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Loggerhead turtle eggshells as a source of maternal nuclear genomic DNA for population genetic studies
Abstract
Tagging studies on nesting beaches are commonly used to estimate nesting frequency, remigration interval and nesting population size for marine turtle rookeries. Estimates of these demographic parameters from tagging projects may be biased because of the small scale of tagging efforts relative to female nest site fidelity and the logistical difficulty of intercepting all nesting females. Therefore, alternative and supplemental means of individual identification of nesting females are required. We demonstrate that maternal nuclear microsatellite DNA can be isolated from unincubated eggshells of the loggerhead sea turtle (Caretta caretta) through comparison of DNA extracted from 59 eggs collected within 15 h of oviposition and DNA derived from skin samples from respective nesting females. Scorable microsatellite genotypes were produced in 897 of 994 (90.2%) single-locus egg amplifications attempted. Among eggs from known females, 730 of 748 (97.6%) single-locus, egg-derived genotypes matched the respective skin-derived genotypes. Allelic dropout was the most common type of error, followed by the presence of nonmaternal, presumably paternal, alleles. Genotypes derived from unincubated eggshells permit individual assignment of nests and therefore demographic parameter estimates for loggerhead turtle nesting populations, despite genotyping errors that require further optimization. Although sampling unincubated eggs is destructive, this technique is noninvasive to nesting females and is applicable in marine turtle population genetics studies when individual resolution is required but direct interception of nesting females is undesirable or logistically infeasible.
... Like many other animal groups, recent studies showed the successful use of DNA barcoding in identifying herpetofauna [21][22][23]. Hence, we hypothesized that eggshells can be used as a source of DNA and in identifying freshwater turtle nests and species, including P. peninsularis, which is already well-practiced in birds and sea turtles [24][25][26]. ...
... We found two P. peninsularis turtle nests in Jeonpyeongje Neighborhood Park, Maewol-dong, Seo-gu, Gwangju, South Korea (Fig 1B-1D), and successfully identified them by combining the egg characteristics, morphological features of the hatchlings, and phylogenetic analysis of partial mitochondrial DNA from the eggshells. This was the first initiative to extract DNA from eggshells, which is already well-practiced in sea turtles [25,26]. Although our initiative was successful, extracting DNA from eggshells is challenging as it is expected to get a lower concentration than that may yield from muscle or blood [40]. ...
... Eggshell DNA in identifying freshwater turtle species turtles [26,43]. Furthermore, freshwater turtle nests are difficult to find and are also difficult and time-consuming to hatch the eggs to identify the nests [15]. ...
Alien invasive species are posing conservation challenges worldwide. Pet trade, one of the many ways, is worsening the situation. Especially, pet turtles have been released into nature due to their longer life span and peoples’ religious and traditional beliefs. In addition, unwanted and undesired pets are also released. While information on the successful local establishment and subsequent dispersal into new habitats is required to designate an invasive and ecosystem-disturbing species, alien freshwater turtle nests have always been hard to find and identify in nature. Because one should identify nests by the eggs, which do not always guide properly, as adults abandon the sites quickly. We thought the recent advancement in DNA technology may help improve the situation. We studied Pseudemys peninsularis, one of the most traded freshwater turtle pet species, which has already been reported from a wide range of wild areas in South Korea. Yet, it is not designated as ecosystem-disturbing species due to a lack of adequate information on their local reproduction and establishment. We conducted surveys and found two nests in Jeonpyeongje Neighborhood Park, Maewol-dong, Seo-gu, Gwangju. We developed the methodology for extracting DNA from the eggshells and successfully identified the nests by phylogenetic analysis and verified through egg characteristics and morphological features of artificially hatched juveniles. This was the first successful initiative to extract DNA from freshwater turtle eggshells. We believe it will help future researchers identify the alien invasive turtle nests and develop their control and management policies. In addition, our study also included comparative descriptions and schematic diagrams of the eggs of eight freshwater turtles, including a native and three ecosystem-disturbing species, from South Korea. We urged an immediate designation of P. peninsularis as an ecosystem-disturbing species considering its local establishment, distribution range, and potential negative impact on native ecosystems.
... Environmental approaches enable the recovery of informative genetic material from study species without requiring difficult in-water siting and capture or having to be physically present during nesting events. Environmental DNA-based detection of sea turtle species in marine environments has been limited to just a few studies in limited circumstances (Farrell, Harper et al., 2020;Kelly et al., 2014;Shamblin et al., 2011;Yetsko et al., 2020Yetsko et al., , 2021, and detection and "genetic fingerprinting" of sea turtle species from beach sand traversed by nesting females and hatchlings has not yet been explored ( Figure 1a). ...
... Therefore, positive amplification of one or more technical replicates per sample was counted as positive detection. Amplification ratio (the proportion of positive amplification detection relative to attempted technical replicate reactions Shamblin et al., 2011]) is reported for all study samples (Tables S1, 2, 4-8). ...
... Current population genetics approaches require invasive tissue sampling or blood draws -for sea turtles this frequently is conducted during nesting, physically interacting with nesting females and probably inducing additional stress -alternatively one egg needs to be sacrificed per clutch to obtain maternal DNA without direct tissue/blood sampling (Adams et al., 2019;Calmanovici et al., 2018;Gadagkar et al., 2005;Gatto et al., 2018;Jensen et al., 2019;Komoroske et al., 2018;Long & Azmi, 2017;Shamblin et al., 2011). ...
Elusive aquatic wildlife, such as endangered sea turtles, are difficult to monitor and conserve. As novel molecular and genetic technologies develop, it is possible to adapt and optimize them for wildlife conservation. One such technology is environmental (e)DNA – the detection of DNA shed from organisms into their surrounding environments. We developed species‐specific green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtle probe‐based qPCR assays, which can detect and quantify sea turtle eDNA in controlled (captive tank water and sand samples) and free ranging (oceanic water samples and nesting beach sand) settings. eDNA detection complemented traditional in‐water sea turtle monitoring by enabling detection even when turtles were not visually observed. Furthermore, we report that high throughput shotgun sequencing of eDNA sand samples enabled sea turtle population genetic studies and pathogen monitoring, demonstrating that non‐invasive eDNA techniques are viable and efficient alternatives to biological sampling (e.g. biopsies and blood draws). Genetic information was obtained from sand many hours after nesting events, without having to observe or interact with the target individual. This greatly reduces the sampling stress experienced by nesting mothers and emerging hatchlings, and avoids sacrificing viable eggs for genetic analysis. The detection of pathogens from sand indicates significant potential for increased wildlife disease monitoring capacity and viral variant surveillance. Together, these results demonstrate the potential of eDNA approaches to ultimately help understand and conserve threatened species such as sea turtles.
... The Caretta Research Project has monitored loggerhead nesting activity on Wassaw Island since 1973 (Williams and Frick 2001;Pfaller et al. 2013), representing one of the longest continuously running CMR programs for marine turtles in the world. More recently, the discovery that freshly laid eggshells contain maternal DNA has allowed for genetic CMR of individuals across almost the entire NRU nesting range, including Wassaw Island (Shamblin et al. 2011b(Shamblin et al. , 2017(Shamblin et al. , 2021. This scenario provides the unique opportunity to evaluate how weak or variable site fidelity to a focal terrestrial breeding site can affect demographic estimates in a threatened marine species. ...
... Starting in 2008, genetic samples were collected from every female each season as part of larger regional and subpopulation-wide genetic studies (Shamblin et al. 2011b(Shamblin et al. , 2017(Shamblin et al. , 2021. Following oviposition or failed nesting, one 6-mm skin biopsy was collected from the shoulder region of each female one time per season (i.e., genetic samples were not collected during within-season recaptures of previously sampled females identified based on tags; see above). ...
... Starting in 2010, one eggshell sample was collected from every loggerhead clutch detected on nesting beaches between Georgia and Maryland, USA (~ 1000 km)-approximately 93% of ocean-facing nesting habitat was surveyed from May to August (80% daily). In most cases, freshly laid eggs were collected, emptied of their contents, and stored in 95% ethanol within 15 h of oviposition to preserve the maternal DNA and avoid embryonic contamination (Shamblin et al. 2011b). A small proportion of nests (5%) were detected greater than 15 h post-oviposition following nest depredation or hatchling emergence. ...
In migratory marine species, demographic estimates are often generated from capture-mark-recapture (CMR) studies conducted at terrestrial breeding sites. However, when logistical difficulties limit the geographic area of these surveys, demographic estimates are vulnerable to biases. We compared demographic rates generated from CMR data of nesting loggerhead turtles (Caretta caretta) collected between 2010 and 2017 at one focal site (Wassaw Island, Georgia, USA; 31.89° N, 80.97° W) with estimates generated from the same group of turtles but including all other nesting events from adjacent sites in Georgia, South Carolina, and North Carolina. We found that estimates of annual recruitment at the focal site were overestimated: each year, 29–45% putative first-time nesters at the focal beach had, in fact, nested on a different beach in a previous season. Estimates of clutch frequency and breeding frequency generated at the focal site were biased low and skewed towards values of one, while estimates for remigration interval were not significantly over- or underestimated. Additionally, estimates of annual and total population productivity in terms of clutches, eggs, and hatchlings were underestimated by more than half at the focal site. Our results show how weak fidelity to a focal nesting/tagging site can affect demographic estimates in marine turtle populations and highlight the need to reconsider estimates from other populations that might be vulnerable to similar biases.
... Advances in genetic techniques and modeling approaches have provided a novel opportunity to assess marine turtle demographic parameters. Genetic tagging through sampling a single egg from each clutch offers an approach to identify individual females responsible for each nest, which alleviates the need to directly intercept nesting females (Shamblin et al. 2011b). Freshly laid eggs that have incubated less than a day yield maternal genomic DNA based on comparisons between DNA derived from skin biopsies and egg shells of respective females (Shamblin et al. 2011b). ...
... Genetic tagging through sampling a single egg from each clutch offers an approach to identify individual females responsible for each nest, which alleviates the need to directly intercept nesting females (Shamblin et al. 2011b). Freshly laid eggs that have incubated less than a day yield maternal genomic DNA based on comparisons between DNA derived from skin biopsies and egg shells of respective females (Shamblin et al. 2011b). We employed this method to genetically tag female loggerhead turtles representing the Northern Recovery Unit (NRU), the northernmost subpopulation of loggerhead turtles nesting from Georgia to Maryland on the Atlantic coast of the United States. ...
... Raccoon depredation was extensive on islands that were monitored at intervals greater than three days, increasing the probability that a clutch deposited within the previous week would be easily detected and sampled. The pilot study demonstrated that eggshells from freshly laid eggs yielded high-quality maternal DNA; however eggshells from partially or fully incubated eggs represented embryonic fingerprints, a mix of maternal and embryonic DNA, and/or had high allele dropout and amplification failure rates (Shamblin et al. 2011b). Therefore, projects that monitored daily attempted to collect a single viable egg from each clutch within 15 h of oviposition to provide the best opportunity for direct individual identification. ...
Background/Question/Methods:
Population monitoring for marine turtles is often limited to assessing trends in nest counts, but individual reproductive data are required to better characterize the population dynamics underlying these nesting trends. Nest site fidelity can be low relative to the scale of tagging effort at individual nesting beaches, resulting in sparse recapture data and potentially biased reproductive parameter estimates. Genetic tagging via genotyping of maternal genomic DNA from egg samples offers an alternative approach of identifying individuals that alleviates the requirement of physically intercepting nesting females. From 2010 through 2012, we collected a single egg from approximately 20,000 loggerhead clutches detected on beaches from Georgia to Maryland, encompassing the nesting range of the Northern Recovery Unit (NRU) subpopulation. We used genotypes from 18 microsatellites to assign clutches to unique females. We quantified nest site fidelity at the subpopulation level as well as in a finer scale, spatially explicit context by assigning females to 50 km latitudinal bins based on their median nesting latitude. We estimated annual nesting female population size and clutch frequency using an open robust design framework. Finally, we compared clutch frequencies estimated from single-island physical tagging (Wassaw and Jekyll Islands, Georgia) and regional genetic tagging.
Results/Conclusions:
Genetic tagging identified 5,681 unique females nesting over the study period. Mean distance between the most distant clutches laid by individual females (nest spread) was 33.4 (± 74.6) km at the NRU scale. Nesting spread at the latitudinal bin scale ranged from 12.3 (± 23.8) to 157.0 (± 213.0) km, with Georgia and South Carolina females exhibiting significantly higher nest site fidelity than North Carolina and Virginia females. The best supported models all contained time dependence in entry and persistence probabilities, with annual estimated clutch frequencies ranging from 3.9 (± 0.04) to 4.5 (± 0.09) clutches per female. Of females that were physically tagged while nesting on Wassaw or Jekyll Island, 50% also nested on another island that season. Of those, 80% did not return to their initial nesting sites over the remainder of each nesting season. Clutch frequencies estimated from physical tagging data were biased low by 16% to 38% relative to those generated from the genetic capture-recapture data. This bias indicates that “permanent” emigration at the scale of individual tagging beaches cannot be fully accounted for through standard modeling, and that regional approaches that consider the scale of nest site fidelity are critical for generating robust parameter estimates.
... Advances in genetic techniques and modeling approaches have provided a novel opportunity to assess marine turtle demographic parameters. Genetic tagging through sampling a single egg from each clutch offers an approach to identify individual females responsible for each nest, which alleviates the need to directly intercept nesting females (Shamblin et al. 2011b). Freshly laid eggs that have incubated less than a day yield maternal genomic DNA based on comparisons between DNA derived from skin biopsies and egg shells of respective females (Shamblin et al. 2011b). ...
... Genetic tagging through sampling a single egg from each clutch offers an approach to identify individual females responsible for each nest, which alleviates the need to directly intercept nesting females (Shamblin et al. 2011b). Freshly laid eggs that have incubated less than a day yield maternal genomic DNA based on comparisons between DNA derived from skin biopsies and egg shells of respective females (Shamblin et al. 2011b). We employed this method to genetically tag female loggerhead turtles representing the Northern Recovery Unit (NRU), the northernmost subpopulation of loggerhead turtles nesting from Georgia to Page 3 of 14 138 ...
... Raccoon depredation was extensive on islands that were monitored at intervals greater than three days, increasing the probability that a clutch deposited within the previous week would be easily detected and sampled. The pilot study demonstrated that eggshells from freshly laid eggs yielded high-quality maternal DNA; however eggshells from partially or fully incubated eggs represented embryonic fingerprints, a mix of maternal and embryonic DNA, and/or had high allele dropout and amplification failure rates (Shamblin et al. 2011b). Therefore, projects that monitored daily attempted to collect a single viable egg from each clutch within 15 h of oviposition to provide the best opportunity for direct individual identification. ...
Nest counts are often used as indices for nesting female abundance in marine turtle monitoring, but accurately interpreting nest count trends requires context on the scale of demographic connectivity and estimates of reproductive parameters. Weak nest site fidelity (NSF) relative to the scale of tagging effort may bias parameter estimates. The reproductive ecology of Northern Recovery Unit (NRU) loggerhead turtles (Caretta caretta) was assessed through subpopulation-scale genetic capture-recapture via clutch sampling from approximately 1000 km of coastline from Georgia to Maryland, USA (30.75–38.06°N and 75.24–81.45°W). Of 20,682 clutches recorded from 2010 to 2012, 20,222 sampled clutches were assigned to 5684 unique females through microsatellite genotyping. Approximately 73% of females detected laying multiple clutches deposited them within a 20-km span, suggesting the possibility of demographic structuring across NRU rookeries that warrants further investigation. Estimated clutch frequencies (ECF) generated from open robust design modeling were 4.28 (4.02–4.54) in 2010, 4.63 (4.45–4.80) in 2011, and 4.57 (4.28–4.77) in 2012, and were significantly higher than observed clutch frequencies. ECF generated from single-island data were biased low by 23–50% relative to those from regional genetic tagging. Among females that nested at least once on a physical tagging beach, 54% also nested elsewhere, with 81% of these “permanently” emigrating during the nesting season. This pattern of relatively strong NSF, but distributed across multiple nearby islands, confounded modeling of detection in single-island datasets and highlights the need for regional coverage for generating robust estimates of demographic parameters for marine turtles.
... Additionally, Wallace et al. (2010) established, based on mtDNA, that the Atlantic-Mediterranean lineage is composed of four Regional Management Units (RMU) being two on the North Atlantic (Northeast of the USA coast and one on the Northwest of the north coast of Africa), one on the Mediterranean-Greece coast, and another on the Southwest Atlantic coast (SWA). On the other hand, only a few studies are using nuclear DNA loci (nDNA; biparental inheritance) like microsatellite loci (SSRs: Simple Sequences Repeats) which have provided greater sensitivity and fine-scale at the population level than D-loop (e.g., Moore and Ball 2002;Shamblin et al. 2007Shamblin et al. , 2011Monzón-Argüello et al. 2010;Carreras et al. 2018). ...
... To check if there was any contamination, we added negative (containing only PCR's mix) and positive controls (samples with a positive result that has been amplified before) for each reaction. The genotyping of the 15 microsatellite loci: Cc7G11, Cc1F01, Cc1G02, Cc1G03, CcP7D04, CcP2F11, CcP7C06, CcP8D06, CcP1F09, CcP5C11, CcP1F01, CcP1G03, CcP1B03, CcP5C08, and CcP5H07 (Shamblin et al. 2007(Shamblin et al. , 2009 following the PCR cycling profile conditions of Shamblin et al. (2009), which were directly labeled with fluorescent FAM, PET, NED, and VIC dyes (Shamblin et al. 2011). ...
The loggerhead sea turtle (Caretta caretta) is a cosmopolitan sea turtle species and is listed by IUCN as Vulnerable globally.
The Southwest Atlantic is an important regional management unit of C. caretta worldwide due to the distinctive mitochondrial
DNA (mtDNA) lineage promoted by recent radiation within the Atlantic-Mediterranean region. However, due to the low
resolution of mtDNA, the population structure of C. caretta SWA has not been well understood in the previous studies using
only mtDNA. Our study encloses data from literature and a long-term genetic survey (1999 to 2021) distributed through four
great nesting areas for the Southwest Atlantic to assess the genetic diversity and the population structure of the C. caretta,
using both mtDNA and 15 microsatellite loci. The results demonstrate that the genetic diversity indexes of the Southwest
Atlantic C. caretta refect distinct compositions at a population level due to variation at an individual level. The SSRs results
identifed well-established and signifcant spatial population structure between nesting areas. Unique genetic patterns were
identifed for those females from studied areas of the Southwest Atlantic, and it may be related to their philopatric behavior
and high relatedness. Thus, this study deeply evaluated the molecular ecology of Southwest Atlantic C. caretta and provides,
for the frst time, a fne-scale and long-term resolution of the genetic diversity and population structure due to the use of
microsatellite data that must be considered for further studies.
... Understanding how maternal experience (i.e., neophyte vs remigrant) affects hatching success can be useful for sea turtle management, as well as understanding their reproductive biology. New advances now allow maternal identity to be incorporated into management by using mitochondrial DNA extracted from freshly laid eggshells sampled from nests (Shamblin et al. 2011). The resulting data can help determine whether certain mothers have more success than others and track how hatching success changes within and across nesting seasons for individual mothers. ...
... Many state agencies already track sea turtle nesting and hatching success, and long-term monitoring of tagged females has demonstrated impacts of maternal identity and nest choice (i.e., sex-ratios [Reneker and Kamel 2016], hatching success [Rafferty et al. 2011]), and these long-term data sets are critical to distinguish between maternal and environmental effects. Further, the Northern Recovery Unit Loggerhead DNA Project has been building a genetic repository for sea turtles for a decade (Shamblin et al. 2011), which allows many more mothers to be tracked through time and space than using traditional methods (i.e., tagging). By leveraging data collected by local management agencies and linking it to genetic information over a long-enough timespan to capture multiple reproductive remigrations across many individual females, we can fully explore the role maternal identity plays in hatching success. ...
Caretta caretta (Loggerhead Sea Turtle) is a globally threatened sea turtle species that nests on United States Atlantic coast beaches. While several environmental factors influence egg incubation and hatching success, maternal identity may also play an important role and has been overlooked. New molecular advances, such as ongoing genetic capture–recapture using eggshells, allow the identification of individual females using fresh
eggshells, which can then be used to track nesting and hatching success across individual females. Across 2 consecutive nesting seasons, we monitored 170 nests for environmental conditions as part of a larger Georgia Department of Natural Resources monitoring program. Mothers had been identified using molecular data and classified as either neophytes or remigrants. We identified 34 of the nests as belonging to neophyte Loggerhead mothers, which exhibited a 61% hatching success rate, compared to only 46% success for remigrant mothers (P < 0.01). Further analysis suggested no differences in environmental parameters
between neophyte and remigrant nests. Thus, while it is possible that different-aged mothers make different nesting-site choices that could affect the nest environment, it is likely that Loggerhead reproductive biology, such as maternal investment, may change over time. More data are required to fully investigate maternal effects on hatching success and to elucidate the mechanisms, both within this species and across multiple species of sea turtles.
... Here, we extended this approach by assigning clutches to individual females from 2013 through 2015. We extracted maternal genomic DNA from eggshells using a modified Qiagen DNEasy tissue extraction protocol (Shamblin et al. 2011b) and genotyped samples at 18 microsatellite loci (Shamblin et al. 2007(Shamblin et al. , 2009 (Table S-2 in Online Resource 1). Maternal genomic DNA was amplified in 10-μl reactions using PCR cycling conditions previously described (Shamblin et al. 2017). ...
... We assigned clutches to individual females through a matching protocol using the program CERVUS (Kalinowski et al. 2007) (Table S-4 in Online Resource 1). The pilot study identified two common sources of mismatches between eggshell-derived and skin-derived maternal DNA: allele dropout and the presence of non-maternal (presumably paternal) alleles (Shamblin et al. 2011b). We, therefore, chose a threshold of acceptable genotyping error that avoided the need for extensive reanalysis but still provided strong individual resolution. ...
Capture–mark–recapture (CMR) studies on marine turtle nesting beaches provide data on reproductive periodicity that inform population trends and models. Annual survival is estimated from observations of remigration, the return of females in subsequent nesting seasons. However, a significant proportion of tagged females are never encountered remigrating in many studies, presumably due to weak nest site fidelity (NSF). We employed a genetic CMR approach based on subpopulation-scale clutch sampling to conduct a 5-year evaluation of inter-seasonal recapture rates and NSF for Northern Recovery Unit loggerhead turtles (Caretta caretta). Of 1770 females genetically tagged from Georgia through Maryland in 2010, 1156 (65%) remigrated between 2011 and 2015. Inter-seasonal NSF, measured as shifts in median latitude nesting locations between years, was highly variable among individuals but strong overall (mean: 15.08 (± 44.61) km, median: 1.84 km). Among three focal beaches with nocturnal tagging projects, 69 of 173 females (40%) remigrated onsite whereas 115 (66%) were detected overall. Regional genetic sampling therefore yielded significantly higher inter-seasonal recapture rates, which may improve precision in future survival analyses. However, despite sampling ~ 1000 km with high annual detection probabilities (p* ≥ 0.94), 35% of 2010-females were not detected remigrating. Several non-exclusive hypotheses to explain these remaining “missing” remigrants should be considered: longer remigration intervals, imperfect detection within the study area, emigration to Florida, anthropogenic mortality, and natural mortality or senescence. This genetic tagging approach can be applied over large spatial scales where nesting densities permit, better characterizing inter-seasonal dispersal.
... At these two sites, tissue samples-yolk from WAS or epidermis from JEK-were collected from each female one time per season (i.e., samples were not collected during within-season recaptures of previously sampled females identified by their tags). At the five sites in SC and NC where nocturnal tagging patrols are not conducted (ORE, PEA, STH, KWH and HHI), yolk samples were identified to individual females using the aforementioned multilocus genetic tags extracted from maternal DNA found in the shell of freshly laid eggs [32]. See Shamblin et al. [32][33] for details on genetic tagging across this subpopulation. ...
... At the five sites in SC and NC where nocturnal tagging patrols are not conducted (ORE, PEA, STH, KWH and HHI), yolk samples were identified to individual females using the aforementioned multilocus genetic tags extracted from maternal DNA found in the shell of freshly laid eggs [32]. See Shamblin et al. [32][33] for details on genetic tagging across this subpopulation. All samples were included in subsequent stable isotope analysis (SIA) to crosscheck foraging-ground assignments when individual females were sampled at more than one site within the same season or within more (B) Previously published foraging-area assignments and proportions for 273 skin samples collected from NRU loggerheads nesting at one additional site (BHI) [14] and six additional years from WAS [15,29]. ...
Population assessments conducted at reproductive sites of migratory species necessitate understanding the foraging-area origins of breeding individuals. Without this information, efforts to contextualize changes in breeding populations and develop effective management strategies are compromised. We used stable isotope analysis of tissue samples collected from loggerhead sea turtles (Caretta caretta) nesting at seven sites in the Northern Recovery Unit (NRU) of the eastern United States (North Carolina, South Carolina and Georgia) to assign females to three separate foraging areas in the Northwest Atlantic Ocean (NWA). We found that the majority of the females at NRU nesting sites (84.4%) use more northern foraging areas in the Mid-Atlantic Bight, while fewer females use more proximate foraging areas in the South Atlantic Bight (13.4%) and more southerly foraging areas in the Subtropical Northwest Atlantic (2.2%). We did not find significant latitudinal or temporal trends in the proportions of NRU females originating from different foraging areas. Combining these findings with previous data from stable isotope and satellite tracking studies across NWA nesting sites showed that variation in the proportion of adult loggerheads originating from different foraging areas is primarily related differences between recovery units: individuals in the NRU primarily use the Mid-Atlantic Bight foraging area, while individuals from the three Florida recovery units primarily use the Subtropical Northwest Atlantic and Eastern Gulf of Mexico foraging areas. Because each foraging area is associated with its own distinct ecological characteristics, environmental fluctuations and anthropogenic threats that affect the abundance and productivity of individuals at nesting sites, this information is critical for accurately evaluating population trends and developing effective region-specific management strategies.
... In these cases, one viable egg was collected from her nest immediately. The yolk and albumin were discarded and the eggshell and its eggshell membrane (which are maternally derived) were stored in 70% ethanol (extraction based upon protocols established by [51]). ...
... Blood: 5 μL of whole blood was added to 50 μL of lysis buffer (10mM TRIS, pH8.3, 40mM KCl, 0.5% Tween20 and 200 μg/mL Proteinase K) and incubated at 65˚C for 1 h, followed by 100˚C for 15 min. Skin/eggshell: a DNEasy blood and tissue kit (Qiagen, Valencia, CA USA) was used following manufacturer 0 s protocol (for eggshell, incubation was longer than manufacturer's instructions per [51]). Samples were genotyped with 7 microsatellite loci following published protocols: CcP7E05, CcP2F11, CcP7D04, CcP5H07, CcP7C06, CcP7B07 and CcP8D06 [52]. ...
Species that display temperature-dependent sex determination are at risk as a result of increasing global temperatures. For marine turtles, high incubation temperatures can skew sex ratios towards females. There are concerns that temperature increases may result in highly female-biased offspring sex ratios, which would drive a future sex ratio skew. Studying the sex ratios of adults in the ocean is logistically very difficult because individuals are widely distributed and males are inaccessible because they remain in the ocean. Breeding sex ratios (BSR) are sought as a functional alternative to study adult sex ratios. One way to examine BSR is to determine the number of males that contribute to nests. Our goal was to evaluate the BSR for loggerhead turtles (Caretta caretta) nesting along the eastern Gulf of Mexico in Florida, from 2013–2015, encompassing three nesting seasons. We genotyped 64 nesting females (approximately 28% of all turtles nesting at that time) and up to 20 hatchlings from their nests (n = 989) using 7 polymorphic microsatellite markers. We identified multiple paternal contributions in 70% of the nests analyzed and 126 individual males. The breeding sex ratio was approximately 1 female for every 2.5 males. We did not find repeat males in any of our nests. The sex ratio and lack of repeating males was surprising because of female-biased primary sex ratios. We hypothesize that females mate offshore of their nesting beaches as well as en route. We recommend further comparisons of subsequent nesting events and of other beaches as it is imperative to establish baseline breeding sex ratios to understand how growing populations behave before extreme environmental effects are evident.
... Genotyping utilized 15 loci and had a minimum nonexclusion probability of identity of 2.07 × 10 −24 (Shamblin et al. 2007(Shamblin et al. , 2009. OSS samples were eggshells collected within 12 h of oviposition and genotyped at 15 or 17 loci to assign individual identity (DNA extraction and sample assignment methods detailed in Shamblin et al. 2011b). Nest samples were dead hatchlings or hatched eggshells collected during post-emergence nest evaluations or blood samples from live hatchlings, and each nest was represented by a single sample. ...
... Genomic DNA was extracted using a standard phenol-chloroform isolation or the DNeasy blood and tissue kit (QIAGEN) following standard protocols for tissue and with modifications previously described for egg shells (Shamblin et al. 2011b). Polymerase chain reaction (PCR) amplifications of an 817 bp fragment of the mitochondrial control region were carried out using primers LCM15382 and H950g (Abreu-Grobois et al. 2006). ...
... Even though the authors obtained good quality DNA, they still recommend extraction from tissues such as blood and skin to obtain high quality DNA. DNA extraction and analysis of microsatellite markers from eggshell samples of non-viable C. caretta hatchlings was efficient in terms of quantity and quality of genetic material (Shamblin et al., 2011). Environmental DNA samples from sea turtles can also be extracted from water and sand samples in nesting areas (Farrell et al., 2022). ...
Sea turtles have a tropical and subtropical distribution and can be found in nearly all seas and oceans. They have been the subject of considerable genetic research. However, it does not yet appear that the molecular techniques used for these genetic studies follow a consensus or universal set of tools to be followed for subsequent studies. This is not desirable since it may preclude data exchange and use among studies. Thus, the aim of this review was to survey the main genetic and molecular methods used for sea turtle research worldwide. To achieve this goal, a total of 95 scientific papers were compiled from online databases. We considered articles that used molecular tools for genetic analysis and provided detailed locality data. The following aspects were assessed: species studied, local of sample collection, type of tissue used for molecular studies and type of genetic material used. The seven known sea turtle species have different distribution patterns, with some overlapping of occurrence. Chelonia mydas has been studied genetically along the coasts of all continents. Skin is the most common type of tissue used for molecular analyses. From genetic studies on sea turtles, it is possible to verify the occurrence of hybridism. This phenomenon is relevant to the conservation of the species and was reported in six articles on this topic. Therefore, it is considered that genetic and molecular assays in sea turtles are important tools for biological evaluation and protection. The aim was to survey the main methods used in genetic and molecular research on marine turtles. Keywords: DNA, Chelonian, Testudines, marine turtles
... Options for obtaining eggs include searching for deposited nests in likely nesting locations, such as the aprons of Gopher Tortoise burrows (Quinn et al. 2016), sea turtle nesting beaches (Sims et al. 2008), riverine sand bars (Dieter et al. 2014), or artificial nesting mounds placed in situ near freeranging females (Buhlmann and Osborn 2011;Quinn et al. 2015). However, nest searching is time consuming and often precludes researchers from knowing the identity of the female who deposited the clutch without genetic tests (see Shamblin et al. 2011). Further, nest searching may not be practical for species that do not produce conspicuous nests, such as Keeled Box Turtles (Cuora mouhotii; Wang et al. 2011), Mississippi Mud Turtles, (Kinosternon subrubrum hippocrepis; Anderson and Horne 2009), and Common Musk Turtles (Sternotherus odoratus; Cagle 1937), which have been observed depositing eggs on or near the soil surface. ...
This article in Herpetological Review details a combined mesocosm and oxytocin approach we used to obtain viable eggs from eastern box turtles. Please contact me for the full text.
... Other turtle species, such as Loggerhead (Caretta caretta), Olive ridley (Lepidochelys olivacea), and Kemp's Ridley turtles (Lepidochelys kempii), are known for aggregating in nearshore waters before the nesting season (Henwood, 1987;Kalb, 1999;Pandav and Choudhury, 2000;Ram, 2000;Kumar, 2015). Loggerhead turtles, for instance, are observed to aggregate along the Florida coast and in the Mediterranean Shamblin et al., 2011;Arendt et al., 2012). Olive ridley and Kemp's Ridley turtles are particularly noted for their distinctive reproductive behaviour, specifically their synchronized mass nesting events known as arribada (Hughes and Richard, 1974;Carr, 1984). ...
... Farrell et al. 40 ), positive amplification of one or more technical replicates per sample was counted as positive detection. Amplification ratio (the proportion of positive amplification detection relative to attempted technical replicate reactions 40,41 is reported for all study samples (Supplemental Table 1S). Thus, here replicates were used not as a certification of "genuine presence", but rather a) in order to increase the chance of detecting rare molecules and b) for quantifying signal abundance. ...
Animal conservation relies on assessing the distribution and habitat use of species, but for endangered/elusive animals this can prove difficult. The Monk Seal, Monachus monachus, is one of the world's most endangered species of pinniped, and the only one endemic to the Mediterranean Sea. During recent decades, direct observations have been few and scattered, making it difficult to determine its distribution away from the Aegean Sea (core distribution area of the post-decline relict population). This study relies on environmental DNA (eDNA) analysis to detect the presence of the Monk Seal in 135 samples collected in 120 locations of the central/western Mediterranean Sea, spanning about 1500 km longitudinally and 1000 km latitudinally. A recently described species-specific qPCR assay was used on marine-water samples, mostly collected during 2021 by a Citizen Science (CS) project. Positive detections occurred throughout the longitudinal range, including the westernmost surveyed area (Balearic archipelago). The distribution of the positive detections indicated six “hotspots”, mostly overlapping with historical Monk Seal sites, suggesting that habitat-specific characteristics play a fundamental role. We applied single-season occupancy models to correct for detection probability and to assess the importance of site-specific characteristics. The distance from small islets and protected (or access-restricted) areas was correlated negatively with the detection probability. This novel molecular approach, applied here for the first time in an extensive CS study, proved its potential as a tool for monitoring the distribution of this endangered/elusive species.
... While not considered for environmental factors affecting hatch success, maternity could be an additional factor which influences nest hatch success (Ditmer and Stapleton 2012). Of the 170 nests used in this study, 160 have been assigned maternity through maternal DNA present in freshly-laid egg shells (Shamblin et al. 2011). These 160 nests were laid by 106 unique females. ...
The loggerhead turtle (Caretta caretta) is a species federally listed as “threatened” whose global populations are declining. Georgia Department of Natural Resources conservation protocols for this species require the daily monitoring of nesting activity and permit physical relocation of nests which are at risk of being eroded or flooded by storms and high tides in order to increase hatch success--the proportion of hatched to unhatched eggs. Relocated nests are moved to an area with higher elevation in order to avoid flooding, but other variables such as increased temperature and decreased moisture are introduced when relocating. For years temperature and moisture have been regarded as the most important factors that contribute to hatch success but these variables are not always directly considered when relocating nests. It is likely that other environmental variables have an effect on hatch success and influence temperature and moisture.
The hypothesis that a combination of geological and biological factors better predicts hatch success compared to temperature and/or moisture alone was tested. Secondly the environmental variables which influence temperature, moisture, and likelihood of tidal washover were also examined to evaluate their impact on hatch success. Loggerhead nests on Ossabaw Island, Georgia were monitored throughout incubation; upon incubation completion, hatch success was calculated. For all nests, temperature, moisture, vegetation cover and composition, elevation, dune morphology, and tidal washovers were recorded. These variables were analyzed to assess their individual and combined influences on nest conditions and ultimately on hatch success. In addition to number of washover events, temperature, and moisture, nest vegetation and elevation were important predictors of hatch success in loggerhead sea turtle nests and should be considered when nest relocation is required.
... To overcome assay sensitivity targeted sequencing, droplet digital PCR, and low input template DNA qPCR-based approaches such as amplification ratio-based detection can be implemented. The latter was used in marine turtle population genetics and FP related eDNA analysis [59,67]. ...
Simple Summary
Characterised by benign tumours, fibropapillomatosis is a debilitating disease that predominantly afflicts the endangered green turtle (Chelonia mydas). A growing body of evidence has associated these tumours with a herpesvirus. However, a recent study detected both herpesvirus and papillomavirus in these tumours. This result challenged the idea that the herpesvirus is the sole virus associated with this disease. The present study aimed to better understand the co-occurrence of these viruses in turtles with fibropapillomatosis (in both tumour samples and non-tumoured skin samples), in addition to samples from non-tumoured turtles. Both viruses were detected in all sample types, with the 43.5% of tumours containing both herpesvirus and papillomavirus. Tumour samples were found to contain the most herpesvirus while the highest amount of papillomavirus was detected in non-tumoured skin from turtles with tumours. Collectively, these results pivot the way we think about this disease; as an infectious disease where two separate viruses may be at play.
Abstract
Characterised by benign tumours, fibropapillomatosis (FP) is a debilitating disease that predominantly afflicts the endangered green turtle (Chelonia mydas). A growing body of histological and molecular evidence has associated FP tumours with Chelonid alphaherpesvirus 5 (ChHV5). However, a recent study which detected both ChHV5 and Chelonia mydas papillomavirus 1 (CmPV1) DNA in FP tumour tissues has challenged this hypothesis. The present study aimed to establish a probe-based qPCR to assess the wider prevalence of CmPV1 and co-occurrence with ChHV5 in 275 marine turtles foraging in waters adjacent to the east coast of Queensland, Australia: three categories: Group A (FP tumours), Group B (non-tumoured skin from FP turtles) and Group C (non-tumoured skin from turtles without FP). Concurrent detection of ChHV5 and CmPV1 DNA is reported for all three categories, where Group A had the highest rate (43.5%). ChHV5 viral loads in Group A were significantly higher than loads seen in Group B and C. This was not the case for CmPV1 where the loads in Group B were highest, followed by Group A. However, the mean CmPV1 load for Group A samples was not significantly different to the mean load reported from Group B or C samples. Collectively, these results pivot the way we think about FP; as an infectious disease where two separate viruses may be at play.
... This group is known collectively as the Georgia Sea Turtle Cooperative raccoons were used on all nests, while secondary smaller mesh screens (60 x 60 cm; 1.5 x 1.5 cm mesh) were used for additional protection against ghost crabs or to reduce partial predation of nests by raccoons. All located nests had a single egg removed for an ongoing genetic tagging study on the Northern Recovery Unit (loggerheads originating from nesting beaches from Florida-Georgia border through southern Virginia; Shamblin et al. 2011, Shamblin & Nairn 2015. To reduce egg loss from tidal inundation, clutches deposited below the spring high tide line were relocated to new sites nearer to the primary dune within 12 hours of deposition. ...
The nine-banded armadillo (Dasypus novemcinctus) has become a species of local abundance in many southeastern habitats and is viewed as a nuisance and invasive by many land managers. My objective was to examine both negative and positive effects of armadillos on the Georgia coastal islands by 1) quantifying armadillo predation of sea turtle nests and comparing it to other predators; and 2) quantifying behavior and activity of armadillo burrow associates. I found that while armadillos do indeed predate sea turtle nests, they are not a major contributor to total egg loss across the coast. I recorded 33 armadillo burrow associates, including 26 species not previously reported in the literature and multiple species of conservation concern. This research provides a data-driven basis for management of armadillos and provides a template for objectively evaluating the ecosystem effects of other "invasive" species.
... To overcome assay sensitivity targeted sequencing, droplet digital PCR, and low input template DNA qPCR-based approaches such as amplification ratio-based detection can be implemented. The latter was used in marine turtle population genetics and FP related eDNA analysis [59,67]. ...
Characterised by the growth of benign tumours, fibropapillomatosis (FP) is a debilitating disease that predominantly afflicts the endangered green turtle (Chelonia mydas). A growing body of histological and molecular evidence has consistently associated FP tumours with Chelonid alphaherpesvirus 5 (ChHV5), leading this virus to be considered the most likely aetiological agent of FP. However, a recent study which detected both ChHV5 and Chelonia mydas papillomavirus 1 (CmPV1) DNA in FP tumour tissues has challenged this hypothesis. The present study aimed to establish the wider prevalence of CmPV1 and co-occurrence with ChHV5 in marine turtles in waters adjacent to the east coast of Queensland, Australia. This comprehensive molecular survey screened a total of 353 samples from 275 foraging turtles using probe-based qPCR. Three sample categories were used in this study: Group A (FP tumours), Group B (non-tumoured skin from turtles with FP tumours) and Group C (non-tumoured skin from turtles without FP tumours). Concurrent detection of ChHV5 and CmPV1 DNA is reported for all three categories, with the highest rate of concurrent detection reported for Group A samples (43.5%). Collectively, these results pivot the way we think about FP; as an infectious disease where two separate viruses may be at play.
... For the extraction, we followed a modified protocol (Shamblin et al., 2011) using the DNeasy R Blood & Tissue kit by completing the following steps. ...
Hawksbill turtles (Eretmochelys imbricata) are exploited for their beautiful shell known as tortoiseshell or bekko, making them extremely vulnerable in the illegal global trade of tortoiseshell products. In this study, we developed an effective, standardized method using a commercially available kit to extract DNA and obtain informative mitochondrial DNA (mtDNA) control region sequences (~800 bp) from hawksbill turtle products in order to trace the sample back to a likely stock origin. We also sequenced additional skin samples from nesting beaches of Milman Island, Australia and Arnavon Island, Solomon Islands to add to the baseline data for hawksbill turtles in the Indo-Pacific. Our results indicate that nine of the 13 tortoiseshell products obtained from Papua New Guinea and Solomon Islands were from turtles with haplotypes found primarily at the Solomon Islands rookery and did not match those from nesting populations in Australia or SE Asia, with the exception of one haplotype also found in 3% of turtles at Milman Island. We also found that 23% of the market samples have haplotypes only documented in foraging populations, which illustrates the urgent need for more extensive sampling of rookeries to fill gaps in the reference baseline database. Nevertheless, our study results demonstrate an effective methodology for obtaining DNA of sufficient quantity and quality from hawksbill turtle products.
... DNA from the caudal fin cuttings of the six species was extracted using the Qiagen DNeasy Tissue Kit (Qiagen) [17]. The purity and concentration of all of the extracted DNA was determined using a UV-VI spectrophotometer (Biochrom Libra S70, Biochrom Ltd, Cambridge, UK) based on absorbance at A260/280. ...
Invasive alien fish species have become a silent treat towards the ecosystem especially the native fish population in Malaysia. There has been a need to develop rapid identification methods that can aid management teams in identifying fish species that are not native to our ecosystem. Current visual identification methods are highly tedious and require time, delaying action towards curbing the invasion. The LAMP assay successfully identified six popular invasive fish species in Malaysia. None of the LAMP assays showed false positives and the Limit of Detection of the LAMP primers were highly sensitive and could detect DNA samples up to 1 × 10 − 15 ng/μl. The LAMP primers designed were highly specific to the target species and did not amplify non target species. DNA sequencing was done to ensure the accuracy of LAMP assay results. This study demonstrates that LAMP is a suitable tool in species identification efforts of invasive fish species in Malaysia.
... Large mesh screens (1.22 Â 1.22 m; 4.1 Â 4.1 cm mesh; MasterNet MN-L77) designed to protect against raccoons were used on all nests, while secondary smaller mesh screens (60 Â 60 cm; 1.5 Â 1.5 cm mesh) were used for additional protection against ghost crabs or to reduce partial predation of nests by raccoons. All located nests had a single egg removed for an ongoing genetic tagging study on the Northern Recovery Unit (loggerheads originating from nesting beaches from Florida-Georgia border through southern Virginia; Shamblin et al., 2011;Shamblin and Nairn, 2015). To reduce egg loss from tidal inundation, clutches deposited below the spring high tide line were relocated to new sites nearer to the primary dune within 12 h of deposition. ...
Nesting beach management is a vital element of the population recovery efforts for the vulnerable loggerhead sea turtle (Caretta caretta) across the globe. In the southeast United Sates, turtle nests are threatened by numerous anthropogenic and natural threats, including predation of eggs by native and non-native predators. We analyzed loggerhead nest predation and other egg ; loss using an exceptional 10-year data set (2009-2018) that covered nesting beaches on 12 islands on the Georgia coast. Our objectives were to 1) determine which predators cause the greatest loss of loggerhead sea turtle eggs, 2) evaluate whether non-native species have a higher ; rate of predation than native species, and 3) compare predation rates to other major sources of egg loss across these islands. Our results show that under current strategies for nest management: 1) non-native feral hogs and native raccoons have the greatest impact as predators on sea turtle eggs; 2) non-native predators have caused significantly more egg loss across Georgia’s coast than native species, but the impact varies greatly by species; and 3) losses to predation are similar in magnitude to post-management losses from tides and storms over the last decade. We recommend the continued use of multiple management techniques, including nest screening and ; targeted predator management, but caution that predator controls should be focused on those demonstrated to cause significant losses in order to prioritize conservation funding.
... Eggshell samples were collected as part of an ongoing loggerhead turtle genetic capture-recapture project aimed at identifying individual females nesting north of Florida for the estimation of population size, clutch frequency, remigration intervals, and annual survival (www.seaturtle.org/nestdb/genetics). Eggshell samples were processed in accordance with the standard eggshell DNA extraction and genotyping protocols as previously described (Shamblin et al. 2011). Briefly, a single fresh egg is collected from each nest. ...
... One reencountered turtle in this study was identified in absentia by genetic detection of her eggs. This methodology, should it become widely utilized, may be a powerful method to detect the presence of individual females for years after an initial encounter (Shamblin et al. 2011). Additional technological advances will likely improve our ability to detect long-term sea turtle movements and habitat use. ...
A survey of sea turtle rehabilitation facilities in the United States revealed that 34 facilities released 11,417 sea turtles through 2016. The number of turtles released per time period increased over time, with 80% of releases occurring between 2007 and 2016, 15% between 1997 and 2006, and 5% prior to 1997. Twenty facilities reported a total of 314 first re-encounters and 6 second re-encounters of turtles that had been previously released, including 12 turtles encountered while successfully nesting. Results revealed substantial efforts to rehabilitate sea turtles in the United States, with some rehabilitated turtles surviving for extended periods after release, but with the fate of most remaining unknown. Greater efforts to determine the long-term outcome for a larger proportion of rehabilitation cases are warranted.
... They obtained genetics samples from a majority of these clutches, either as a single egg collected during the morning survey following oviposition or as dead hatchling tissue and/or an eggshell from an undeveloped egg collected during nest evaluation inventories following hatchling emergence. We conducted sample processing and DNA extractions as previously described for loggerhead turtle eggs (Shamblin et al. 2011b). We genotyped DNA samples at 15 microsatellite loci originally isolated from loggerhead turtles (Shamblin et al. 2007) as previously described (Shamblin et al. 2017a). ...
Green turtle nesting has been recorded in North Carolina, since 1980, but how these nesting females fit into the broader regional context genetically has not been determined. Genetic tagging through microsatellite genotyping of clutches laid in northern South Carolina, North Carolina, and Delaware from 2010 through 2014 identified at least 52 individual nesting females. The mitochondrial control region haplotype frequencies of these individuals were significantly different from all northern Greater Caribbean subpopulations, including those in Florida, suggesting that these northern US females represent an incipient subpopulation that warrants distinct management unit status.
... However, some females may disperse their nests beyond the limits of the areas of beach monitoring for tagging or observation, or there may be insufficient resources to conduct consistent monitoring, leading to missed turtles, sparse recapture data, and biased estimates of these key parameters. Shamblin et al. (2011) developed a technique to address this limitation by extracting maternal genomic DNA from freshly laid loggerhead eggs, permitting individual identification of females without the need to physically intercept them during the nesting process. This type of sampling allows genetic CMR on spatial scales that would be logistically impossible to replicate through traditional tagging approaches. ...
Marine turtles migrate across long distances, exhibit complex life histories, and occupy habitats that are difficult to observe. These factors present substantial challenges to understanding fundamental aspects of their biology or assessing human impacts, many of which are important for the effective conservation of these threatened and endangered species. The early development and application of genetic tools made important contributions to understanding marine turtle population and evolutionary biology, such as providing evidence of regional natal homing by breeding adults, establishing connectivity between rookeries and foraging habitats, and determining phylogeography and broad scale stock structure for most marine turtle species. Recent innovations in molecular technologies, statistical methods, and creative application of genetic tools have significantly built upon this knowledge to address key questions in marine turtle biology and conservation management. Here, we evaluate the latest major advances and potential of marine turtle genetic applications, including improved resolution and large-scale syntheses of population structure, connectivity and phylogeography, estimation of key demographic rates such as age to maturity and operational or breeding sex ratios, insight into reproductive strategies and behavior, and assessment of differential human impacts among populations. We then discuss remaining challenges and emerging capabilities, such as rapid, multiplexed genotyping, and investigation of the genomic underpinnings of adaptive variation afforded by high-throughput sequencing technologies.
... Eggshell samples were collected as part of an ongoing loggerhead turtle genetic capture-recapture project aimed at identifying individual females nesting north of Florida for the estimation of population size, clutch frequency, remigration intervals, and annual survival (www.seaturtle.org/nestdb/genetics). Eggshell samples were processed in accordance with the standard eggshell DNA extraction and genotyping protocols as previously described (Shamblin et al. 2011). Briefly, a single fresh egg is collected from each nest. ...
... DNA from a total of 11 eggs amplified at four or more loci; only four eggs successfully amplified at all six loci ( Table 2). The low amplification success rate may be attributed to lower concentrations of template DNA in egg material as compared to tissue samples (Shamblin et al. 2011). The area of the yolk from which samples were taken may also impact results. ...
Genetic analyses of the endangered Alabama red-bellied turtle (Pseudemys alabamensis) have the potential to answer questions regarding the species’ population dynamics and reproductive ecology. Here we used oviductal eggs from road-kill female turtles to obtain DNA from P. alabamensis specimens. Six microsatellite markers were successfully amplified in egg yolks/developing embryos. DNA taken from multiple eggs in the same clutch showed variation in allele sizes at multiple loci, indicating amplification of both maternally and paternally contributed alleles. These results introduce DNA extraction from oviductal eggs from road-kill mortalities as a novel approach for examining the reproductive habits and population dynamics of P. alabamensis and similar turtle species. This approach may be particularly effective in exploring mating patterns and monitoring population stability in endangered species where traditional sampling methods are difficult to perform.
... Genomic DNA was extracted using the DNeasy blood and tissue kit (QIAGEN) following standard protocols. Extractions from hatched eggshells were conducted with modifications previously described for extractions from freshly laid eggs (Shamblin et al. 2011b). Polymerase chain reaction (PCR) amplifications of an *818 bp portion of the mitochondrial control region were carried out using primers LCM15382 and H950 g (Abreu- Grobois et al. 2006). ...
... Genomic DNA was extracted using the DNeasy blood and tissue kit (QIAGEN) following standard protocols. Extractions from hatched eggshells were conducted with modifications previously described for extractions from freshly laid eggs (Shamblin et al. 2011b). Polymerase chain reaction (PCR) amplifications of an *818 bp portion of the mitochondrial control region were carried out using primers LCM15382 and H950 g (Abreu- Grobois et al. 2006). ...
Green turtle (Chelonia mydas) nesting has increased dramatically in Florida over the past two decades, ranking the Florida nesting aggregation among the largest in the Greater Caribbean region. Individual beaches that comprise several hundred kilometers of Florida’s east coast and Keys support tens to thousands of nests annually. These beaches encompass natural to highly developed habitats, and the degree of demographic partitioning among rookeries was previously unresolved. We characterized the genetic structure of ten Florida rookeries from Cape Canaveral to the Dry Tortugas through analysis of 817 base pair mitochondrial DNA (mtDNA) control region sequences from 485 nesting turtles. Two common haplotypes, CM-A1.1 and CM-A3.1, accounted for 87 % of samples, and the haplotype frequencies were strongly partitioned by latitude along Florida’s Atlantic coast. Most genetic structure occurred between rookeries on either side of an apparent genetic break in the vicinity of the St. Lucie Inlet that separates Hutchinson Island and Jupiter Island, representing the finest scale at which mtDNA structure has been documented in marine turtle rookeries. Florida and Caribbean scale analyses of population structure support recognition of at least two management units: central eastern Florida and southern Florida. More thorough sampling and deeper sequencing are necessary to better characterize connectivity among Florida green turtle rookeries as well as between the Florida nesting aggregation and others in the Greater Caribbean region.
... Many beaches do not have the resources for such programs, but those already building long-term mark-recapture datasets should continue in order to provide the information needed to estimate population parameters. Genetic studies of eggs from individual nests may be used to supplement mark-recapture analyses where night-time monitoring is not possible (Shamblin et al. 2010(Shamblin et al. , 2011 and will identify nesting activity by marked individuals outside the range of beach-based studies. Nest site fidelity is a factor in determining apparent survival rates, and a combination of mark-recapture and genetic analysis will allow more robust estimates of adult female survival rates. ...
A twenty year mark-recapture dataset from the loggerhead nesting beach on Keewaydin Island, off the southwest coast of Florida, was analyzed using a two-state open robust design model in Program MARK to provide insight into recent nesting declines in the state. A total of 2,292 encounters representing 841 individual tag IDs were used for this analysis. Survival was estimated at 0.73 (95% CI 0.69-0.76), and there was no evidence from remigration rate or clutch frequency to suggest the composition of the nesting assemblage had changed over time. The mark-recapture analysis was supplemented with a satellite tracking component to identify the offshore foraging areas utilized by Keewaydin nesters. Eleven nesting females were outfitted with platform terminal transmitters, which transmitted for 42 to 300+ days including inter-nesting intervals and subsequent migration to foraging grounds. Site fidelity tests and kernel density home range analyses were used to describe foraging habitats. Females foraging in the eastern Gulf of Mexico were within the recent 64 m bottom longline fishery restriction. Areas identified as important habitats during the remigration interval should be used to inform managers in creating targeted management strategies to aid population recovery without the use of broad fishery closures.
Capture–mark–recapture studies that fail to account for the frequency and dynamics of marker loss risk generating biased demographic estimates. In this study, we used permanent multilocus genotypes (i.e., “genetic tags”) and a new enhanced tag loss model to quantify the tag loss dynamics for both passive integrated transponder (PIT) and Inconel metal tags applied to loggerhead turtles (Caretta caretta) nesting on Wassaw Island, GA USA. Our results indicate that tag loss is most likely to occur within the nesting season in which tags were applied and is maximal just after tagging (maximum likelihood estimates): 0.00098 PIT tags day⁻¹ and 0.007 Inconel tags day⁻¹. After that, PIT tag loss was negligible and Inconel tag loss remained low but constant at 0.00028 tags day⁻¹, such that after 5 years, the probability of losing one PIT tag was 0.06 and losing at least one Inconel tag was 0.46. The use of genetic tags in this study makes these the first truly accurate estimates of PIT and Inconel tag loss for marine turtles, and the new model of tag loss described herein represents an important advancement in the analytical methods used to estimate and compare tag loss dynamics.
Diet items and habitat constitute some of the environmental resources that may be used differently by individuals within a population. Long-term fidelity by individuals to particular resources exemplifies individual specialization, a phenomenon that is becoming increasingly recognized across a wide range of species. Less is understood about the consequences of such specialization. Here, we investigate the effects of differential foraging ground use on reproductive output in 183 loggerhead sea turtles (Caretta caretta) nesting at Wassaw Island, Georgia (31.89°N, 80.97°W), between 2004 and 2011 with resulting possible fitness effects. Stable isotope analysis was used to assign the adult female loggerheads to one of three foraging areas in the Northwest Atlantic Ocean. Our data indicate that foraging area preference influences the size, fecundity, and breeding periodicity of adult female loggerhead turtles. We also found that the proportion of turtles originating from each foraging area varied significantly among the years examined. The change in the number of nesting females across the years of the study was not a result of uniform change from all foraging areas. We develop a novel approach to assess differential contributions of various foraging aggregations to changes in abundance of a sea turtle nesting aggregation using stable isotopes. Our approach can provide an improved understanding of the influences on the causes of increasing or decreasing population trends and allow more effective monitoring for these threatened species and other highly migratory species.
The southeastern USA hosts the largest nesting concentration of loggerhead turtles Caretta caretta in the Atlantic. Regionally significant nesting also occurs along the Caribbean coast of Mexico, in Cuba, and in the Bahamas. Previous studies of North Atlantic loggerhead turtle rookeries based on a 380 bp fragment of the mitochondrial control region supported recognition of 8 demographically independent nesting populations (management units) in the Northwest Atlantic in addition to Cape Verde in the eastern Atlantic. Recent analysis of expanded mitochondrial control region sequences revealed additional genetic diversity and increased population structure between western and eastern Atlantic loggerhead turtle rookeries. We sequenced an 817 bp mitochondrial DNA fragment in 2427 samples from nesting beaches in the southeastern USA, Cay Sal Bank, Bahamas, and Quintana Roo, Mexico. Pairwise F-ST comparisons, pairwise exact tests of population differentiation, and analysis of molecular variance support previously proposed management unit designations and additionally indicate that southeastern and southwestern Florida rookeries should be recognized as distinct management units. Therefore, Northwest Atlantic loggerhead turtle rookeries can be subdivided into 10 management units, corresponding to the beaches from (1) Virginia through northeastern Florida, (2) central eastern Florida, (3) southeastern Florida, (4) Dry Tortugas, Florida, (5) Cay Sal, Bahamas, (6) southwestern Cuba, (7) Quintana Roo, Mexico, (8) southwestern Florida, (9) central western Florida, and (10) northwestern Florida. We confirmed increased resolution of stock structure between many Northwest Atlantic management units and the Cape Verde rookery with the expanded control region haplotypes.
Most turtle species suffer high mortality in their first year, have a long juvenile period, and can live for decades once they reach adulthood. Conservationists have implemented a number of recovery plans for threatened turtle populations, including experimental ''headstart'' programs. Headstarting involves the captive rearing of hatchlings from eggs collected in the wild. The hatchlings are held for several months to help them avoid high mortality in their first year. It is hoped that these turtles survive and grow like wild turtles after release. The purpose of our study was to evaluate headstarting as a management tool for threatened turtle populations. We critically examined the population-level effects of headstarting with a series of deterministic matrix models for yellow mud turtles (Kinosternon flavescens), a ''non-threatened,'' well-studied species, and endangered Kemp's ridley sea turtles (Lepidochelys kempi). We show that management efforts focused exclusively on improving survival in the first year of life are unlikely to be effective for long-lived species such as turtles. Population projections for both turtles predict that headstarting can augment increasing populations when adult survival is returned to or maintained at high levels, provided that headstarted juveniles are as vigorous as wild turtles. However, when subadult and adult survival is reduced, headstarting cannot compensate for losses in later stages. Proportional sensitivity (elasticity) analyses of stage-based matrix models indicated that annual survival rates for subadult and adult turtles are most critical; small decreases in the survival of older turtles can quickly overcome any potential benefits of headstarting. In general, the biological benefits of headstarting programs may be overestimated for turtles, and a careful examination of stage-specific mortality sources, demography, and life history can guide us toward more effective management strategies.
Management of many species is currently based on an inadequate under- standing of their population dynamics. Lack of age-specific demographic information, particularly for long-lived iteroparous species, has impeded development of useful models. We use a Lefkovitch stage class matrix model, based on a preliminary life table developed by Frazer (1983a), to point to interim management measures and to identify those data most critical to refining our knowledge about the population dynamics of threatened log- gerhead sea turtles (Caretta caretta). Population projections are used to examine the sen- sitivity of Frazer's life table to variations in parameter estimates as well as the likely response of the population to various management alternatives. Current management practices appear to be focused on the least responsive life stage, eggs on nesting-beaches. Alternative protection efforts for juvenile loggerheads, such as using turtle excluder devices (TEDs), may be far more effective.
Here we report methods for extracting maternal DNA from avian eggshells or offspring DNA from eggshells and embryos. These methods offer alternative techniques for obtaining DNA from oviparous organisms. Using DNA extracted from eggshells, we obtain microsatellite genotypes of the brood parasitic brown-headed cowbird (Molothrus ater) female that laid the eggs and/or her hatched offspring. Using DNA extracted from embryos, we obtain microsatellite genotypes of offspring. We demonstrate that separate extractions performed on the embryo and shell from a single egg can provide DNA from the embryo and its mother, respectively. This single-egg approach for obtaining both maternal and embryonic DNA simplifies paternity analyses because alleles unique to the embryo can be considered paternal in origin. Finally we report two new microsatellite loci and primer sequences for brown-headed cowbirds.
One of the many threats to sea turtlepopulations is the take of turtles and theireggs for consumption and sale. Improved speciesidentification methods for sea turtle eggs andcooked meats would facilitate prosecution ofthose involved. Fatty acid-based methods toidentify eggs cannot resolve loggerheads andthe two ridley species. Protein-based methodsare not applicable to eggs or cooked meat. Wepresent methods to extract DNA from turtle eggand cooked meat and to produce diagnosticrestriction fragment length polymorphismpatterns in the cytochrome b region of themitochondrial DNA. This method works on DNAfrom any tissue, and provides wildlife lawenforcement another tool to combat illegal takeof endangered species.
In this study we developed eight quantitative PCR (qPCR) assays to evaluate the starting copy number of nuclear and mitochondrial
DNA fragments ranging from 75 to 350 base-pairs in DNA extracts from Chinook salmon tissues with varying quality. Samples
were genotyped with 13 microsatellite and 29 SNP assays and average genotyping success for good, intermediate, and poor quality
samples was 96%, 24%, and 24% for microsatellite loci, and 98%, 97%, and 79% for SNPs, respectively. As measured by qPCR,
good quality samples had a consistently high number of starting copies across all fragment sizes with little change between
the smallest and largest size. In contrast, the intermediate and poor quality samples displayed decreases in starting copy
number as fragment size increased, and was most pronounced with poor samples. Logistic regression of genotyping success by
starting copy number indicated that in order to achieve at least 90% genotyping success, approximately 1,000 starting copies
of nuclear DNA are necessary for microsatellite loci, and as few as 14 starting copies for SNP assays (but we recommend at
least 50 copies to reduce genotyping error). While these guidelines apply specifically to Chinook salmon and the genetic markers
included in this study, the principles are transferable to other species and markers due to the underlying process associated
with template quantity and PCR amplification.
Noninvasive samples are useful for molecular genetic analyses of wild animal populations. However, the low DNA content of such samples makes DNA amplification difficult, and there is the potential for erroneous results when one of two alleles at heterozygous microsatellite loci fails to be amplified. In this study we describe an assay designed to measure the amount of amplifiable nuclear DNA in low DNA concentration extracts from noninvasive samples. We describe the range of DNA amounts obtained from chimpanzee faeces and shed hair samples and formulate a new efficient approach for accurate micro-satellite genotyping. Prescreening of extracts for DNA quantity is recommended for sorting of samples for likely success and reliability. Repetition of results remains extensive for analysis of microsatellite amplifications beginning from low starting amounts of DNA, but is reduced for those with higher DNA content.
Population generic analyses have been highly successful in deciphering inter- and intraspecific evolutionary relationships, levels of gene flow, genetic divergence and effective population sizes. Parameters estimated by traditional population genetic analyses are evolutionary averages and thus not necessarily relevant for contemporary ecological or conservation issues. Molecular data can, however, also provide insight into contemporary patterns of divergence, population size and gene flow when a sufficient number of variable loci are analysed to focus subsequent data analyses on individuals rather than populations. Genetic tagging of individuals is an example of such individual-based approaches and recent studies have shown it to be a viable alternative to traditional ragging methods. Owing to the ubiquitous presence of hyper-variable DNA sequences in eukaryote genomes it is in principle possible to tag any eukaryote species and the required DNA can be obtained indirectly from substrates such as faeces, sloughed skin and hair. The purpose of this paper is to present the concept of genetic tagging and to further advocate the extension of individual-based generic analyses beyond the Identification of individuals to other kinds of relationships, such as parent-offspring relations, which more fully exploit the genetic nature of the data. (C) 1999 The Limnean Society of London.
We examined the utility of feathers and egg shell membranes, deposited in the nests of Spectacled Eiders (Somateria fischeri), as a source of DNA for genetic studies at both the population and individual level. The potential for feather DNA contamination as a result of female behavioral interactions (e.g. nest parasitism), reuse of nest sites from previous years, or other unknown occurrences was acknowledged and specifically tested. DNA was successfully extracted from both feathers and egg shell membranes and waterfowl microsatellite loci were used to construct individual genotypes. We found no difference in the genotypes obtained from nest feathers or blood of the incubating female. Detection of nest feather contamination was possible with as little as one feather when samples from multiple females were intentionally mixed. Triplicate DNA extractions from 33 nests provided a means of detecting contamination in 3 nests. Egg membranes proved a viable source of offspring DNA and can contribute valuable data to investigations of parentage when assayed jointly with maternal feather DNA. Nest materials provide an efficient, non-invasive method of genetic sampling that can be readily incorporated into field research. However, the natural history traits and mating strategies of a species must be considered during sample collection to identify the possible sources of nest materials (e.g., paternal, maternal, parasite, etc.). Specific experiments should also be designed to test sampling assumptions.
Individual identification via non-invasive sampling is of prime importance in conservation genetics and in behavioural ecology. This approach allows for genetics studies of wild animals without having to catch them, or even to observe them. The material used as a source of DNA is usually faeces, shed hairs, or shed feathers. It has been recently shown that this material may lead to genotyping errors, mainly due to allelic dropout. In addition to these technical errors, there are problems with accurately estimating the probability of identity (PI, or the probability of two individuals having identical genotypes) because of the presence of close relatives in natural populations. As a consequence, before initiating an extensive study involving non-invasive sampling, we strongly suggest conducting a pilot study to assess both the technical difficulties and the PI for the genetic markers to be used. This pilot study could be carried out in three steps: (i) estimation of the PI using preliminary genetic data; (ii) simulations taking into account the PI and choosing the technical error rate that is sufficiently low for assessing the scientific question; (iii) polymerase chain reaction (PCR) experiments to check if it is technically possible to achieve this error rate.
We studied correlations among traits related to body size and reproductive behavior in marine turtles, using data from 96 different populations representing seven species. Our analyses focused on patterns of phenotypic covariation among species and among populations within species. At the species level, body size correlated positively with several reproductive traits, including egg size and overall reproductive effort. A trade-off between clutch size and egg size was confirmed for marine turtles, after factoring out the effects of body size. Patterns of variation within species were different from those among species. For example, in five out of six species there was a positive relationship between adult body size and clutch size, although this correlation was not found at the interspecific level. We also found important differences among species in the way life-history traits correlated with one another. Four species having a sufficient number of samples exhibited congruent worldwide patterns of body size variation. A comparative approach may prove useful for extending demographic models developed for loggerhead turtles to less well-known species, even though many of the model parameters have not been estimated for other species.
Monitoring trends in loggerhead turtle popu-lations is critical to assessing population status and to developing and assessing conservation strategies. Presently, the most reliable estimates of sea turtle population size come from counts on nesting beaches. In addition to providing population estimates, nesting beach data also provide information on how reproductive effort is focused spatially and temporally. Several mea-sured parameters are key to describing repro-ductive effort and to estimating the number of nesting females from nest count data. Among these parameters are clutch frequency, remigra-tion interval, and nesting site fidelity (collec-tively referred to here as nesting patterns). These three key measures are intimately linked and have great bearing on the accuracy of the simple calculations used to derive nesting population estimates from numbers of nests. Although numerous authors have reported clutch frequency and remigration interval values for loggerheads at nesting beaches around the world, information on nesting site fidelity is frequent in the literature, perhaps because the difficulty of obtaining this measure. Effective conservation programs for turtles require more than an understan nesting patterns and nesting populatio With regard to the adult life stage, info on migratory routes and foraging areas needed. The purpose of this chapter is line the loggerhead reproductive data for guiding conservation progr authors review available data on log nesting patterns, reproductive migra adult foraging areas, and they cies in understanding these aspect head life history.
gmconvert is a platform-independent program provided in GUI (for Apple OS X and Windows XP) and command-line versions (for other platforms). gmconvert allows rapid reformatting of microsatellite data from output files produced by Applied Biosystems genemapper software (version 3.x). The program will re-array data into three formats commonly used in downstream analysis: genepop, cervus, and gerud. gmconvert will greatly increase the speed of data preparation prior to analysis and aid in reducing transpositional errors associated with manual re-arraying and reformatting steps. gmconvert is available from http://gallus.forestry.uga.edu/software/.
We describe primers and polymerase chain reaction conditions to amplify 15 tetranucleotide microsatellite loci from the loggerhead sea turtle (Caretta caretta). The primers were tested on 30 individuals that nested along the Georgia, USA coast. The primer pairs developed in this study yielded an average of 13.9 alleles per locus (range of 10–21), an average observed heterozygosity of 0.91 (range 0.79–1.00), and an average polymorphic information content of 0.88 (range 0.84–0.92).
Individual identification via non-invasive sampling is of prime importance in conservation genetics and in behavioural ecology. This approach allows for genetics studies of wild animals without having to catch them, or even to observe them. The material used as a source of DNA is usually faeces, shed hairs, or shed feathers. It has been recendy shown that this material may lead to genotyping errors, mainly due to allelic dropout. In addition to these technical errors, there are problems with accurately estimating the probability of identity (PI, or the probability of two individuals having identical genotypes) because of the presence of close relatives in natural populations. As a consequence, before initiating an extensive study involving non-invasive sampling, we strongly suggest conducting a pilot study to assess both the technical difficulties and the PI for the genetic markers to be used. This pilot study could be carried out in three steps: (i) estimation of the PI using preliminary genetic data; (ii) simulations taking into account the PI and choosing the technical error rate mat is sufficiently low for assessing the scientific question; (iii) polymerase chain reaction (PCR) experiments to check if it is technically possible to achieve this error rate.
Population genetic analyses have been highly successful in deciphering inter-and intra-specific evolutionary relationships, levels of gene flow, genetic divergence and effective population sizes. Parameters estimated by traditional population genetic analyses are evolu-tionary averages and thus not necessarily relevant for contemporary ecological or conservation issues. Molecular data can, however, also provide insight into contemporary patterns of divergence, population size and gene flow when a sufficient number of variable loci are analysed to focus subsequent data analyses on individuals rather than populations. Genetic tagging of individuals is an example of such individual-based approaches and recent studies have shown it to be a viable alternative to traditional tagging methods. Owing to the ubiquitous presence of hyper-variable DNA sequences in eukaryote genomes it is in principle possible to tag any eukaryote species and the required DNA can be obtained indirectly from substrates such as faeces, sloughed skin and hair. The purpose of this paper is to present the concept of genetic tagging and to further advocate the extension of individual-based genetic analyses beyond the identification of individuals to other kinds of relationships, such as parent-offspring relations, which more fully exploit the genetic nature of the data.
The use of non-destructive sampling methods to collect genetic material from wildlife allows researchers to minimize disturbance. Most avian studies employ capturing and handling of young and parents to draw blood for DNA analysis. In some cases adult female birds are difficult to catch, so maternal genotyping has required collection of contour feathers from nests, or destructive sampling of eggs. Many species do not leave contour feathers in the nest, and destructive sampling has been unreliable due to contamination with embryonic DNA. Alternative field sampling techniques for collection of maternal DNA from birds are therefore desirable. Here we demonstrate that avian maternal DNA can be isolated in a non-invasive and non-destructive way from the external surface of eggs. We used cotton swabs to collect maternal DNA from the external shells of herring gull (Larus argentatus) and Caspian tern (Sterna caspia) eggs. DNA was then amplified by the polymerase chain reaction (PCR) for microsatellite genotyping. We verified that the DNA samples were maternal by comparing microsatellite profiles to those obtained from adults and chicks from the same nests. In 100% of Caspian tern (n=16) and herring gull families (n=12), the egg swabs that amplified matched the maternal microsatellite genotype. In a screening of many nests of both species, we successfully amplified microsatellite markers from 101/115 (88%) egg swabs. Swabs from eggs with blood stains on the shell were more likely to amplify successfully than those from clean eggs. The advantages of this new method include increased parentage assignment/exclusion power, and increased availability of maternal DNA for genotyping of species that do not deposit contour feathers in nests.
Many avian studies, aimed at collecting samples for genetic analysis, rely upon invasive procedures involving the capture and handling of parents and their offspring. Our goal was to develop a nondestructive method for sampling maternal DNA that would not require blood collection from the mother. Herein, we describe a method for isolating genomic DNA from eggshell powder, obtained by filing the outer shell of an avian egg. Comparison of microsatellite profiles, obtained from genomic DNA found within eggshell matrices and their corresponding parents, verified the presence of maternal DNA in the eggshell matrix in 100% of the herring gull nests assessed (n= 11). In addition, the microsatellite profiles of eggshell DNA were identical among eggs from the same clutch. The ability to rapidly obtain a DNA sample from an avian eggshell in a noninvasive manner could aid in a wide range of genetic sampling studies, and in this study, we provide one potential application of this finding: assessing the fertilization status of nonviable herring gull (Larus argentatus) eggs from the Laurentian Great Lakes. Detection of fertilization was successful as the microsatellite profiles of eggshell powder (maternal only) and the fertilized embryonic contents of those eggs did not match. Ideally, the application of such an approach will help to discriminate unfertilized eggs from embryos aborted early in development and provide insights into avian reproductive health.
Basic reproductive data from 21 green turtle ( Chelonia mydas ), 8 leatherback (Dermochelys coriacea), 7 hawksbill ( Eretmochelys imbricata ), 7 olive ridley ( Lepidochelys olivacea ),6 loggerhead ( Caretta caretta ), 1 Kemp's ridley ( Lepidochelys kempi ), and 1 flatback ( Chelonia depressa ) populations are provided. Some intraspecific and interspecific relationships between size of nester and clutch, egg size and hatchling size are analyzed. Measurements of reproductive rates (=numbers of hatchlings per female per year) in 11 populations varied from 35 to 200 in an olive ridley and loggerhead colony, respectively. Nesting behavior of each species is described in terms of type of nesting emergence and time spent on the nesting beach (=chelonery). The relatively large number of yolkless eggs laid by many leatherbacks and by some hawksbills invites further study. Some aspects of sea turtle nesting behavior and reproduction are compared to those of other chelonians.
In the context of a study of wild chimpanzees, Pan troglodytes verus, we found that genotypes based on single PCR amplifications of microsatellite loci from single shed hair have a high error rate. We quantified error rates using the comparable results of 791 single shed hair PCR amplifications of 11 microsatellite loci of 18 known individuals. The most frequent error was the amplification of only one of the two alleles present at a heterozygous locus. This phenomenon, called allelic dropout, produced false homozygotes in 31% of single-hair amplifications. There was no difference in the probability of preferential amplification between longer and shorter alleles. The probability of scoring false homozygotes can be reduced to below 0.05 by three separate amplifications from single hairs of the same individual or by pooling hair samples from the same individual. In this study an additional 5.6% of the amplifications gave wrong genotypes because of contamination, labelling and loading errors, and possibly amplification artefacts. In contrast, amplifications from plucked hair taken from four dead individuals gave consistent results (error rate < 0.01%, n = 120). Allelic dropout becomes a problem when the DNA concentration falls below 0.05 ng/10 microL in the template as it can with shed hair, and extracts from faeces and masticated plant matter.
To test whether plucked hairs are a reliable source of DNA for genotyping microsatellite loci, we carried out experiments using one, three, or 10 hairs per extract for 50 alpine marmots. For each extract, seven independent genotypings were performed for the same locus (multiple-tubes approach). Two types of genotyping errors were recorded: a false homozygote defined as the detection of only one allele of a true heterozygote, and a false allele defined as a PCR-generated allele that was not one of the alleles of the true genotype. Using DNA extracted from one, three, or 10 hairs the overall error rate was 14.00%, 4.86%, and 0.29%, respectively. Based on our results, we conclude that 10 hairs should be used to obtain consistently reliable genotypings using the single-tube approach, and that a single plucked hair could represent a reliable source of DNA if the multiple-tubes approach is used. For future studies of dinucleotide repeat diversity using DNA extracted from one to three shed or plucked hairs, we strongly recommend initiating an appropriate pilot study to quantify the error rate and to determine the reliability of the single-tube approach.
Noninvasive samples are useful for molecular genetic analyses of wild animal populations. However, the low DNA content of such samples makes DNA amplification difficult, and there is the potential for erroneous results when one of two alleles at heterozygous microsatellite loci fails to be amplified. In this study we describe an assay designed to measure the amount of amplifiable nuclear DNA in low DNA concentration extracts from noninvasive samples. We describe the range of DNA amounts obtained from chimpanzee faeces and shed hair samples and formulate a new efficient approach for accurate microsatellite genotyping. Prescreening of extracts for DNA quantity is recommended for sorting of samples for likely success and reliability. Repetition of results remains extensive for analysis of microsatellite amplifications beginning from low starting amounts of DNA, but is reduced for those with higher DNA content.
Determining population sizes can be difficult, but is essential for conservation. By counting distinct microsatellite genotypes, DNA from noninvasive samples (hair, faeces) allows estimation of population size. Problems arise because genotypes from noninvasive samples are error-prone, but genotyping errors can be reduced by multiple polymerase chain reaction (PCR). For faecal genotypes from wolves in Yellowstone National Park, error rates varied substantially among samples, often above the 'worst-case threshold' suggested by simulation. Consequently, a substantial proportion of multilocus genotypes held one or more errors, despite multiple PCR. These genotyping errors created several genotypes per individual and caused overestimation (up to 5.5-fold) of population size. We propose a 'matching approach' to eliminate this overestimation bias.
Genotypes are frequently used to identify parentage. Such analysis is notoriously vulnerable to genotyping error, and there is ongoing debate regarding how to solve this problem. Many scientists have used the computer program CERVUS to estimate parentage, and have taken advantage of its option to allow for genotyping error. In this study, we show that the likelihood equations used by versions 1.0 and 2.0 of CERVUS to accommodate genotyping error miscalculate the probability of observing an erroneous genotype. Computer simulation and reanalysis of paternity in Rum red deer show that correcting this error increases success in paternity assignment, and that there is a clear benefit to accommodating genotyping errors when errors are present. A new version of CERVUS (3.0) implementing the corrected likelihood equations is available at http://www.fieldgenetics.com.
A stage-based population model for loggerhead sea turtles and implications for conservation Touchdown PCR to prevent spur-ious priming during gene amplification
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Crouse D, Crowder L, Caswell H (1987) A stage-based population model for loggerhead sea turtles and implications for conservation. Ecology, 6, 1412–1423. ? 2010 Blackwell Publishing Ltd 114 TECHNICAL ADVANCES rDon RH, Cox PT, Wainwright BJ (1991) Touchdown PCR to prevent spur-ious priming during gene amplification. Nucleic Acids, 6, 968–970
Population Structure of Loggerhead Sea Turtles (Caretta caretta) Nesting in the Southeastern United States Inferred from Mitochon-drial DNA Sequences and Microsatellite Loci Tetranculeotide micro-satellites from the loggerhead sea turtle (Caretta caretta)
- Smithsonian
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Smithsonian, Washington, D.C. Shamblin BM (2007) Population Structure of Loggerhead Sea Turtles (Caretta caretta) Nesting in the Southeastern United States Inferred from Mitochon-drial DNA Sequences and Microsatellite Loci. Masters thesis. University of Georgia, Athens, Georgia. Shamblin BM, Faircloth BC, Dodd M et al. (2007) Tetranculeotide micro-satellites from the loggerhead sea turtle (Caretta caretta). Molecular Ecol-ogy Notes, 7, 784–787.
Touchdown PCR to prevent spur-ious priming during gene amplification
- Don Rh Cox
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- Wainwright
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Don RH, Cox PT, Wainwright BJ (1991) Touchdown PCR to prevent spur-ious priming during gene amplification. Nucleic Acids, 6, 968–970.
Simple biopsy technique for sampling skin for DNA analysis of sea turtles. NOAA Technical Memorandum NMFS-SEFSC-387. National Oceanographic and Atmospheric Service National Marine Fisheries Service
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Dutton PH, Balazs GH (1995) Simple biopsy technique for sampling skin for DNA analysis of sea turtles. NOAA Technical Memorandum NMFS-SEFSC-387. National Oceanographic and Atmospheric Service National Marine Fisheries Service, Miami, Florida.
Population Structure of Loggerhead Sea Turtles (Caretta caretta) Nesting in the Southeastern United States Inferred from Mitochondrial DNA Sequences and Microsatellite Loci
- Bm Shamblin
Shamblin BM (2007) Population Structure of Loggerhead Sea Turtles (Caretta
caretta) Nesting in the Southeastern United States Inferred from Mitochondrial DNA Sequences and Microsatellite Loci. Masters thesis. University of
Georgia, Athens, Georgia.
Results from the long-term monitoring of nesting loggerhead sea turtles (Caretta caretta) on Wassaw Island, Georgia: 1973-2000. NOAA Technical Memorandum NMFS-SEFSC-446. National Oceanographic and Atmospheric Service National Marine Fisheries Service
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Williams KL, Frick MG (2001) Results from the long-term monitoring of
nesting loggerhead sea turtles (Caretta caretta) on Wassaw Island, Georgia: 1973-2000. NOAA Technical Memorandum NMFS-SEFSC-446.
National Oceanographic and Atmospheric Service National Marine
Fisheries Service, Miami, Florida.
An Assessment of the Loggerhead Sea Turtle Population in the Western North Atlantic Ocean
- Turtle Expert Working Group
Results from the long-term monitoring of nesting loggerhead sea turtles (Caretta caretta) on Wassaw Island Georgia: 1973-2000.NOAA Technical Memorandum NMFS-SEFSC-446.National Oceanographic and Atmospheric Service National Marine Fisheries Service Miami Florida
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