An unrecorded specimen of the Javan rhinoceros (Rhinoceros sondaicus)

An hitherto unpuhlicized specimen of the Javan (Sondaic) rhinoceros in the Manchester Museum is briefly described.     

Polymorphic microsatellite loci in the endangered Indian rhinoceros,Rhinoceros unicornis

Eleven novel polymorphic microsatellite loci are presented for the highly endangered Indian (or greater one horned) rhinoceros Rhinoceros unicornis (Mammalia: Rhinocerotidae). These will be used to analyse the genetic variability within and between the two remaining large populations of the Indian rhinoceros and to manage captive breeding.     

Genetic variation and population structure in remnant populations of black rhinoceros, Diceros bicornis, in Africa

Black rhinoceros (Diceros bicornis) are one of the most endangered mammal species in Africa, with a population decline of more than 96% by the end of the last century. Habitat destruction and encroachment has resulted in fragmentation of the remaining populations. To assist in conservation management, baseline information is provided here on relative genetic diversity and population differentiation among the four remaining recognized subspecies. Using microsatellite data from nine loci and 121 black rhinoceros individuals, and comparing the results with those of other African species affected in similar ways, Diceros bicornis michaeli retained the most genetic diversity (heterozygosity 0.675) compared with Diceros bicornis minor (0.459) and Diceros bicornis bicornis (0.505), suggesting that the duration of the known bottlenecks in these populations has only had a limited impact on diversity. Comparable and moderate degrees of population differentiation were found between D. b. minor, D. b. bicornis and D. b. michaeli. Results from the single sample available of the most endangered subspecies, Diceros bicornis longipes, showed the least diversity of all individuals examined. This information should assist conservation management decisions, especially those affecting population viability assessments and selection of individuals for translocations, and will also facilitate subspecies identification for ex situ individuals of uncertain origin.     

Semen cryopreservation in the Indian rhinoceros (Rhinoceros unicornis)

The objective was to identify an extender and cryoprotectant combination for Indian rhinoceros (Rhinoceros unicornis) sperm that yielded high post-thaw sperm quality. Male Indian rhinoceroses (n = 6; 7.5-34 yr old) were anesthetized and subjected to a regimented electroejaculation procedure (75-100 mAmps; 4-10 volts; 7-150 stimuli; total of 10 electroejaculation procedures). High quality semen fractions from each ejaculate were divided into four aliquots and a 2 x 2 factorial design used to compare the effect of two sperm extenders (standard equine [EQ] and skim milk-egg-yolk-sugar [SMEY]), and two cryoprotectants (glycerol and dimethylsulfoxide [DMSO]). Cyropreserved samples were thawed and assessed for motility, viability and acrosome integrity over time. Electroejaculate fractions processed for cryopreservation had high sperm concentration (516 × 10⁶/mL) and motility (79%). Post-thaw sperm characteristics were higher (P < 0.05) when semen was cryopreserved in EQ versus SMEY. Post-thaw motility of sperm cyropreserved in EQ averaged 50-55% compared to 22-37% in SMEY, with no significant differences in sperm characteristics of samples cyropreserved in glycerol and DMSO. In conclusion, sperm collected from Indian rhinoceroses via electroejaculation were cryopreserved using EQ extender with either glycerol or DMSO; post-thaw quality was adequate for use in assisted reproductive procedures.     

Characterization of microsatellite loci in the black rhinoceros (Diceros bicornis) and white rhinoceros (Ceratotherium simum): their use for cross-species amplification and differentiation between the two species

As the population sizes of the black and white rhinoceroses continues to decline, more efforts are needed in multiple areas to help with the conservation efforts. One area being explored is the use of genetic diversity information to aid conservation decisions. In this study, we designed 21 microsatellite primers for white and black rhinoceroses, 16 and 17 of which amplified bands in the white and black rhinoceros, respectively. Out of these primers all 16 were polymorphic in the white rhinoceros and 12 of the 17 were polymorphic in the black rhinoceros. The mean number of alleles was 3.31 and 2.12, the expected heterozygosities were 0.420 and 0.372, and the observed heterozygosities were 0.436 and 0.322 for the white and black rhinoceroses, respectively. Seven of the primers produced different allele sizes and variations that distinguished between black and white rhinoceroses. Further genetic analyses with larger wild population sample sizes and markers are recommended to obtain a better understanding of the genetic structure of the black and white rhinoceros populations in order to be useful in the conservation efforts of these critically endangered species. A. Kilbourn—In memoriam.     

Genetic consequences of Pleistocene fragmentation: Isolation, drift, and loss of diversity in rock iguanas (Cyclura)

Pleistocene fragmentation of the Great BahamaBank resulted in one large and several smallpopulations of rock iguanas (Cycluracychlura). We explore patterns of geneticvariation within and among these islandpopulations using mitochondrial sequence data(partial ND4 to tRNA^Leu) in combinationwith eight polymorphic microsatellite loci (2to 10 alleles). Genetic data support twophylogeographically distinct groups, AndrosIsland and the Exuma cays. This resultconflicts with current subspecific taxonomy inwhich three subspecies are described. Analysesof allelic data indicate that most islandpopulations are currently demographicallyindependent. Pairwise Fst values between eightisland populations range from 0.18 to 0.63, and6 of 135 individuals are misassigned in anassignment test. Population-genetic diversityis characterized using standard measures suchas number of alleles and heterozygosity (H) inaddition to a normalized Shannon-Weaver indexof diversity (D). We find genetic diversity inthe Andros Island population comparable to thatin other non-piscine animals (avg. # ofalleles = 5, avg. H = 0.56, avg. D = 0.66) while inthe Exuma cays populations these measures aremuch lower (avg. # of alleles = 2.75–1.625, avg.H = 0.43–0.17, avg. D = 0.45–0.18). These dataare used to discuss conservation managementstrategies, including prioritization andtranslocation.     

Genetic diversity,differentiation and conservation in <Emphasis Type="Italic">Araucaria bidwillii</Emphasis>(Araucariaceae), Australia's Bunya pine

Habitat fragmentation has resulted in many species becoming geographically restricted, as dispersal among subsequent isolates becomes compromised. This study investigated the effects of historical fragmentation on the genetic diversity and differentiation of Australia's Bunya Pine (Araucaria bidwillii) using random amplified DNA (RAPD) markers. High diversity characterises all Bunya populations sampled, regardless of population size or degree of isolation, which may be either due to similar effective population size among populations, or the lagging effects of the detection of contemporary genetic signal in long-lived conifer species. Large genetic differentiation characterises the northern and southern populations, although all sampled populations are significantly differentiated from each other. The northern population, at Mt Lewis, is responsible for the majority of variation detected among populations, and as such represents a significant genetic reservoir. The conservation of the genotypes of the Mt Lewis population may be imperative for the future of the species, given predicted models of accelerated climate change over the coming decades.     

Optimization of novel polymorphic microsatellites in the endangered Sumatran rhinoceros (Dicerorhinus sumatrensis)

Loss of habitat and poaching have led to a drastic reduction in numbers of the Sumatran rhinoceros (Dicerorhinus sumatrensis). To aid in the conservation management of this species, we isolated and optimized 10 polymorphic Sumatran rhinoceros microsatellite loci. A survey of six individuals yielded a mean number of alleles of 3.7, mean expected heterozygosity of 0.551 and probability of identity of 3.46 × 10^?8. Although this estimate is similar to estimates of microsatellite variability in the Black, Indian and White rhinoceroses, such a conclusion is premature as locus purity, sample size and number of loci surveyed vary significantly among studies.     

Energy and mineral nutrition and water intake in the captive Indian rhinoceros (Rhinoceros unicornis)

In the captive Indian rhinoceros (Rhinoceros unicornis), two disease complexes with a high incidence—chronic foot problems and uterine leiomyomas—may be linked to excess body weight (BW). In this study, intake and digestion trials were conducted (by means of 7‐day weigh‐backs, and 5‐day total fecal collections, respectively) with 11 Indian rhinoceroses at four zoological institutions in Europe and the United States to quantify energy and mineral nutrition on conventional or roughage‐only diets. Diets comprising a variety of forages (grass hay only, a combination of grass hay and grass silage, straw, or a mixture of grass and legume hay) were offered as the roughage source, along with various concentrates, produce, and supplements. Water intake was quantified, and urine samples were obtained opportunistically. The animals consumed 0.5–1.1% of their BW in dry matter (DM) daily, with calculated digestible energy (DE, in megajoules MJ) values ranging from 0.27 to 0.99 MJ DE/kg BW^0.75/day compared to an estimated requirement of 0.49–0.66 MJ DE/kg BW^0.75/day. Seven of 11 rhinos (64%) fed restricted levels of concentrate plus forage consumed DE in excess of this estimate. Even on roughage‐only diets, some individuals consumed energy well above the presumed metabolic requirements. Hence, restriction of both concentrates and roughage may be important for weight management in this species. Water intake ranged from 30 to 49 mL/kg BW daily (3.4–5.2 L/kg ingested DM), similar to values that have been reported for domestic equids. Excretion amounts and patterns also resembled those found in horses. Endogenous fecal losses measured for Ca, P, Cu, Fe, and Zn indicate that the maintenance requirements of these minerals should be met in Indian rhinoceroses by diets that meet recommendations for domestic horses. It is particularly important to evaluate dietary adequacy in mineral nutrition in this species in concert with the need for restricted energy intake, especially with regard to the hypothetical involvement of a low Zn supply in chronic foot problems. Zoo Biol 24:1–14, 2005. © 2005 Wiley‐Liss, Inc.     

Genetic diversity in the rare orchid Gymnadenia odoratissima and a comparison with the more common congener, G. conopsea

Patterns and levels of genetic diversity mayhave significant influence on the long termpersistence of local populations and revealingsuch information is important in protectingrare species. In this study we investigated thegenetic pattern in five microsatellite lociwithin five Swedish populations of the rareorchid species Gymnadenia odoratissima. Thegeographic distribution of G. odoratissima isrestricted to Europe and in Scandinavia it isonly found in three provinces in southernSweden; Östergötland,Västergötland and on the island ofGotland.Compared with the more widespread congener G.conopsea our results indicate lower levels ofgenetic variation within and higher degrees ofgenetic differentiation among populations ofG. odoratissima (H_EL = 0.6–0.8 in G. conopseaand 0.3–0.7 in G. odoratissima; F_ST over allpopulations = 0.06 in G. conopsea and 0.19 inG. odoratissima). Also, we found a cleardistinction among mainland and islandpopulations of G. odoratissima wherepopulations on the island of Gotland seem toexhibit higher levels of gene flow andintragenetic variation, probably as a result ofa larger number of existing populations.Future conservation of this species shouldfocus on facilitation on colonisation events,especially on the mainland, and preservation ofthe genetically more variable Gotlandpopulations.     

Genetic Variation in Remnant Populations of the Woolly Spider Monkey (Brachyteles arachnoides)

The muriqui or woolly spider monkey (Brachyteles arachnoids) is an endangered primate endemic to the Atlantic Forest of Brazil, <5% of which remains. The known muriqui population consists of <700 individuals separated into approximately 15 geographically isolated forest fragments. I present data on the distribution of genetic variation within and between two such remnant populations (FE and FBR) and summarize the implications of these results for long-range management of species genetic diversity. Eleven of 32 allozyme loci were polymorphic, representing an overall level of polymorphism of 34.4% and a mean heterozygosity per locus of 11%. Both values are among the highest reported for New World monkeys. Genetic differentiation between the two localities is highly significant (F_ST = 0.413, p < 0.001). Genetic distance between them is an order of magnitude greater than that between other populations of platyrrhine subspecies, but this could be an artifact of the small sample size from FBR. High levels of genetic diversity apparently characteristic of this species persist because (1) fragmentation and size reduction of muriqui populations has occurred very rapidly relative to the muriqui life span—although both polymorphism and heterozygosity were lost between generations in the largest population, the high genetic diversity present in the parent population was still in evidence; and (2) genetic diversity before population fragmentation by human activity was not distributed uniformly throughout the species' historic distribution. Thus, remnant muriqui populations are important genetic reservoirs of alleles that are unique or rare in the species gene pool as a whole. These results emphasize the need for the integration of conservation management efforts throughout the species range.     

A potential link between lateral semicircular canal orientation,head posture,and dietary habits in extant rhinos (Perissodactyla,Rhinocerotidae)

Extant rhinoceroses share the characteristic nasal horn, although the number and size of horns varies among the five species. Although all species are herbivores, their dietary preferences, occipital shapes, and common head postures vary. Traditionally, to predict the “usual” head posture (the most used head posture of animals during normal unstressed activities, i.e., standing) of rhinos, the occipital shape was used. While a backward inclined occiput implies a downward hanging head (often found in grazers), a forward inclined occiput is related to the horizontal head posture in browsing rhinos. In this study, the lateral semicircular canal (LSC) of the bony labyrinth was virtually reconstructed from µCT‐images in order to investigate a possible link between LSC orientation and head posture in extant rhinoceroses. The usual head posture was formerly reconstructed for several non‐rhinoceros taxa with the assumption that the LSC of the inner ear is held horizontal (parallel to the ground) during normal activity of the living animal. The current analysis of the LSC orientation resulted in a downward inclined usual head posture for the grazing white rhinoceros and a nearly horizontal head posture in the browsing Javan rhinoceros. The other three browsing or mixed feeding species show subhorizontal (closer to horizontal than a downgrade inclination) head postures. The results show that anatomical and behavioral aspects, like occipital shape, presence and size of horns/tusk‐like lower incisors, as well as feeding and feeding height preferences influence the usual head posture. Because quantitative behavioral data are lacking for the usual head postures of the extant rhinos, the here described relationship between the LSC orientation and the resulting head posture linked to feeding preferences gives new insights. The results show, that the inner ear provides additional information to interpret usual head postures linked to feeding preferences that can easily be adapted to fossil rhinoceroses.     

Genetic diversity and structure of the cycad Zamia loddigesii Miq. (Zamiaceae): implications for evolution and conservation

The genetic diversity and structure of four populations of the cycad Zamia loddigesii were studied throughout its range in Mexico. Allozyme electrophoresis of 15 loci was conducted. The mean number of alleles per locus was 1.80 ± 0.09, the percentage of polymorphic loci was 66.6 ± 5.4, and the expected heterozygosity was 0.266 ± 0.02. The results indicated that the genetic diversity was relatively higher, with respect to tropical tree species and other cycads. The genetic variation explained by differences among populations was 18%. On average, gene flow between paired populations was similar ( Nm  = 1.6) to other tropical forest trees and cycad species. Our results indicated that the geographical isolation among populations of Z. loddigesii generated allele loss, as well as a clinal variation in the frequencies of two loci ( MDH and MNR2 ), in relation to the latitudinal distribution of populations. The populations have become fragmented due to increasingly higher pressure of habitat conversion and disturbance. The importance of the establishment of sanctuaries and protected areas and a reduction in deforestation is highlighted in this research as a way of preserving the high genetic diversity of this and other endemic species.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 152 , 533–544.     

Identity of an endangered grasshopper (Acrididae: Brachaspis): Taxonomy, molecules and conservation

Brachaspis robustus is an endangeredgrasshopper endemic to South Island, NewZealand. It is both rare and localised;occupying low altitude floodplain terraces andbraided riverbeds of the Mackenzie Basin. Thisis in stark contrast to the two other speciesin this genus (B. nivalis and B.collinus) which occupy montane habitats.Mitochondrial and nuclear sequence data wereemployed to explore genetic diversity andphylogenetic relationships of populations ofBrachaspis with a view to establishingthe status of B. robustus. Molecularevidence indicates that Brachaspisprobably radiated during the Pliocene and thatdivisions within the genus relate more tospatial distribution developed during thePleistocene than to ecology. The mitochondrial(Cytochrome oxidase I) and nuclear (ITS)sequence data indicate that Brachaspisnivalis is divided into northern and southernpopulations. The northern clade is furthersubdivided geographically. The southern cladecomprises alpine populations of B.nivalis and includes the lowland B.robustus. Additionally, it is observed thatsome morphological features previously thoughtto be specific to B. robustus also occurin members of the southern B. nivalisclade. It is suggested that the taxon B.robustus should include all of the southernBrachaspis populations. But it is arguedthat the absence of genetic evidencedistinguishing the endangered population doesnot preclude it from conservation effort. Acombination of morphological and habitatpeculiarities indicate that the survival ofB. robustus (sensu lato) isimportant to the maintenance of diversity.     

Complex evolutionary history in the brown trout: Insights on the recognition of conservation units

We screened genetic variation in a polytypicorganism, whose populations are oftendistributed into numerous isolated habitats,and integrated the results into a critique ofdefining ``units' of conservation for organismswith highly fragmented populations. Sixteenpopulations of brown trout Salmo truttaL. across 8 Portuguese river basins werescreened for variation at 5 loci (mtDNA andallozymes). Population history based on mtDNArevealed a mosaic pattern driven by pastfragmentation and restricted gene flow withlittle correspondence to major river drainagesor recently proposed OCUs on the IberianPeninsula. Such patterns of variation offer achallenge to conservation strategies that basethemselves on defining units of conservation,particularly if such units intend to reflect ahierarchical evolutionary structure. Wesuggest that geographically mosaic patterns ofevolutionary lineages, as well as adaptivelysignificant traits are common characteristicsof many freshwater organisms. Thus,large-scale units, even if diagnosed by mtDNAclades, are often too heterogeneous to considera ``unit' of conservation. Alternatively, abottom-up perspective that prioritizespopulations or metapopulations is both morepractical and more effective in recognizing andpreserving evolutionary diversity.     

A survey of equid mitochondrial DNA: Implications for the evolution, genetic diversity and conservation of Equus

The evolution, taxonomy and conservation of the genus Equuswere investigated by examining the mitochondrial DNA sequences of thecontrol region and 12S rRNA gene. The phylogenetic analysis of thesesequences provides further evidence that the deepest node in thephylogeny of the extant species is a divergence between twolineages; one leading to the ancestor of modern horses (E.ferus, domestic and przewalskii) and the other to thezebra and ass ancestor, with the later speciation events of the zebrasand asses occurring either as one or more rapid radiations, or withextensive secondary contact after speciation. Examination of the geneticdiversity within species suggested that two of the E. hemionussubspecies (E. h. onager and E. h. kulan) onlyrecently diverged, and perhaps, are insufficiently different to beclassified as separate subspecies. The genetic divergence betweendomestic and wild forms of E. ferus (horse) and E.africanus (African ass) was no greater than expected within anequid species. In E. burchelli (plains zebra) there was anindication of mtDNA divergence between populations increasing withdistance. The implications of these results for equid conservation arediscussed and recommendations are made for conservation action.     

Therps16 intron and the phylogeny of theRubioideae (Rubiaceae)

The phylogeny of the subfamilyRubioideae (Rubiaceae) was estimated from sequence variation in therps16 intron (cpDNA) in 143 ingroup and 5 outgroup taxa. The analysis largely confirms a recent one based onrbcL sequences, but branch support is often much stronger. Three of the traditional subfamilies are supported,Rubioideae, Cinchonoideae s. str., andIxoroideae s. l. while there is no support forAntirheoideae. TheRubioideae are the sister group of all otherRubiaceae and comprise the tribesAnthospermeae, Coccocypseleae, Cruckshanksieae, Coussareeae, Gaertnereae, Hedyotideae, Knoxieae, Morindeae, Ophiorrhizeae, Paederieae, Pauridiantheae, Perameae, Psychotrieae, Rubieae, Spermacoceae, Theligoneae, andUrophylleae. TheHamelieae andHillieae belong to theCinchonoideae. Rachicallis andSiemensia should be transferred from theHedyotideae to theCinchonoideae. ThePauridiantheae, Urophylleae, Ophiorrhizeae, andRaritebe form the basalmost subclade of theRubioideae. The second basalmost clade consists of the generaLasianthus andPerama. The third basalmost clade consists of the tribesCoussareeae, Coccocypseleae andCruckshanksieae, and the generaDeclieuxia andHindsia. The tribesKnoxieae, Anthospermeae, Argostemmateae, Paederieae, Theligoneae, Rubieae, Hedyotideae, andSpermacoceae are members of one clade. TheKnoxieae are monophyletic ifOtiophora, Otomeria, andPentas are included. The tribeAnthospermeae is supported as monophyletic, but its subtribes are not. ThePaederieae, together withTheligonum, form a paraphyletic grade basal to theRubieae. TheHedyotideae, includingSchismatoclada, form a grade at the base of theSpermacoceae. TheGaertnereae are monophyletic and distinct from thePsychotrieae. TheMorindeae are monophyletic and includeDamnacanthus andMitchella. Schradera is the sister group of theMorindeae. ThePsychotrieae are monophyletic when theGaertnereae, Lasianthus, andDeclieuxia are excluded. The recognition of a subtribeHydnophytineae leaves the rest of thePsychotrieae paraphyletic.Psychotria is paraphyletic with respect to all other genera of the tribe. Approximately 50 genera are here classified for the first time based on molecular data.     

Genetic analysis and conservation of the endangered Canary Island woody sow-thistle, Sonchus gandogeri (Asteraceae)

Sonchus gandogeri, a woody sow-thistle, is an endangered Canary Island endemic with only two known populations, one in the El Golfo and another in the Las Esperillas of El Hierro. Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic variation within and among populations. The mean genetic diversity of two populations was estimated to be 0.380, and the El Golfo population (0.380) had higher genetic diversity than the southeastern one (0.268). The unbiased Neis genetic identity between the two populations was 0.846. The mean genetic diversity of S. gandogeri was much higher than that of the other endangered plant species. This is perhaps due to breeding system, life form, extinction, and/or introgressive hybridization and hybrid origin of the taxon. This study also indicates that the two populations are not strongly differentiated (G_ST=0.149). This study suggests that S. gandogeri is more likely to become extinct due to environmental or demographic forces than genetic factors, such as inbreeding depression. More strict control of introduced herbivores is necessary to protect these populations, and germplasm collection for ex situ conservation is needed.     

Genetic diversity and conservation of Ipomoea microdactyla (Convolvulaceae): an endemic vine from the Bahamas,Cuba, and southeastern Florida

Ipomoea microdactyla Griseb. (Convolvulaceae) is restricted to the Bahamian archipelago, Cuba, and southeastern Florida. The species is listed as a state endangered species in Florida, where it is mostly restricted to the hyperfragmented pine rockland of Miami‐Dade County. Using seven DNA microsatellite loci, we assessed levels of genetic diversity for 12 populations of this species from Andros Island in the Bahamas (six sites), Cuba (one site), and Florida (five sites). We found significantly greater mean numbers of alleles, and higher mean values for both observed and expected heterozygosity in populations from the continuous forest on Andros than those from the habitat fragments in Florida. It is unknown if these patterns of genetic diversity in the Florida populations are the result of habitat fragmentation or founder effects. The population from Cuba exhibited relatively high levels of genetic variation, suggesting that this island is a major center of diversity and dispersal for this species. It appears that hybrid introgression for I. carolina alleles within I. microdactyla individuals occurred at a single site on Andros Island. Overall, the mean inbreeding coefficient value was 0.089, suggesting low levels of inbreeding. The highest inbreeding coefficient values were mostly recorded in Florida. Two groups were revealed, one containing the populations from Florida, and the second one encompassing those from the Bahamas and Cuba. Our results highlight the negative genetic consequences of habitat fragmentation and support initiatives recently established to establish corridors to connect the remnants of the pine forest of the Miami‐Dade County.