Melanie Culver

Long-term survival despite low genetic diversity in the critically endangered Madagascar fish-eagle

The critically endangered Madagascar fish‐eagle (Haliaeetus vociferoides) is considered to be one of the rarest birds of prey globally and at significant risk of extinction. In the most recent census, only 222 adult individuals were recorded with an estimated total breeding population of no more than 100–120 pairs. Here, levels of Madagascar fish‐eagle population genetic diversity based on 47 microsatellite loci were compared with its sister species, the African fish‐eagle (Haliaeetus vocifer), and 16 of these loci were also characterized in the white‐tailed eagle (Haliaeetus albicilla) and the bald eagle (Haliaeetus leucocephalus). Overall, extremely low genetic diversity was observed in the Madagascar fish‐eagle compared to other surveyed Haliaeetus species. Determining whether this low diversity is the result of a recent bottleneck or a more historic event has important implications for their conservation. Using a Bayesian coalescent‐based method, we show that Madagascar fish‐eagles have maintained a small effective population size for hundreds to thousands of years and that its low level of neutral genetic diversity is not the result of a recent bottleneck. Therefore, efforts made to prevent Madagascar fish‐eagle extinction should place high priority on maintenance of habitat requirements and reducing direct and indirect human persecution. Given the current rate of deforestation in Madagascar, we further recommend that the population be expanded to occupy a larger geographical distribution. This will help the population persist when exposed to stochastic factors (e.g. climate and disease) that may threaten a species consisting of only 200 adult individuals while inhabiting a rapidly changing landscape.

Novel primers for complete mitochondrial cytochrome b gene sequencing in mammals

Sequence‐based species identification relies on the extent and integrity of sequence data available in online databases such as GenBank. When identifying species from a sample of unknown origin, partial DNA sequences obtained from the sample are aligned against existing sequences in databases. When the sequence from the matching species is not present in the database, high‐scoring alignments with closely related sequences might produce unreliable results on species identity. For species identification in mammals, the cytochrome b (cyt b) gene has been identified to be highly informative; thus, large amounts of reference sequence data from the cyt b gene are much needed. To enhance availability of cyt b gene sequence data on a large number of mammalian species in GenBank and other such publicly accessible online databases, we identified a primer pair for complete cyt b gene sequencing in mammals. Using this primer pair, we successfully PCR amplified and sequenced the complete cyt b gene from 40 of 44 mammalian species representing 10 orders of mammals. We submitted 40 complete, correctly annotated, cyt b protein coding sequences to GenBank. To our knowledge, this is the first single primer pair to amplify the complete cyt b gene in a broad range of mammalian species. This primer pair can be used for the addition of new cyt b gene sequences and to enhance data available on species represented in GenBank. The availability of novel and complete gene sequences as high‐quality reference data can improve the reliability of sequence‐based species identification.

Concurrent speciation in the eastern woodland salamanders (Genus Plethodon): DNA sequences of the complete albumin nuclear and partial mitochondrial 12s genes

Salamanders of the North American plethodontid genus Plethodon are important model organisms in a variety of studies that depend on a phylogenetic framework (e.g., chemical communication, ecological competition, life histories, hybridization, and speciation), and consequently their systematics has been intensively investigated over several decades. Nevertheless, we lack a synthesis of relationships among the species. In the analyses reported here we use new DNA sequence data from the complete nuclear albumin gene (1818 bp) and the 12s mitochondrial gene (355 bp), as well as published data for four other genes (Wiens et al., 2006), up to a total of 6989 bp, to infer relationships. We relate these results to past systematic work based on morphology, allozymes, and DNA sequences. Although basal relationships show a strong consensus across studies, many terminal relationships remain in flux despite substantial sequencing and other molecular and morphological studies. This systematic instability appears to be a consequence of contemporaneous bursts of speciation in the late Miocene and Pliocene, yielding many closely related extant species in each of the four eastern species groups. Therefore we conclude that many relationships are likely to remain poorly resolved in the face of additional sequencing efforts. On the other hand, the current classification of the 45 eastern species into four species groups is supported. The Plethodon cinereus group (10 species) is the sister group to the clade comprising the other three groups, but these latter groups (Plethodon glutinosus [28 species], Plethodon welleri [5 species], and Plethodon wehrlei [2 species]) probably diverged from each other at approximately the same time.

Development of nine new microsatellite loci for the American beaver, Castor canadensis (Rodentia: Castoridae), and cross-species amplification in the European beaver, Castor fiber.

We developed nine new nuclear dinucleotide microsatellite loci for Castor canadensis. All loci were polymorphic, except for one. The number of alleles ranged from two to four and from five to 12 in populations from Arizona and Wisconsin, respectively. Average heterozygosity ranged from 0.13 to 0.86 per locus. Since cross‐species amplification in Castor fiber was successful only in four loci, we tested also nine recently published C. canadensis loci in the Eurasian species. Eight of the published loci amplified; however, three were monomorphic. The number of alleles was lower in C. fiber than in C. canadensis at all loci tested.

Eleven new microsatellite loci for the tiger rattlesnake (Crotalus tigris)

Eleven microsatellite loci were isolated from an enriched genomic library from the tiger rattlesnake Crotalus tigris. Average observed heterozygosities in two populations were 0.456 and 0.427, respectively, and mean number of alleles were 7.54 (range 2–14) and 4.72 (range 2–13) respectively. No evidence of linkage disequilibrium was found across pairs of loci. The markers will be used in a long‐term study examining the potential effects of urbanization on population dynamics and connectivity of this species in the mountain ranges surrounding Tucson, Arizona.

Genetic Analysis of Scats Reveals Minimum Number and Sex of Recently Documented Mountain Lions

Abstract Recent records of mountain lions Puma concolor and concurrent declines in desert bighorn sheep Ovis canadensis mexicana on Kofa National Wildlife Refuge in Arizona, United States, have prompted investigations to estimate the number of mountain lions occurring there. We performed noninvasive genetic analyses and identified species, individuals, and sex from scat samples collected from the Kofa and Castle Dome Mountains. From 105 scats collected, we identified a minimum of 11 individual mountain lions. These individuals consisted of six males, two females and three of unknown sex. Three of the 11 mountain lions were identified multiple times over the study period. These estimates supplement previously recorded information on mountain lions in an area where they were historically considered only transient. We demonstrate that noninvasive genetic techniques, especially when used in conjunction with camera-trap and radiocollaring methods, can provide additional and reliable information to wildlife man...

A new panel of SNP markers for the individual identification of North American pumas

Abstract Pumas Puma concolor are one of the most studied terrestrial carnivores because of their widespread distribution, substantial ecological impacts, and conflicts with humans. Over the past decade, managing pumas has involved extensive efforts including the use of genetic methods. Microsatellites have been the most commonly used genetic markers; however, technical artifacts and little overlap of frequently used loci render large-scale comparison of puma genetic data across studies challenging. Therefore, a panel of genetic markers that can produce consistent genotypes across studies without the need for extensive calibrations is essential for range-wide genetic management of puma populations. Here, we describe the development of PumaPlex, a high-throughput assay to genotype 25 single nucleotide polymorphisms in pumas. We validated PumaPlex in 748 North American pumas Puma concolor couguar, and demonstrated its ability to generate reproducible genotypes and accurately identify individuals. Furthermore...

Diagnostic Genetic Marker That Differentiates Eastern Fox Squirrels From Eastern Gray Squirrels

Abstract The Delmarva fox squirrel (Sciurus niger cinereus) has been listed as endangered by the United States Department of Interior since 1967. A high-priority task for the recovery of this taxon is to determine its current geographic distribution. Toward this end, we have identified a microsatellite locus that unambiguously differentiates Delmarva fox squirrels from eastern gray squirrels (S. carolinensis), which frequently co-occur with Delmarva fox squirrels. Analysis of this marker in noninvasively collected hair samples will allow unequivocal identification of localities occupied by Delmarva fox squirrels with a minimum investment of funds, time, and effort because handling individuals will be unnecessary. This protocol will expedite site review in connection with the Endangered Species Act consultation process.

Characterization of microsatellite loci from two-spotted octopus Octopus bimaculatus Verrill 1883 from pyrosequencing reads

We characterized 22 novel microsatellite loci in the two-spotted octopus Octopus bimaculatus using 454 pyrosequencing reads. All loci were polymorphic and will be used in studies of marine connectivity aimed at increasing sustainability of the resource. The mean number alleles per locus was 13.09 (range 7–19) and observed heterozygosities ranged from 0.50 to 1.00. Four loci pairs were linked and three deviated from Hardy–Weinberg equilibrium. Eighteen and 12 loci were polymorphic in Octopus bimaculoides and Octopus hubbsorum, respectively.

Isolation of microsatellite loci from the lesser long-nosed bat ( Leptonycteris yerbabuenae )

Leptonycteris yerbabuenae is a nectarivore (subfamily: Glossophaginae, family: Phyllostomidae), is found from southern Arizona/southwestern New Mexico to southern Mexico including the Baja California peninsula (Ceballos et al. 1997; Cockrum 1991). Leptonycteris yerbabuenae is listed as endangered in the United States (Shull 1988) and threatened in Mexico (SEMARNAT 2002). They migrate up to 1,800 km between wintering and breeding grounds (Fleming and Eby 2001). Females mate in the winter in southern Mexico, and migrate to maternity roosts in northern Mexico/southern Arizona to give birth in late spring-early summer (Ceballos et al. 1997; Cockrum 1991). Wilkinson and Fleming (1996) have suggested two separate migration corridors where bats occupy southeastern Arizona and southwestern Arizona arrive and leave through separate corridors. We isolated 12 microsatellites loci to estimate gene flow between southwestern and southeastern Arizona roosts, as well as Sonora, Baja California, and Jalisco, Mexico. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen). Approximately 5 lg of genomic DNA was digested with RsaI (NEB) and fragments were ligated to double-stranded SuperSNX-24 linkers to construct an enriched genomic DNA library using a modified version of Glenn and Schable (2005). Linker-ligated fragments ranging from 300–1400 bp were recovered using the polymerase chain reaction (PCR) with a SuperSNX-24 forward primer and Platinum high-fidelity Taq DNA polymerase (Invitrogen), and were hybridized to 50-biotinylated microsatellite oligonucleotide probes (GT)15, (CT)15, (GATA)10 and (GACA)8. Hybridized fragments were captured on streptavidin-coated paramagnetic beads (Dynal) and then recovered by PCR. These fragments were ligated into the vector PCR4-TOPO (Invitrogen), and transformed into TOP10 chemically competent E. coli cells (Invitrogen) following the manufacturer’s protocol. Clones were amplified by PCR with M13 primers and visualized in 2% agarose gels to select clones for sequencing that amplified. Ninety-six clones were directly sequenced in both directions with the M13 primers on an Applied Biosystems 3730XL DNA Analyzer using the BigDye Terminator Cycle Sequencing Kit. Forty-six (47.9%) of the clones had recognizable microsatellite sequences and enough flanking regions to create primer pairs. Using PRIMER3 software (Rozen and Skaletsky 2000) we designed primer pairs. All forward primers were individually fluorescentlylabeled. The reverse primer of Leye3a was designed with a ‘‘pig-tail’’ at the 5’ end to reduce variability in adenylation of amplification products (Brownstein et al. 1996). Polymerase chain reactions were performed in 10 ll volumes containing *40 ng genomic DNA, 19 PCR buffer, 0.2 mM each dNTP, 0.4 lM of reverse primer, 0.4 lM fluorescently-labeled forward primer, 0.4 U taq DNA polymerase (Invitrogen), 0.75% BSA, and 1.5 mM MgCl2 for Leye3a, Leye4, LeyeGT2, LeyeL67; 2.5 mM MgCl2 for LeyeCT2, Leye23, Leye11; 3.0 mM MgCl2 for Leye27, Leye17, Leye7, Leye21, LeyeL71. All loci were amplified with PCR that included an initial denaturation at 94 C for 5 min, 40 cycles at 94 C for 30 s, annealing for 30 s at 55 C for Leye3a; 53 C for Leye11 and Leye21; 58 C for Leye17 and Leye23; 60 C for Leye27, LeyeCT2 and LeyeGT2; 61 C for LeyeL67 and LeyeL71; 67 C for Leye4 and Leye7; followed by 72 C for 30 s and a final extension at 72 C for J. Ramirez (&) A. Munguia-Vega M. Culver Conservation Genetics Laboratory, Arizona Cooperative Fish and Wildlife Research Unit USGS, School of Natural Resources and the Environment, The University of Arizona, BioSciences East 325, Tucson, AZ 85721, USA e-mail: judithrm@email.arizona.edu