Jesús E. Maldonado

Tidal Marshes: A Global Perspective on the Evolution and Conservation of Their Terrestrial Vertebrates

Abstract Globally, tidal marshes are found in small pockets or narrow bands totaling only approximately 45,000 square kilometers. The combination of salinity, low floristic and structural complexity, and regular tidal inundation, as well as unpredictable catastrophic flooding, provides a unique selective environment that shapes local adaptations, including those that are morphological, physiological, demographic, and behavioral. Although tidal marshes support a low diversity of nonaquatic vertebrate species, a high proportion of these inhabitants, at least along North American coastlines, are restricted to or have subspecies restricted to tidal marshes. Tidal marshes and their endemic fauna face broad threats from a variety of human-caused environmental changes. Future research should focus on global inventories, intercontinental comparative work, and investigation to determine why almost all presently described endemic taxa appear to be found in North America.

Detection and accuracy rates of dogs trained to find scats of San Joaquin kit foxes (Vulpes macrotis mutica)

Specially trained detection dogs have been used to locate faeces (scats) for faecal analyses but their effectiveness has not been quantified. We evaluated detection and accuracy rates of dogs trained to find scats of endangered San Joaquin kit foxes (Vulpes macrotis mutica). Four dogs found from 0.43 to 5.37 presumptive kit fox scats per km of transect searched in two field sites where kit foxes and coyotes (Canis latrans) but not non-native red foxes (V. vulpes) were present. The unusually low detection rate (0.43 scats per km) by one dog (probably due to excessive panting in hot weather) was still similar to the average scat detection rate of two experienced humans. DNA tests of 1298 scats showed that all dogs were 100% accurate at distinguishing kit fox scats under our field conditions. Because red foxes are sympatric with kit foxes in some areas, we also conducted controlled discrimination experiments to see if trained dogs could distinguish between scats from kit and red foxes. Four dogs were 100% accurate at choosing a kit fox scat when red fox scats were present (n = 64 trials), but were less accurate at ignoring red fox scats in trials where a kit fox scat was absent.

Conservation genetics of the endangered Pampas deer (Ozotoceros bezoarticus)

The Pampas deer (Ozotoceros bezoarticus L. 1758) is the most endangered neotropical cervid, and in the past occupied a wide range of open habitats including grassland, pampas, savanna, and cerrado (Brazil) from 5° to 41° S. To better understand the effect of habitat fragmentation on gene flow and genetic variation, and to uncover genetic units for conservation, we examined DNA sequences from the mitochondrial control region of 54 individuals from six localities distributed throughout the present geographical range of the Pampas deer. Our results suggest that the control region of the Pampas deer is one of the most polymorphic of any mammal. This remarkably high variability probably reflects large historic population sizes of millions of individuals in contrast to numbers of fewer than 80 000 today. Gene flow between populations is generally close to one migrant per generation and, with the exception of two populations from Argentina, all populations are significantly differentiated. The degree of gene flow was correlated with geographical distance between populations, a result consistent with limited dispersal being the primary determinant of genetic differentiation between populations. The molecular genetic results provide a mandate for habitat restoration and reintroduction of Pampas deer so that levels of genetic variation can be preserved and historic patterns of abundance can be reconstructed. However, the source of individuals for reintroduction generally should be from populations geographically closest to those now in danger of extinction.

Behavioural inbreeding avoidance in wild African elephants

The costs of inbreeding depression, as well as the opportunity costs of inbreeding avoidance, determine whether and which mechanisms of inbreeding avoidance evolve. In African elephants, sex‐biased dispersal does not lead to the complete separation of male and female relatives, and so individuals may experience selection to recognize kin and avoid inbreeding. However, because estrous females are rare and male–male competition for mates is intense, the opportunity costs of inbreeding avoidance may be high, particularly for males. Here we combine 28 years of behavioural and demographic data on wild elephants with genotypes from 545 adult females, adult males, and calves in Amboseli National Park, Kenya, to test the hypothesis that elephants engage in sexual behaviour and reproduction with relatives less often than expected by chance. We found support for this hypothesis: males engaged in proportionally fewer sexual behaviours and sired proportionally fewer offspring with females that were natal family members or close genetic relatives (both maternal and paternal) than they did with nonkin. We discuss the relevance of these results for understanding the evolution of inbreeding avoidance and for elephant conservation.

The surprising evolutionary history of South American deer

To clarify the systematic relationships and evolutionary history of South American deer, we conducted a comprehensive phylogenetic analysis using representative species of all of the genera of Neotropical deer. Our results revealed high levels of molecular and cytogenetic divergence between groups of morphologically similar species of brockets (Mazama), and suggest a polyphyletic origin. At least eight ancestral forms of deer invaded South America during the late Pliocene (2.5-3 MYA), and members of the red brockets had an independent early explosive diversification soon after their ancestor arrived there, giving rise to a number of morphologically cryptic species.

Male dominance, paternity, and relatedness in the Jamaican fruit‐eating bat (Artibeus jamaicensis)

We analysed variation at 14 nuclear microsatellite loci to assess the genetic structure, relatedness, and paternity of polygynous Jamaican fruit‐eating bats. A total of 84 adults captured in two caves exhibited little genetic differentiation between caves (FST = 0.008). Average relatedness among adult females in 10 harem groups was very low (R = 0.014 ± 0.011), providing no evidence of harem structure. Dominant and subordinate males shared paternity in large groups, while dominant and satellite males shared paternity in smaller groups. However, our results suggest that male rank influences paternity. Dominant males fathered 69% of 40 offspring, followed by satellite (22%) and subordinate males (9%). Overall adult male bats are not closely related, however, in large harem groups we found that subordinate and dominant males exhibited relatedness values consistent with a father‐offspring relationship. Because dominant and subordinate males also sired all the pups in large groups, we propose that their association provides inclusive fitness to them.

Tripartite genetic subdivisions in the ornate shrew (Sorex ornatus)

We examined cytochrome b sequence variation in 251 ornate shrews (Sorex ornatus) from 20 localities distributed throughout their geographical range. Additionally, vagrant (S. vagrans) and montane (S. monticolus) shrews from four localities were used as outgroups. We found 24 haplotypes in ornate shrews from California (USA) and Baja California (Mexico) that differed by 1–31 substitutions in 392 bp of mitochondrial DNA (mtDNA) sequence. In a subset of individuals, we sequenced 699 bp of cytochrome b to better resolve the phylogeographic relationships of populations. The ornate shrew is phylogeographically structured into three haplotype clades representing southern, central and northern localities. Analysis of allozyme variation reveals a similar pattern of variation. Several other small California vertebrates have a similar tripartite pattern of genetic subdivision. We suggest that topographic barriers and expansion and contraction of wetland habitats in the central valley during Pleistocene glacial cycles account for these patterns of genetic variation. Remarkably, the northern ornate shrew clade is phylogenetically clustered with another species of shrew suggesting that it may be a unique lowland form of the vagrant shrew that evolved in parallel to their southern California counterparts.

Ancient wolf lineages in India

All previously obtained wolf (Canis lupus) and dog (Canis familiaris) mitochondrial (mt) DNA sequences fall within an intertwined and shallow clade (the ‘wolf–dog’ clade). We sequenced mtDNA of recent and historical samples from 45 wolves from throughout lowland peninsular India and 23 wolves from the Himalayas and Tibetan Plateau and compared these sequences with all available wolf and dog sequences. All 45 lowland Indian wolves have one of four closely related haplotypes that form a well–supported, divergent sister lineage to the wolf–dog clade. This unique lineage may have been independent for more than 400 000 years. Although seven Himalayan wolves from western and central Kashmir fall within the widespread wolf–dog clade, one from Ladakh in eastern Kashmir, nine from Himachal Pradesh, four from Nepal and two from Tibet form a very different basal clade. This lineage contains five related haplotypes that probably diverged from other canids more than 800 000 years ago, but we find no evidence of current barriers to admixture. Thus, the Indian subcontinent has three divergent, ancient and apparently parapatric mtDNA lineages within the morphologically delineated wolf. No haplotypes of either novel lineage are found within a sample of 37 Indian (or other) dogs. Thus, we find no evidence that these two taxa played a part in the domestication of canids.

A technique for sampling ancient DNA that minimizes damage to museum specimens.

Because of the utility of ancient DNA to conservation genetics (Baker 1994), the number of requests to collect tissue from museum specimens has increased. A drawback of consumptive sampling is that it requires removal and destruction of part of the specimen. Epithelium, hair, dried skeletal muscle, and bone have been used as sources of ancient DNA taken from skulls, postcranial material or study skins (Herrmann and Hummel 1994). Although many museums permit consumptive sampling of collections, the request is generally contrary to the goal of collection managers, which is long-term care and maintenance of specimens. We propose a collection method that attempts to satisfy goals of both loan requestor and collection manager, the use of maxilloturbinal bone material. Maxilloturbinates are thin bones attached anteriorly to ridges inside the nasal cavity (Hillenius 1992). In this study we compare success rates in amplification of genomic DNA taken from epithelium and maxilloturbinates ranging in age from 17 to 111 years old. We sampled 5–20 mg epithelial or 10–20 mg maxilloturbinal bone tissue of black-footed ferrets (Mustela nigripes) from six collections. Curatorial staff collected epithelial tissue from study skins. We instructed staff to sterilize collection tools before and in between handling specimens using 50% bleach solution and to deposit samples in sterile containers. Epithelial samples included material from the ventral incision of study skins and clippings from the inner ear. All maxilloturbinal samples were collected by one author (SMW). Forceps sterilized with 50% bleach were inserted into the nasal cavity of the skull, and bone material was carefully dislodged. No force was applied to retrieve 20 mg of material. Fragments were collected onto sterile aluminum foil and poured into a sterile 2 ml screw cap tube. We extracted and amplified DNA from epithelial and turbinal samples in an isolated ancient DNA laboratory. Epithelium was cut into small pieces; turbinates were already fragmentary. Samples were incubated at 58 ◦ C for 24 hrs in lysis buffer (10 mM Tris, 2 mM EDTA, 10 mM NaCl, 1% SDS, 10 mg/ml DTT and 10 mg/ml protienase K), extracted with phenol twice and chloroform once. Three ml of deionized water were added to the final supernate and cleaned with a Centricon 30 dialysis filter (Amicon). Deionized sterile water was added so that the final volume was 200 µl. The extracted DNA solution was

Assessing reliability of microsatellite genotypes from kit fox faecal samples using genetic and GIS analyses

Noninvasive faecal DNA sampling has the potential to provide a wealth of information necessary for monitoring and managing endangered species while eliminating the need to capture, handle or observe rare individuals. However, scoring problems, and subsequent genotyping errors, associated with this monitoring method remain a great concern as they can lead to misidentification of individuals and biased estimates. We examined a kit fox scat data set (353 scats; 80 genotypes) for genotyping errors using both genetic and GIS analyses, and evaluated the feasibility of combining both approaches to assess reliability of the faecal DNA results. We further checked the appropriateness of using faecal genotypes to study kit fox populations by describing information about foxes that we could deduce from the ‘acceptable’ scat genotypes, and comparing it to information gathered with traditional field techniques. Overall, genetic tests indicated that our data set had a low rate of genotyping error. Furthermore, examination of distributions of scat locations confirmed our data set was relatively error free. We found that analysing information on sex primer consistency and scat locations provided a useful assessment of scat genotype error, and greatly limited the amount of additional laboratory work that was needed to identify potentially ‘false’ scores. ‘Acceptable’ scat genotypes revealed information on sex ratio, relatedness, fox movement patterns, latrine use, and size of home range. Results from genetic and field data were consistent, supporting the conclusion that our data set had a very low rate of genotyping error and that this noninvasive method is a reliable approach for monitoring kit foxes.