subpopulation (0.399–0.633), and a lack of private alleles (AP = 0.09–0.27) at microsatellite loci. Mode shifts in microsatellite allele frequencies and positive M-ratio tests provide evidence of bottlenecks within a GP subpopulation. The effective population size for GP (Ne = 9.1) highlights the effects of genetic drift on this isolated population. We suggest conservation efforts that could maintain these last extant populations of a native species in urban Los Angeles.

Considering the Influence of Nonadaptive Evolution on Primate Color Vision.

Color vision in primates is variable across species, and it represents a rare trait in which the genetic mechanisms underlying phenotypic variation are fairly well-understood. Research on primate color vision has largely focused on adaptive explanations for observed variation, but it remains unclear why some species have trichromatic or polymorphic color vision while others are red-green color blind. Lemurs, in particular, are highly variable. While some species are polymorphic, many closely-related species are strictly dichromatic. We provide the first characterization of color vision in a wild population of red-bellied lemurs (Eulemur rubriventer, Ranomafana National Park, Madagascar) with a sample size (87 individuals; NX chromosomes = 134) large enough to detect even rare variants (0.95 probability of detection at ≥ 3% frequency). By sequencing exon 5 of the X-linked opsin gene we identified opsin spectral sensitivity based on known diagnostic sites and found this population to be dichromatic and monomorphic for a long wavelength allele. Apparent fixation of this long allele is in contrast to previously published accounts of Eulemur species, which exhibit either polymorphic color vision or only the medium wavelength opsin. This unexpected result may represent loss of color vision variation, which could occur through selective processes and/or genetic drift (e.g., genetic bottleneck). To indirectly assess the latter scenario, we genotyped 55 adult red-bellied lemurs at seven variable microsatellite loci and used heterozygosity excess and M-ratio tests to assess if this population may have experienced a recent genetic bottleneck. Results of heterozygosity excess but not M-ratio tests suggest a bottleneck might have occurred in this red-bellied lemur population. Therefore, while selection may also play a role, the unique color vision observed in this population might have been influenced by a recent genetic bottleneck. These results emphasize the need to consider adaptive and nonadaptive mechanisms of color vision evolution in primates.

Genetic variability and bottleneck detection of four Tricholoma matsutake populations from northeastern and southwestern China.

The excessive commercial collection of matsutake mushrooms can lead to extreme reduction of population size, which may cause genetic bottleneck and decrease genetic diversity of Tricholoma matsutake. Here, six polymorphic microsatellite loci markers were used to examine the genetic diversity of four natural T. matsutake populations from two main producing regions of China. The minimum combinations of four loci were able to discriminate total 86 sampled individuals with distinctive multilocus genotypes. Our analysis of molecular variance (AMOVA) revealed that about 80% and 20% of the overall genetic variation were respectively partitioned within and among populations. The principal-coordinate analyses (PCA) distinguished the four tested populations into three genetic clusters, each of which was correlated with respective endemic host plants on a geographical basis. The AMOVA, PCA and pairwise population FST estimates consistently displayed the same genetic divergence patterns and spatial structure of T. matsutake mediated by host plants in China. The significant heterozygosity excesses demonstrated that a recent genetic bottleneck occurred in each population tested. The complementary M-ratio test indicated past genetic bottleneck events over longer periods. Only four individuals were identified as putative first generation migrants within northeastern China, which implies restricted interpopulation gene flow in T. matsutake. We discuss that the significant genetic differentiation among populations of T. matsutake is most likely a function of host adaptation, host specificity, genetic bottleneck, limited dispersal and habitat fragmentation.

Genetics at the verge of extinction: insights from the Iberian lynx

Population viability might become compromised by the loss of genetic diversity and the accumulation of inbreeding resulting from population decline and fragmentation. The Iberian lynx (Lynx pardinus) provides a paradigmatic example of a species at the verge of extinction, and because of the well-documented and different demographic histories of the two remaining populations (Doñana and Andújar), it provides the opportunity to evaluate the performance of analytical methods commonly applied to recently declined populations. We used mitochondrial sequences and 36 microsatellite markers to evaluate the current genetic status of the species and to assess the genetic signatures of its past history. Mitochondrial diversity was extremely low with only two haplotypes, alternatively fixed in each population. Both remnant populations have low levels of genetic diversity at microsatellite markers, particularly the population from Doñana, and genetic differentiation between the two populations is high. Bayesian coalescent-based methods suggest an earlier decline starting hundreds of years ago, while heterozygosity excess and M-ratio tests did not provide conclusive and consistent evidence for recent bottlenecks. Also, a model of gene flow received overwhelming support over a model of pure drift. Results that are in conflict with the known recent demography of the species call for caution in the use of these methods, especially when no information on previous demographic history is available. Overall, our results suggest that current genetic patterns in the Iberian lynx are mainly the result of its recent decline and fragmentation and alerts on possible genetic risks for its persistence. Conservation strategies should explicitly consider this threat and incorporate an integrated genetic management of wild, captive and re-introduced populations, including genetic restoration through translocations.

Genetic structure and admixture between the Posavina and Croatian Coldblood in contrast to Lipizzan horse from Croatia

The Posavina and Croatian Coldblood are Croatian autochthonous horse breeds with interwoven breeding histories for which studbooks have only recently been established. The Lipizzan breed has the oldest formalized breeding and no record of recent genetic introgression from other breeds in Croatia. We analyzed the genetic structure, interbreeding, and breed characteristics by genotyping nine dinucleotide microsatellite loci for 53 Posavina, 37 Croatian Coldblood, and 33 Lipizzan horses and showed that differing breeding schemes and histories have had a strong and measurable impact on the population genetic structure within and between the three breeds. A Bayesian clustering method demonstrated that two population clusters best explain the genetic structure. Samples from the pre-defined breeds of the Posavina and Croatian Coldblood were assigned to a separate genetic cluster, while Lipizzan specimens were assigned to another distinct genetic group. Twelve samples of the Posavina/Croatian Coldblood cluster (13%) showed admixed ancestry with Lipizzan horses. A test for heterozygosity excess, allele frequency distribution mode-shift, and M-ratio test were used to detect genetic evidence of recent population bottlenecks, none of which provided evidence for bottlenecks in the Posavina and Croatian Coldblood populations. In contrast, although somewhat ambiguous, evidence suggests a genetic bottleneck in the Lipizzan population in Croatia.

Population history, genetic variation and conservation status of the endangered birch species Betula nana L. in Poland

The effective conservation of species requires data on the levels and distribution of genetic diversity within and among populations. In this study, we estimated the genetic variation in three isolated populations of Betula nana in Poland. An analysis of 11 nuclear microsatellites revealed moderate mean heterozygosities (H=0.556, H=0.562), low mean number of alleles per locus (A=4.57) and no inbreeding in the total sample. An M-ratio test indicated that each population had experienced a severe bottleneck in the past. Tests for heterozygosity excess revealed that a significant decrease in the numbers of individuals in two populations had occurred quite recently. The large number of private alleles and very restricted number of migrants between populations (Nm=0.35) strongly suggest that genetic drift and geographic isolation are the primary factors responsible for the reduction of genetic variation in the Polish populations of B. nana. We detected two cpDNA haplotypes in the study populations, which can be explained in terms of either the genetic drift acting on the relict localities or a postglacial recolonisation from distinct refugia. Palynological data indicated that one refugium could be located in the Carpathians and their northern foreland. The primary threat to B. nana in Poland is the overgrowth of its habitats by competing species, which has likely resulted in a lack of generative reproduction in the mountain populations.OE

Inferring Population Decline and Expansion From Microsatellite Data: A Simulation-Based Evaluation of the Msvar Method

Reconstructing the demographic history of populations is a central issue in evolutionary biology. Using likelihood-based methods coupled with Monte Carlo simulations, it is now possible to reconstruct past changes in population size from genetic data. Using simulated data sets under various demographic scenarios, we evaluate the statistical performance of Msvar, a full-likelihood Bayesian method that infers past demographic change from microsatellite data. Our simulation tests show that Msvar is very efficient at detecting population declines and expansions, provided the event is neither too weak nor too recent. We further show that Msvar outperforms two moment-based methods (the M-ratio test and Bottleneck) for detecting population size changes, whatever the time and the severity of the event. The same trend emerges from a compilation of empirical studies. The latest version of Msvar provides estimates of the current and the ancestral population size and the time since the population started changing in size. We show that, in the absence of prior knowledge, Msvar provides little information on the mutation rate, which results in biased estimates and/or wide credibility intervals for each of the demographic parameters. However, scaling the population size parameters with the mutation rate and scaling the time with current population size, as coalescent theory requires, significantly improves the quality of the estimates for contraction but not for expansion scenarios. Finally, our results suggest that Msvar is robust to moderate departures from a strict stepwise mutation model.

Siberian tiger's recent population bottleneck in the Russian Far East revealed by microsatellite markers

Because size reduced, or bottlenecked, populations are more prone to adverse events, the detection of genetic bottleneck signatures in wildlife species is highly relevant for conservation. Here we applied 11 microsatellite markers to the endangered Siberian tiger ( Panthera tigris altaica) using tissue and blood samples of animals from the Primorsky region of the Russian Far East. Excess heterozygosity and mode shift in allele frequencies tests were positive, while the M-ratio test was negative, indicating the likelihood of a contemporary rather than a historical population bottleneck. The recent genetic population bottleneck could be attributed to the well-documented demographic collapse of the Siberian tiger population in the 1940s, when population size hit bottom with 20–30 surviving animals. The mean effective population size N e was 14 Siberian tigers (CI 95: 12–25 animals), and the effective population size/census size ratio ( N e / N ratio) was 0.028. This is the first molecular evidence of a recent Siberian tiger population bottleneck, which is of great interest for further conservation and management plans of the highly endangered largest felid species, while the worryingly low effective population size challenges the optimism for the recovery of the huge Siberian cat.

Signatures of demographic bottlenecks in European wolf populations

Monitoring the loss of genetic diversity in wild populations after a bottleneck event is a priority in conservation and management plans. Here, we used diverse molecular markers to search for signatures of demographic bottlenecks in two wolf populations; an isolated population from the Iberian Peninsula and a non-isolated population from European Russia. Autosomal, mtDNA and Y-chromosomal diversity and the effective population size (Ne) were significantly lower in the Iberian population. Neutrality tests using mtDNA sequences, such as R2, Fu and Li’s F*, Tajima’s D and Fu’s Fs, were positively significant in the Iberian population, suggesting a population decline, but were not significant for the Russian population, likely due to its larger effective population size. However, three tests using autosomal data confirmed the occurrence of the genetic bottleneck in both populations. The M-ratio test was the only one providing significant results for both populations. Given the lack of consistency among the different tests, we recommend using multiple approaches to investigate possible past bottlenecks. The small effective population size (about 50) in the Iberian Peninsula compared to the presumed extant population size could indicate that the bottleneck was more powerful than initially suspected or an overestimation of the current population. The risks associated with small effective population sizes suggest that the genetic change in this population should be closely monitored in the future. On the other hand, the relatively small effective population size for Russian wolves (a few hundred individuals) could indicate some fragmentation, contrary to what is commonly assumed.

No evidence for a recent genetic bottleneck in the endangered Sheko cattle breed (African Bos taurus) revealed by microsatellite analysis

AbstractSheko is African taurine cattle, valued for its milk yield, adaptation to humid tsetse infested environment and trypanotolerance. We used 30 microsatellite markers in analyzing 30 DNA samples. We found high genetic diversity and no genetic bottlenecks in endangered Sheko cattle. Sheko cattle have not undergone recent genetic bottlenecks, in spite of drastic reduction in its overall demographic population size. The results were supported by three statistical methods: (i) detection of heterozygosity excess (ii) a mode-shift indicator of allele distribution pattern (iii) the ratio of the number of alleles to the range of allele size, M-ratio test. This breed reflects historical and cultural identity of local communities and represents a unique component of the global domestic animal biodiversity that deserve priority for conservation.

Microsatellite variation, population structure, and bottlenecks in the threatened copperbelly water snake

Habitat loss and isolation is pervasive in the Midwest U.S. Wetlands are experiencing particularly dra- matic declines, yet there is a paucity of information on the genetic impacts of these losses to obligate wetland verte- brates. We quantified the genetic variation of extant populations of a shallow wetland specialist and evaluated potential reductions in population size (i.e. bottlenecks) using seven polymorphic microsatellite DNA markers. We analyzed 228 copperbelly water snakes (Nerodia eryth- rogaster neglecta), representing populations from three states. Moderate genetic differentiation exists among all three regions (FST = 0.12, P \ 0.001), with evidence for low levels of differentiation within the federally protected Ohio region (FST = 0.025, P = 0.007), and moderate to strong differentiation within the Indiana region (FST = 0.23, P \ 0.001). Furthermore, Bayesian clustering (i.e. STRUCTURE) supports the separation of the Indiana sites, both from each other and from all other sampling sites. However, it does not support the separation of the Ohio sites from the Kentucky sites. Differentiation among sampling sites did not appear to be related to geographic distance, but rather depended on the quality of terrestrial corridors used for dispersal. Mode shifts in allele frequencies and excess heterozygosity tests were negative, while M-ratio tests were nearly all positive, indicating the likelihood of historical rather than contemporary population bottlenecks. However, potential subspecific intergradation in the Kentucky region may have artificially lowered the M-ratio, and we suggest caution when using the M-ratio approach if intergradation is suspected. Our results have conservation implications for wetland management and management of the copperbelly populations, and emphasizes the importance of protecting wetland complexes.

Recent demographic bottlenecks are not accompanied by a genetic signature in banner‐tailed kangaroo rats (Dipodomys spectabilis)

Single-sample methods of bottleneck detection are now routine analyses in studies of wild populations and conservation genetics. Three common approaches to bottleneck detection are the heterozygosity excess, mode-shift, and M-ratio tests. Empirical groundtruthing of these methods is difficult, but their performances are critical for the accurate reconstruction of population demography. We use two banner-tailed kangaroo rat (Dipodomys spectabilis) populations from southeastern Arizona (USA) that are known to have experienced recent demographic reductions to search for genetic bottleneck signals with eight microsatellite loci. Over eight total sample-years, neither population showed a genetic bottleneck signature. M-ratios in both populations were large, stable, and never fell below a critical significance value (Mc). The mode shift test did not detect any distortion of allele frequencies, and tests of heterozygosity excess were not significant in postbottleneck samples when we used standard microsatellite mutation models. The genetic effects of bottlenecks like those experienced by our study populations should be strongly influenced by rates of mutation and migration. We used genetic parentage data to estimate a relatively high mutation rate in D. spectabilis (0.0081 mutants/generation/locus), but mutation alone is unlikely to explain the temporal distribution of rare alleles that we observed. Migration (gene flow) is a more likely explanation, despite prior mark-recapture analysis that estimated very low rates of interpopulation dispersal. We interpret our kangaroo rat data in light of the broader literature and conclude that in natural populations connected by dispersal, demographic bottlenecks may prove difficult to detect using molecular genetic data.