Spatial, climate and ploidy factors drive genomic diversity and resilience in the widespread grass Themeda triandra.

Global climate change poses a significant threat to natural communities around the world, with many plant species showing signs of climate stress. Grassland ecosystems are not an exception, with climate change compounding contemporary pressures such as habitat loss and fragmentation. In this study, we assess the climate resilience of Themeda triandra, a foundational species and the most widespread plant in Australia, by assessing the relative contributions of spatial, environmental and ploidy factors to contemporary genomic variation. Reduced-representation genome sequencing on 472 samples from 52 locations was used to test how the distribution of genomic variation, including ploidy polymorphism, supports adaptation to hotter and drier climates. We explicitly quantified isolation by distance (IBD) and isolation by environment (IBE) and predicted genomic vulnerability of populations to future climates based on expected deviation from current genomic composition. We found that a majority (54%) of genomic variation could be attributed to IBD, while an additional 22% (27% when including ploidy information) could be explained by two temperature and two precipitation climate variables demonstrating IBE. Ploidy polymorphisms were common within populations (31/52 populations), indicating that ploidy mixing is characteristic of T. triandra populations. Genomic vulnerabilities were found to be heterogeneously distributed throughout the landscape, and our analysis suggested that ploidy polymorphism, along with other factors linked to polyploidy, reduced vulnerability to future climates by 60% (0.25-0.10). Our data suggests that polyploidy may facilitate adaptation to hotter climates and highlight the importance of incorporating ploidy in adaptive management strategies to promote the resilience of this and other foundation species.

Genomic impact of severe population decline in a nomadic songbird.

Uncovering the population genetic histories of non-model organisms is increasingly possible through advances in next generation sequencing and DNA sampling of museum specimens. This new information can inform conservation of threatened species, particularly those for which historical and contemporary population data are unavailable or challenging to obtain. The critically endangered, nomadic regent honeyeater Anthochaera phrygia was abundant and widespread throughout south-eastern Australia prior to a rapid population decline and range contraction since the 1970s. A current estimated population of 250-400 individuals is distributed sparsely across 600,000 km2 from northern Victoria to southern Queensland. Using hybridization RAD (hyRAD) techniques, we obtained a SNP dataset from 64 museum specimens (date 1879-1960), 102 'recent' (1989-2012) and 52 'current' (2015-2016) wild birds sampled throughout the historical and contemporary range. We aimed to estimate population genetic structure, genetic diversity and population size of the regent honeyeater prior to its rapid decline. We then assessed the impact of the decline on recent and current population size, structure and genetic diversity. Museum sampling showed population structure in regent honeyeaters was historically low, which remains the case despite a severe fragmentation of the breeding range. Population decline has led to minimal loss of genetic diversity since the 1980's. Capacity to quantify the overall magnitude of both genetic diversity loss and population decline was limited by the poorer quality of genomic data derived from museum specimens. A rapid population decline, coupled with the regent honeyeater's high mobility, means a detectable genomic impact of this decline has not yet manifested. Extinction may occur in this nomadic species before a detectable genomic impact of small population size is realised. We discuss the implications for genetic management of endangered mobile species and enhancing the value of museum specimens in population genomic studies.

Global Diversity of the Brachypodium Species Complex as a Resource for Genome-Wide Association Studies Demonstrated for Agronomic Traits in Response to Climate.

The development of model systems requires a detailed assessment of standing genetic variation across natural populations. The Brachypodium species complex has been promoted as a plant model for grass genomics with translation to small grain and biomass crops. To capture the genetic diversity within this species complex, thousands of Brachypodium accessions from around the globe were collected and genotyped by sequencing. Overall, 1897 samples were classified into two diploid or allopolyploid species, and then further grouped into distinct inbred genotypes. A core set of diverse B. distachyon diploid lines was selected for whole genome sequencing and high resolution phenotyping. Genome-wide association studies across simulated seasonal environments was used to identify candidate genes and pathways tied to key life history and agronomic traits under current and future climatic conditions. A total of 8, 22, and 47 QTL were identified for flowering time, early vigor, and energy traits, respectively. The results highlight the genomic structure of the Brachypodium species complex, and the diploid lines provided a resource that allows complex trait dissection within this grass model species.

Landscape genomic prediction for restoration of a Eucalyptus foundation species under climate change.

As species face rapid environmental change, we can build resilient populations through restoration projects that incorporate predicted future climates into seed sourcing decisions. Eucalyptus melliodora is a foundation species of a critically endangered community in Australia that is a target for restoration. We examined genomic and phenotypic variation to make empirical based recommendations for seed sourcing. We examined isolation by distance and isolation by environment, determining high levels of gene flow extending for 500 km and correlations with climate and soil variables. Growth experiments revealed extensive phenotypic variation both within and among sampling sites, but no site-specific differentiation in phenotypic plasticity. Model predictions suggest that seed can be sourced broadly across the landscape, providing ample diversity for adaptation to environmental change. Application of our landscape genomic model to E. melliodora restoration projects can identify genomic variation suitable for predicted future climates, thereby increasing the long term probability of successful restoration.

Species trees from consensus single nucleotide polymorphism (SNP) data: Testing phylogenetic approaches with simulated and empirical data.

Datasets of hundreds or thousands of SNPs (Single Nucleotide Polymorphisms) from multiple individuals per species are increasingly used to study population structure, species delimitation and shallow phylogenetics. The principal software tool to infer species or population trees from SNP data is currently the BEAST template SNAPP which uses a Bayesian coalescent analysis. However, it is computationally extremely demanding and tolerates only small amounts of missing data. We used simulated and empirical SNPs from plants (Australian Craspedia, Asteraceae, and Pelargonium, Geraniaceae) to compare species trees produced (1) by SNAPP, (2) using SVD quartets, and (3) using Bayesian and parsimony analysis with several different approaches to summarising data from multiple samples into one set of traits per species. Our aims were to explore the impact of tree topology and missing data on the results, and to test which data summarising and analyses approaches would best approximate the results obtained from SNAPP for empirical data. SVD quartets retrieved the correct topology from simulated data, as did SNAPP except in the case of a very unbalanced phylogeny. Both methods failed to retrieve the correct topology when large amounts of data were missing. Bayesian analysis of species level summary data scoring the two alleles of each SNP as independent characters and parsimony analysis of data scoring each SNP as one character produced trees with branch length distributions closest to the true trees on which SNPs were simulated. For empirical data, Bayesian inference and Dollo parsimony analysis of data scored allele-wise produced phylogenies most congruent with the results of SNAPP. In the case of study groups divergent enough for missing data to be phylogenetically informative (because of additional mutations preventing amplification of genomic fragments or bioinformatic establishment of homology), scoring of SNP data as a presence/absence matrix irrespective of allele content might be an additional option. As this depends on sampling across species being reasonably even and a random distribution of non-informative instances of missing data, however, further exploration of this approach is needed. Properly chosen data summary approaches to inferring species trees from SNP data may represent a potential alternative to currently available individual-level coalescent analyses especially for quick data exploration and when dealing with computationally demanding or patchy datasets.

Isolation by distance and isolation by environment contribute to population differentiation in Protea repens (Proteaceae L.), a widespread South African species.

PREMISE OF THE STUDY: The Cape Floristic Region (CFR) of South Africa is renowned for its botanical diversity, but the evolutionary origins of this diversity remain controversial. Both neutral and adaptive processes have been implicated in driving diversification, but population-level studies of plants in the CFR are rare. Here, we investigate the limits to gene flow and potential environmental drivers of selection in Protea repens L. (Proteaceae L.), a widespread CFR species. METHODS: We sampled 19 populations across the range of P. repens and used genotyping by sequencing to identify 2066 polymorphic loci in 663 individuals. We used a Bayesian FST outlier analysis to identify single-nucleotide polymorphisms (SNPs) marking genomic regions that may be under selection; we used those SNPs to identify potential drivers of selection and excluded them from analyses of gene flow and genetic structure. RESULTS: A pattern of isolation by distance suggested limited gene flow between nearby populations. The populations of P. repens fell naturally into two or three groupings, which corresponded to an east-west split. Differences in rainfall seasonality contributed to diversification in highly divergent loci, as do barriers to gene flow that have been identified in other species. CONCLUSIONS: The strong pattern of isolation by distance is in contrast to the findings in the only other widespread species in the CFR that has been similarly studied, while the effects of rainfall seasonality are consistent with well-known patterns. Assessing the generality of these results will require investigations of other CFR species.

Genomic diversity guides conservation strategies among rare terrestrial orchid species when taxonomy remains uncertain.

Background and Aims: Species are often used as the unit for conservation, but may not be suitable for species complexes where taxa are difficult to distinguish. Under such circumstances, it may be more appropriate to consider species groups or populations as evolutionarily significant units (ESUs). A population genomic approach was employed to investigate the diversity within and among closely related species to create a more robust, lineage-specific conservation strategy for a nationally endangered terrestrial orchid and its relatives from south-eastern Australia. Methods: Four putative species were sampled from a total of 16 populations in the Victorian Volcanic Plain (VVP) bioregion and one population of a sub-alpine outgroup in south-eastern Australia. Morphological measurements were taken in situ along with leaf material for genotyping by sequencing (GBS) and microsatellite analyses. Key Results: Species could not be differentiated using morphological measurements. Microsatellite and GBS markers confirmed the outgroup as distinct, but only GBS markers provided resolution of population genetic structure. The nationally endangered Diuris basaltica was indistinguishable from two related species ( D. chryseopsis and D. behrii ), while the state-protected D. gregaria showed genomic differentiation. Conclusions: Genomic diversity identified among the four Diuris species suggests that conservation of this taxonomically complex group will be best served by considering them as one ESU rather than separately aligned with species as currently recognized. This approach will maximize evolutionary potential among all species during increased isolation and environmental change. The methods used here can be applied generally to conserve evolutionary processes for groups where taxonomic uncertainty hinders the use of species as conservation units.

Genotyping of whole genome amplified reduced representation libraries reveals a cryptic population of Culicoides brevitarsis in the Northern Territory, Australia.

BACKGROUND: The advent of genotyping by Next Generation Sequencing has enabled rapid discovery of thousands of single nucleotide polymorphism (SNP) markers and high throughput genotyping of large populations at an affordable cost. Genotyping by sequencing (GBS), a reduced representation library sequencing method, allows highly multiplexed sequencing of genomic subsets. This method has limitations for small organisms with low amounts of genomic DNA, such as the bluetongue virus (BTV) vectors, Culicoides midges. RESULTS: This study employed the GBS method to isolate SNP markers de novo from whole genome amplified Culicoides brevitarsis genomic DNA. The individuals were collected from regions representing two different Australian patterns of BTV strain distribution: the Northern Territory (NT) and the east coast. We isolated 8145 SNPs using GBS. Phylogenetic analysis conducted using the filtered 3263 SNPs revealed the presence of a distinct C. brevitarsis sub-population in the NT and this was confirmed by analysis of mitochondrial DNA. Two loci showed a very strong signal for selection and were unique to the NT population. Bayesian analysis with STRUCTURE indicated a possible two-population cluster. CONCLUSIONS: The results suggest that genotyping vectors with high density markers in combination with biological and environmental data is useful. However, more extensive sampling over a wider spatial and temporal range is needed. The presence of sub-structure in populations and loci under natural selection indicates the need for further investigation of the role of vectors in shaping the two Australian systems of BTV transmission. The described workflow is transferable to genotyping of small, non-model organisms, including arthropod vectors of pathogens of economic and medical importance.

Population and phylogenomic decomposition via genotyping-by-sequencing in Australian Pelargonium.

Species delimitation has seen a paradigm shift as increasing accessibility of genomic-scale data enables separation of lineages with convergent morphological traits and the merging of recently diverged ecotypes that have distinguishing characteristics. We inferred the process of lineage formation among Australian species in the widespread and highly variable genus Pelargonium by combining phylogenomic and population genomic analyses along with breeding system studies and character analysis. Phylogenomic analysis and population genetic clustering supported seven of the eight currently described species but provided little evidence for differences in genetic structure within the most widely distributed group that containing P. australe. In contrast, morphometric analysis detected three deep lineages within Australian Pelargonium; with P. australe consisting of five previously unrecognized entities occupying separate geographic ranges. The genomic approach enabled elucidation of parallel evolution in some traits formerly used to delineate species, as well as identification of ecotypic morphological differentiation within recognized species. Highly variable morphology and trait convergence each contribute to the discordance between phylogenomic relationships and morphological taxonomy. Data suggest that genetic divergence among species within the Australian Pelargonium may result from allopatric speciation while morphological differentiation within and among species may be more strongly driven by environmental differences.

Surgical quality in colorectal cancer.

OBJECTIVE: To determine the quality of surgical management offered to patients with colorectal cancer (CRC) as measured by adequacy of nodal resections and compare variations across the major hospitals in Jamaica. METHOD: Data was obtained from the CRC Registry of patients diagnosed and treated surgically for CRC during the 3-year period commencing January 1, 2011. Variables analyzed included tumor site, stage and number of lymph nodes resected across hospitals. RESULTS: During the period under review 60% (349) of 586 patients had resections and formed the basis of this study. Of these 49% were treated at the UHWI, 27% from the KPH and STH, 15% from CRH and MRH and 8% from a private laboratory (DPS). Patient distribution was similar at UHWI compared to the others with mean age (61 vs 62) and with slightly more women having surgery (53% Vs 54%) (UHWI vs Others). For tumor grade, margin status, lymphovascular and depth of invasion (majority T3) there was no difference between UHWI and the other sites, although a smaller percentage of tumors treated at UHWI had Crohn's like reaction (p = 0.01). There was a larger proportion of sigmoid cancer at UHWI while the reverse trend was seen in cancers of the rectum (p = 0.027). The tumors treated at UHWI have a larger median number of regional nodes when compared to the other facilities (14 vs 10; p < 0.001) and also more likely to have positive nodes, as were women and younger patients. Comparison across facilities revealed that the proportion of tumors classed as well differentiated, circumferential margin involvement, and having lymphovascular invasion were higher for specimens processed at the private facility (p = 0.021, 0.035, 0.01 respectively). Histopathology reports of tumors treated at UHWI and DPS had median 14 and 18 nodes respectively while at NPH laboratory and CRH they were 9 and 10 respectively (p < 0.001), whilst those of the ascending, descending, sigmoid colon and rectum had median 15, 11, 13, 11 nodes respectively (p < 0.001). CONCLUSIONS: This review demonstrates measurable differences in the surgery and histopathological reports for CRC patients treated across the island. Given adjuvant treatment and prognostic implications there is room for improvement.

Creating new evolutionary pathways through bioinvasion: the population genetics of brushtail possums in New Zealand.

Rapid increases in global trade and human movement have created novel mixtures of organisms bringing with them the potential to rapidly accelerate the evolution of new forms. The common brushtail possum (Trichosurus vulpecula), introduced into New Zealand from Australia in the 19th century, is one such species having been sourced from multiple populations in its native range. Here, we combine microsatellite DNA- and GIS-based spatial data to show that T. vulpecula originating from at least two different Australian locations exhibit a population structure that is commensurate with their introduction history and which cannot be explained by landscape features alone. Most importantly, we identify a hybrid zone between the two subspecies which appears to function as a barrier to dispersal. When combined with previous genetic, morphological and captive studies, our data suggest that assortative mating between the two subspecies may operate at a behavioural or species recognition level rather than through fertilization, genetic incompatibility or developmental inhibition. Nevertheless, hybridization between the two subspecies of possum clearly occurs, creating the opportunity for novel genetic combinations that would not occur in their natural ranges and which is especially likely given that multiple contact zones occur in New Zealand. This discovery has implications for wildlife management in New Zealand because multiple contact zones are likely to influence the dispersal patterns of possums and because differential susceptibility to baiting with sodium fluoroacetate between possums of different origins may promote novel genetic forms.

Complicações pós-operatórias menores relacionadas à anestesia em pacientes de cirurgias eletivas ginecológicas e ortopédicas em um hospital universitário de Kingston, Jamaica / Minor postoperative complications related to anesthesia in elective gynecological and orthopedic surgical patients at a teaching hospital in Kingston, Jamaica / Complicaciones postoperatorias menores relacionadas con la anestesia en pacientes para cirugías electivas ginecológicas y ortopédicas en un hospital universitario de Kingston, Jamaica

JUSTIFICATIVA E OBJETIVOS: As complicações anestésicas pós-operatórias menores podem aumentar o desconforto e a insatisfação do paciente e retardar sua recuperação. Este trabalho procurou determinar a frequência das complicações menores relatadas nas primeiras 48 horas do período pós-operatório por pacientes de cirurgias eletivas (ginecológicas e ortopédicas) no University Hospital of the West Indies, Jamaica. A satisfação geral com os cuidados anestésicos e os possíveis fatores de risco para desenvolver complicações também foram avaliados. MÉTODOS: Um estudo prospectivo e descritivo de coorte foi realizado por meio de entrevistas com pacientes operados 24 e 48 horas após a anestesia. Os dados foram analisados usando SPSS versão 12 e avaliados pelo teste do χ2-quadrado e modelos de regressão logística múltipla. RESULTADOS: Foram incluídos 505 pacientes, sendo 374 do sexo feminino (74 por cento). A maioria era ASA I (55 por cento) ou ASA II (38 por cento) e foi submetida à anestesia geral (80 por cento). Um total de 419 (83 por cento) pacientes relataram pelo menos uma complicação pós-operatória. As complicações mais relatadas foram dor de garganta (44 por cento), náusea (30 por cento), vômito (24 por cento) e tromboflebite (20 por cento). A moda do Índice de Classificação Numérica Verbal (ICNV) para cada complicação variou entre 2 e 5, sugerindo que a maioria não causa desconforto grave. Idade inferior a 45 anos (OR 2,22, IC de 95 por cento 1,34-3,69, p = 0,002) e sexo feminino (OR 3,64, IC de 95 por cento 2,14-6,20, p < 0,001) foram identificados como variáveis independentes significativas. A maioria dos pacientes considerou sua experiência anestésica como excelente (51 por cento) ou muito boa (22 por cento). CONCLUSÃO: Este estudo mostrou uma incidência relativamente alta de complicações menores pós-operatórias (83 por cento), mas baixa gravidade dos sintomas relatados e um alto grau de satisfação geral. Deve ser dada atenção especial à redução dessas complicações menores por meio de técnicas anestésicas mais meticulosas.

BACKGROUND AND OBJECTIVES: Minor postoperative anesthetic complications may increase patient discomfort and dissatisfaction and delay recovery. This paper sought to determine the frequency of minor complications in the first 48 hours postoperatively reported by elective gynecological and orthopedic surgical patients at the University Hospital of the West Indies, Jamaica. Overall satisfaction with anesthetic care and possible risk factors for developing complications were also assessed. METHODS: A prospective, descriptive cohort study was undertaken with patient interviews 24 to 48 hours after anesthesia. Data were analyzed using SPSS version 12 and assessed using the χ2-square test and multiple logistic regression models. RESULTS: Five hundred and five (505) patients were included, with 374 females (74 percent). Most were ASA I (55 percent) or ASA II (38 percent) and had general anesthesia (80 percent). A total of 419 (83 percent) patients reported at least one complication postoperatively. The most frequently reported complications were sore throat (44 percent), nausea (30 percent), vomiting (24 percent), and thrombophlebitis (20 percent). The mode of the Verbal Numerical Rating Score (VNRS) for each complication ranged between 2 and 5, suggesting that most did not cause severe distress. Age less than 45 years (OR 2.22, 95 percent CI 1.34-3.69, p = 0.002) and female gender (OR 3.64, 95 percent CI 2.14-6.20, p < 0.001) were identified as significant independent variables. Most patients regarded their anesthetic experience as excellent (51 percent) or very good (22 percent). CONCLUSION: This study showed a comparatively high incidence of minor postoperative complications (83 percent), but low reported severity of symptoms and a high overall satisfaction rate. Special attention should be paid to reduce these minor complications through more meticulous anesthetic technique.

JUSTIFICATIVA Y OBJETIVOS: Las complicaciones anestésicas postoperatorias menores pueden aumentar la incomodidad y la insatisfacción del paciente y retardar la recuperación. Este trabajo intentó determinar la frecuencia de las complicaciones menores relatadas en las primeras 48 horas del período postoperatorio por pacientes de cirugías electivas (ginecológicas y ortopédicas), en el University Hospital of the West Indies, Jamaica. También se evaluaron, la satisfacción general con los cuidados anestésicos y los posibles factores de riesgo para desarrollar complicaciones. MÉTODOS: Un estudio prospectivo y descriptivo de cohorte fue realizado por medio de entrevistas con pacientes operados 24 y 48 horas después de la anestesia. Los datos fueron analizados usando SPSS versión 12 y evaluados por el test del χ2-cuadrado y modelos de regresión logística múltiple. RESULTADOS: Se incluyeron 505 pacientes, siendo que 374 eran del sexo femenino (74 por ciento). La mayoría era ASA I (55 por ciento) o ASA II (38 por ciento) y se sometió a la anestesia general (80 por ciento). Un total de 419 (83 por ciento) pacientes relataron por lo menos una complicación postoperatoria. Las complicaciones más relatadas fueron el dolor de garganta (44 por ciento), náusea (30 por ciento), vómito (24 por ciento) y tromboflebitis (20 por ciento). La moda del Índice de Clasificación Numérica Verbal (ICNV), para cada complicación varió entre 2 y 5, lo que sugiere que la mayoría no causa una grave incomodidad. La edad inferior a 45 años (OR 2,22, IC de 95 por ciento 1,34-3,69, p = 0,002) y el sexo femenino (OR 3,64, IC de 95 por ciento 2,14-6,20, p < 0,001), fueron identificados como variables independentes significativas. La mayoría de los pacientes consideró su experiencia anestésica como excelente (51 por ciento) o muy buena (22 por ciento). CONCLUSIONES: Este estudio mostró una incidencia relativamente alta de complicaciones menores postoperatorias (83 por ciento), pero con una baja gravedad de los síntomas relatados y un alto grado de satisfacción general. Debemos darle una atención especial a la reducción de esas complicaciones menores por medio de técnicas anestésicas más meticulosas.

Minor postoperative complications related to anesthesia in elective gynecological and orthopedic surgical patients at a teaching hospital in Kingston, Jamaica.

BACKGROUND AND OBJECTIVES: Minor postoperative anesthetic complications may increase patient discomfort and dissatisfaction and delay recovery. This paper sought to determine the frequency of minor complications in the first 48 hours postoperatively reported by elective gynecological and orthopedic surgical patients at the University Hospital of the West Indies, Jamaica. Overall satisfaction with anesthetic care and possible risk factors for developing complications were also assessed. METHODS: A prospective, descriptive cohort study was undertaken with patient interviews 24 to 48 hours after anesthesia. Data were analyzed using SPSS version 12 and assessed using the χ(2)-square test and multiple logistic regression models. RESULTS: Five hundred and five (505) patients were included, with 374 females (74%). Most were ASA I (55%) or ASA II (38%) and had general anesthesia (80%). A total of 419 (83%) patients reported at least one complication postoperatively. The most frequently reported complications were sore throat (44%), nausea (30%), vomiting (24%), and thrombophlebitis (20%). The mode of the Verbal Numerical Rating Score (VNRS) for each complication ranged between 2 and 5, suggesting that most did not cause severe distress. Age less than 45 years (OR 2.22, 95% CI 1.34-3.69, p=0.002) and female gender (OR 3.64, 95% CI 2.14-6.20, p<0.001) were identified as significant independent variables. Most patients regarded their anesthetic experience as excellent (51%) or very good (22%). CONCLUSION: This study showed a comparatively high incidence of minor postoperative complications (83%), but low reported severity of symptoms and a high overall satisfaction rate. Special attention should be paid to reduce these minor complications through more meticulous anesthetic technique.

Determining microsatellite genotyping reliability and mutation detection ability: an approach using small-pool PCR from sperm DNA.

Microsatellite genotyping from trace DNA is now common in fields as diverse as medicine, forensics and wildlife genetics. Conversely, small-pool PCR (SP-PCR) has been used to investigate microsatellite mutation mechanisms in human DNA, but has had only limited application to non-human species. Trace DNA and SP-PCR studies share many challenges, including problems associated with allelic drop-out, false alleles and other PCR artefacts, and the need to reliably identify genuine alleles and/or mutations. We provide a framework for the validation of such studies without a multiple tube approach and demonstrate the utility of that approach with an analysis of microsatellite mutations in the tammar wallaby (Macropus eugenii). Specifically, we amplified three autosomal microsatellites from somatic DNA to characterise efficiency and reliability of PCR from low-template DNA. Reconstruction experiments determined our ability to discriminate mutations from parental alleles. We then developed rules to guide data interpretation. We estimated mutation rates in sperm DNA to range from 1.5 × 10(-2) to 2.2 × 10(-3) mutations per locus per generation. Large multi-step mutations were observed, providing evidence for complex mutation processes at microsatellites and potentially violating key assumptions in the stepwise mutation model. Our data demonstrate the necessity of actively searching for large mutation events when investigating microsatellite evolution and highlight the need for a thorough understanding of microsatellite amplification characteristics before embarking on SP-PCR or trace DNA studies.

Single nucleotide polymorphism (SNP) discovery in mammals: a targeted-gene approach.

Single nucleotide polymorphisms (SNPs) have rarely been exploited in nonhuman and nonmodel organism genetic studies. This is due partly to difficulties in finding SNPs in species where little DNA sequence data exist, as well as to a lack of robust and inexpensive genotyping methods. We have explored one SNP discovery method for molecular ecology, evolution, and conservation studies to evaluate the method and its limitations for population genetics in mammals. We made use of 'CATS' (or 'EPIC') primers to screen for novel SNPs in mammals. Most of these primer sets were designed from primates and/or rodents, for amplifying intron regions from conserved genes. We have screened 202 loci in 16 representatives of the major mammalian clades. Polymerase chain reaction (PCR) success correlated with phylogenetic distance from the human and mouse sequences used to design most primers; for example, specific PCR products from primates and the mouse amplified the most consistently and the marsupial and armadillo amplifications were least successful. Approximately 24% (opossum) to 65% (chimpanzee) of primers produced usable PCR product(s) in the mammals tested. Products produced generally high but variable levels of readable sequence and similarity to the expected genes. In a preliminary screen of chimpanzee DNA, 12 SNPs were identified from six (of 11) sequenced regions, yielding a SNP on average every 400 base pairs (bp). Given the progress in genome sequencing, and the large numbers of CATS-like primers published to date, this approach may yield sufficient SNPs per species for population and conservation genetic studies in nonmodel mammals and other organisms.