Phylogeography and conservation genetics of the Columbia spotted frog (Rana luteiventris; Amphibia, Ranidae)

The Columbia spotted frog (Rana luteiventris) has a widespread distribution in western Canada and the western US, although the southern reach of its range is highly fragmented into several isolated populations. Threats from various factors have raised concerns regarding the long-term survival of many small, isolated populations. Here, we report a study designed to determine the phylogeographic and conservation genetic parameters of R. luteiventris in the western US. Mitochondrial DNA (mtDNA) sequences were examined for phylogeographic structuring using phylogenetic reconstruction methods, coupled with networking and nested clade analyses. These methods permitted a distinction to be made between historic and demographic forces acting to generate geographical patterning of genetic variation. Phylogenetic analysis revealed four geographically correlated monophyletic clades. Three of these clades correspond to well-defined, nonoverlapping geographical locations in the fragmented portion of the range. The other is comprised of all samples collected from the contiguous range and includes one isolate from northern Wyoming. Networking and nested clade analyses confirmed these results and revealed that historical processes, such as range expansion and vicariance, rather than recurrent gene flow are likely responsible for observed patterns of genetic variation. A measure of genetic variation (theta = 4N(e)mu) revealed that R. luteiventris populations in Utah have a relatively low amount of genetic variation compared with populations in the continuous portion of the range.     

报春花属的保育遗传学及亲缘地理学研究进展

现存物种的分布格局、遗传结构是当前因素和历史因素共同作用的结果。人类的过度开发、全球变暖等当前因素造成的生境片段化是目前许多报春花属植物濒危的一大原因。该文总结了近年来报春花属内的保育遗传学研究进展,期望以此为更好地保护报春花属植物提供一定的理论基础。应用亲缘地理学研究方法可以弥补古生物学难以研究报春花植物历史成因的不足,因此也总结该方法在报春花属内的研究进展,并初步整理不同地区间的报春花属植物的分化式样,同时期望这些研究成果能为为研究报春花属植物在应对全球气候变化的响应机制方面提供一些参考。     

Rhodanese (thiosulfate:cyanide sulfurtransferase) from frog Rana temporaria

The molecular mass of rhodanese from the mitochondrial fraction of frog Rana temporaria liver, equaling 8.7 kDa, was determined by high-performance size exclusion chromatography (HP-SEC). The considerable difference in molecular weight and the lack of common antigenic determinants between frog liver rhodanese and bovine rhodanese suggest the occurrence of different forms of this sulfurtransferase in the liver of these animals.     

Physiological significance of behavioral hypothermia in hypoglycemic frogs (Rana catesbeiana)

Hypoxia elicits a number of compensatory responses in animals, including behavioral hypothermia. The hypothesis that hypoglycemia induces hypothermia in the bullfrog Rana catesbeiana was tested and that this behavioral response would be beneficial. Frogs equipped with a temperature probe were tested in a thermal gradient (10–40°C). Insulin (15 IU kg⁻¹) caused significant reduction of body temperature, from 25.0 to 17.8°C. A non-metabolizable glucose analogue, 2-deoxy-d-glucose (2-DG, 50 mg kg⁻¹),which blocks intracellular glucose utilization, was also injected and caused a similar drop in body temperature, despite an increase in plasma glucose levels. To assess the possible benefits of hypoglycemia-induced hypothermia, the effects of insulin and 2-DG injections were measured on plasma glucose concentration and on oxygen consumption of frogs equilibrated at 10, 20 and 30°C. The plasma glucose was elevated at higher temperatures and so was oxygen consumption. The insulin caused a significant reduction of plasma glucose concentration (about 1.22 μMol ml⁻¹) whereas 2-DG caused a significant increase (about 0.70 μMol ml⁻¹) at 30°C. Both drugs caused a reduction of oxygen consumption (approximately 0.388 and 0.382 ml min⁻¹ kg at 30°C after insulin and 2-DG injection, respectively). No effect of either insulin or 2-DG was observed when the animals were equilibrated at 10°C. In conclusion, hypothermia may be a beneficial response to hypoglycemia in frogs.     

Rangewide phylogeography in the greater horseshoe bat inferred from microsatellites: implications for population history, taxonomy and conservation

The distribution of genetic variability across a species' range can provide valuable insights into colonization history. To assess the relative importance of European and Asian refugia in shaping current levels of genetic variation in the greater horseshoe bats, we applied a microsatellite-based approach to data collected from 56 localities ranging from the UK to Japan. A decline in allelic richness from west Asia to the UK and analyses of F(ST) both imply a northwestward colonization across Europe. However, sharp discontinuities in gene frequencies within Europe and between the Balkans and west Asia (Syria/Russia) are consistent with suture zones following expansion from multiple refugia, and a lack of recent gene flow from Asia Minor. Together, these results suggest European populations originated from west Asia in the ancient past, and experienced a more recent range expansion since the Last Glacial Maximum. Current populations in central Europe appear to originate from the Balkans and those from west Europe from either Iberia and/or Italy. Comparisons of R(ST )and F(ST) suggest that stepwise mutation has contributed to differentiation between island and continental populations (France/UK and China/Japan) and also among distant samples. However, pairwise R(ST) values between distant populations appear to be unreliable, probably due to size homoplasy. Our findings also highlight two priorities for conservation. First, stronger genetic subdivision within the UK than across 4000 km of continental Eurasia is most likely the result of population fragmentation and highlights the need to maintain gene flow in this species. Second, deep splits within China and between Europe and China are indicative of cryptic taxonomic divisions which need further investigation.     

Population genetics of the endangered Canarian endemic Atractylis arbuscula (Asteraceae): implications for taxonomy and conservation

We use the information provided by 36 RAPD loci and 15 morphological traits to describe and construe the population differentiation in A. arbuscula, a Canarian endemic Asteraceae threatened with extinction that exhibits a disjunct distribution in the islands of Gran Canaria (var. schizogynophylla) and Lanzarote (var. arbuscula). Our evaluation of morphological characters and the extent of RAPD differentiation found sets the stage for a taxonomic restructuring to hoist both var. arbuscula and var. schizogynophylla to subspecies category. Our genetic results suggest that fragmentation and generally low population sizes are jeopardising the survival of this species through a predominance of stochastic processes in microevolutionary dynamics, especially in Gran Canaria, where subpopulations exhibit the lowest levels of genetic variation and gene flow. If, as most Asteraceae, these plants have a sporophytic self-incompatibility system, the scarce reproductive turnover observed in the much smaller subpopulations from Gran Canaria (ssp. schizogynophylla) is possibly more influenced by a deficiency of S-alleles (that would be provoking the unavailability of compatible mating types, and a cascade of deleterious effects associated with the Allee effect) than by a reduced pollinator visitation frequency. Based on the depauperated values of genetic parameters for this subspecies, urgent mixed subpopulation reinforcements and reintroductions (with specimens belonging to the same subspecies) seem advisable on the grounds that they might allow the isolated habitat remnants to retrieve from inbreeding through the introduction of a new stock of S-alleles and the subsequent production of genotypes that may have a higher selective value.     

Genetic diversity of the endangered Brazilian endemic herb Cunila menthoides Benth. (Lamiaceae) and its implications for conservation

Cunila menthoides is classified as a xylopodiferous subshrub found in fragmented environments threatened by local agriculture and pasture activities. The species presents strong and pleasant smell, and is commonly used in south Brazilian folk medicine. Based on the species’ medicinal and aromatic potential, the aim of this work was to examine the genetic variation within and among C. menthoides populations, focusing on the conservation of this species. In this context, six C. menthoides populations collected in Rio Grande do Sul, Brazil, were analyzed by ISSR. The populations were sorted as four clusters according to geographic distribution. The six C. menthoides populations analyzed presented low genetic variability, indicating that each population derives from a limited number of plants in a low gene flow scenario. In situ and ex situ conservation approaches may be useful in this species. Conservation strategies for the species should be based on in situ and ex situ approaches.     

Glycation of wood frog (Rana sylvatica) hemoglobin and blood proteins: In vivo and in vitro studies

The effects of in vivo freezing and glucose cryoprotectant on protein glycation were investigated in the wood frog, Rana sylvatica. Our studies revealed no difference in the fructoselysine content of blood plasma sampled from control, 27 h frozen and 18 h thawed wood frogs. Glycated hemoglobin (GHb) decreased slightly with 48 h freezing exposure and was below control levels after 7 d recovery, while glycated serum albumin was unchanged by 48 h freezing but did increase after 7 d of recovery. In vitro exposure of blood lysates to glucose revealed that the GHb production in wood frogs was similar to that of the rat but was lower than in leopard frogs. We conclude that wood frog hemoglobin was glycated in vitro; however, GHb production was not apparent during freezing and recovery when in vivo glucose is highly elevated. It is possible that wood frog blood proteins have different in vivo susceptibilities to glycation.     

不同程度的尾损伤对镇海林蛙(Rana zhenhaiensis)蝌蚪游泳速度的影响

无尾两栖类蝌蚪的尾巴通过产生强大的游泳速度在反捕食中起到了重要的作用。以镇海林蛙(Rana zhenhaiensis)蝌蚪为实验动物来评估断尾的运动代价。以74尾具有完整尾蝌蚪作为实验组,通过截去不同尾长片段,人为分成轻微尾损伤组(30%)和严重尾损伤组(30%)并测定两组蝌蚪在断尾前后的游泳速度。以16尾完整尾蝌蚪作为对照组在实验组断尾前后同时进行游泳速度的测定。实验结果显示断尾影响蝌蚪的游泳速度,但仅在尾损伤程度达到尾长的30%以上时才产生不利的影响。这表明轻微尾损伤并不对镇海林蛙蝌蚪的游泳速度产生严重影响。在断尾前后实验组蝌蚪的游泳速度均与尾长呈正相关。在相同尾长状态下,尾损伤蝌蚪的相对游泳速度明显快于完整尾蝌蚪。因此,尾损伤的镇海林蛙蝌蚪有可能通过改变尾和身体的摆动频次等方式在断尾后对游泳速度进行了一定的补偿。尾损伤在野外频繁发生于蝌蚪的尾远端,据此推测镇海林蛙蝌蚪在自然条件下的尾损伤并不会产生严重运动代价。     

Genetic diversity in mitochondrial 12S rDNA of western Palearctic water frogs (Anura, Ranidae) and implications for their systematics

The nucleotide sequence of a part of the mitochondrial 12S rRNA gene of eight western Palearctic water frog species was analysed. The results are consistent with the species status of Rana bedriagae, Rana bergeri, Rana epeirotica, Rana lessonae, Rana perezi, Rana ridibunda, Rana saharica and Rana shqiperica . The obtained DNA data suggest that lake frogs from Greece and Yugoslavia on the one hand and lake frogs from Georgia, Uzbekistan and Turkmenistan on the other hand represent two distinct species. However, it is not yet clear whether lake frogs from Georgia, Uzbekistan and Turkmenistan belong to R. ridibunda or represent a new species. The very high similarity between the analysed 12S rDNA segments of German R. ridibunda and R. lessonae confirm the finding that mtDNA of R. lessonae was transmitted into the mitochondrial gene pool of R. ridibunda probably as a result of backcrosses with the hybridogenetic hybrid R. kl. esculenta . The results of parsimony analyses speak in favour of very close phylogenetic relations between R. perezi and R. saharica ; with a high probability these species represent an adelphotaxon. Furthermore, the clades ( R. lessonae + R. shqiperica + R. bergeri ) and ( R. ridibunda + R. bedriagae ) are considered to be sister groups. According to the mt 12S rDNA data R. epeirotica seems to be more closely related to the supraspecific taxon ( R. ridibunda + R. bedriagae ) than to ( R. lessonae + R. shqiperica + R. bergeri ). Thus, it can be excluded that R. shqiperica and R. epeirotica represent sister species.     

The disappearing northern leopard frog (Lithobates pipiens): conservation genetics and implications for remnant populations in western Nevada

Global amphibian declines suggest a major shift in the amount and quality of habitat for these sensitive taxa. Many species that were once widespread are now experiencing declines either in part of or across their historic range. The northern leopard frog (Rana [Lithobates] pipiens] has undergone significant declines particularly in the western United States and Canada. Leopard frog population losses in Nevada are largely due to habitat fragmentation and the introduction of nonnative fish, amphibian, and plant species. Only two populations remain in the Truckee and Carson River watersheds of western Nevada which represents the western boundary of this species range. We used sequence data for an 812 base pair fragment of the mitochondrial NADH dehydrogenase 1 (ND1) gene to support a native origin for western Nevada populations. All frogs had a single haplotype (W07) from the distinct western North America ND1 haplotype clade. Data from seven polymorphic microsatellite loci show that Truckee and Carson River populations are highly differentiated from each other and from leopard frogs collected from eastern Nevada sites. Lack of gene flow among and distinct color morphs among the western Nevada populations likely predates the current geographical isolation. Comparisons with other peripheral L. pipiens populations show western Nevada populations have similar levels of gene diversity despite their contemporary isolation (H(E) 0.411, 0.482). Restoration of leopard frog populations in these watersheds will be challenging given well-entrenched nonnative bullfrog populations and major changes to the riparian zone over the past century. Declines of once common amphibian species has become a major conservation concern. Contemporary isolation of populations on a species range periphery such as the leopard frog populations in the Truckee and Carson rivers further exacerbate extirpation risk as these populations are likely to have fewer genetic resources to adaptively respond to rapidly changing biotic and abiotic environments.     

Localization of prolactin receptor in the dorsal and ventral skin of the frog (Rana ridibunda)

The dorsal and ventral skin in amphibians plays an important role in osmoregulation. Prolactin hormone is involved in regulation of amphibian skin functions, such as water and electrolyte balance. Therefore, amphibians may be useful as a model for determining the sites of the prolactin receptor. In this study, prolactin receptor was detected in frog dorsal and ventral skin using immunohistochemical staining method. Prolactin receptor immunoreactivity was localized in all epidermal layers except stratum corneum of dorsal skin epidermis, stratum germinativum layer of ventral skin epidermis, myoepithelial cells, secretory epithelium and secretory channel cells of granular glands in both skin regions. The mucous glands and secretory granules of granular glands did not show immunoreactivity for the prolactin receptor. According to our immunohistochemical results, the more widespread detection of prolactin receptor in dorsal skin epidermis indicates that prolactin is more effective in dorsal skin. Presence of prolactin receptors in epidermis points out its possible osmoregulatory effect. Moreover, detection of receptor immunoreactivity in various elements of poison glands in the dermis of both dorsal and ventral skin regions suggests that prolactin has a regulatory effect in gland functions.     

Mitochondrial phylogeography and genetic diversity of Tibetan gazelle (Procapra picticaudata): implications for conservation

The Tibetan gazelle (Procapra picticaudata) is a threatened species and distributed on the Qinghai-Tibet Plateau of China (Qinghai Province, Tibet Autonomous Region and the adjacent Gansu Province, Sichuan Province, and Xinjiang Uigur Autonomous Region). Small peripheral populations of Tibetan gazelle were once found in northern Sikkim and Ladakh, but now these are close to extinction. To describe the evolutionary history and to assess the genetic diversity within this monotypic species and population structure among different geographic locations in China, we sequenced mitochondrial DNA from the control region (CR) and cytochrome (cyt) b gene for 46 individuals from 12 geographic localities in Qinghai, Tibet, Xinjiang, Gansu, and Sichuan. A total of 25 CR haplotypes and 16 cyt b haplotypes were identified from these gazelle samples. CR haplotype diversity (0.98+/-0.01) and nucleotide diversity (0.08+/-0.009) were both high. Phylogenetic trees indicate that the Tibetan gazelle in China can be divided into three main clades: Tibet, Sichuan (SCH) and Qinghai-Arjin Shan-Kekexili (QH-ARJ-KKXL). Analysis of molecular variance (AMOVA) and network analysis consistently support this geographic structure in both datasets. Significant differentiation between populations argues for the presence of management units (MUs). Such differentiation may reflect a geographic separation resulting from the uplift of the Qinghai-Tibet Plateau during the Late Pliocene and Pleistocene. Mismatch distribution analysis implies that Tibetan gazelle has undergone complex population changes. We suggest that the present population structure has resulted from habitat fragmentation during the recent glacial period on the Qinghai-Tibet Plateau and population expansion from glacial refugia after the glacial period. It is likely that the present populations of Tibetan gazelle exhibit a pattern reminiscent of several bottlenecks and expansions in the recent past.     

Mitochondrial phylogeography and subspecific variation in the red panda (Ailurus fulgens): implications for conservation

The red panda (Ailurus fulgens) is an endangered species and its present distribution is restricted to isolated mountain ranges in western China (Sichuan, Yunnan, and Tibet provinces) and the Himalayan Mountains chain of Nepal, India, Bhutan, and Burma. To examine the evolutionary history across its current range, and to assess the genetic divergence among current subspecies and population structure among different geographic locations, we sequenced mitochondrial DNA from the control region (CR) and cytochrome (cyt) b gene for 41 individuals in Sichuan, Yunnan, Tibet of China, and Burma. 25 CR haplotypes (10 for cyt b) were identified from 11 geographic locations. Only three haplotypes were shared among sample localities, including one among current subspecies. Nine haplotypes were shared with the study of Su et al. [Mol. Biol. Evol. 18 (2001) 1070]. CR haplotype diversity was high (0.95+/-0.02) and nucleotide diversity among all haplotypes was relatively low (0.018+/-0.009). Phylogenetic confirmed trees show a shallow pattern with very little structure or statistical robustness. The application of two coalescent-based tests for population growth allowed us to interpret this phylogeny as the result of a recent population expansion. Analysis of molecular variance and nested clade analysis failed to detect significant geographic structure in both data sets. The lack of significant differentiation between subspecies does not indicate the presence of evolutionary significant units. We suggest that the present population structure has resulted from habitat fragmentation and expansion from glacial refugia. Due to its habitat requirements it is likely that the red panda has undergone bottlenecks and population expansions several times in the recent past. The present population may exhibit a pattern reminiscent of a relatively recent population expansion.     

Biological properties of the endemic and threatened shrub in Iberia Vella pseudocytisus subsp. paui Gómez Campo (Cruciferae) and implications for its conservation

We present new data about chorology, seed bank, dispersal, seedling biology and population structure of the threatened endemic shrub Vella pseudocytisus subsp. paui Gómez Campo in Spain. In addition, we investigate human activities that are linked with the fine-scale distribution of the taxon to offer conservation recommendations.Currently, the plant occurs in two populations close to the city of Teruel (East central Spain), growing in an open shrubby community on patches of gypsum substrate surrounded by agricultural landscape. The plant has disappeared from two reported locations dated from early 20th Century likely due to human activities.The seed bank seems to be poor. A ballistic dispersal mechanism appears to move seeds between 0 and 4 m. We did not find a clear seasonal pattern of seedlings emergence although spring cohorts are the largest. Also, this period was where leaf seedling growth was more prominent. During the 3 years of our survey, the cumulative mortality was 94.2% of all seedlings plants. Plants are estimated to remain in the seedling stage for approximately 5 years. The demographic structure of both populations shows no apparent gaps in the distribution of size classes. Adult plants greater than 200 dm³ represent the most important stage for seed production and persistence.We point out two key factors for conservation purposes, first the weakness of some of the plant biological properties for natural establishment and second its ability to colonise artificial environments. We propose a mitigation of clonal spread in those sites and the protection of juvenile stages in more natural locations.     

Evolution,phylogeography and taxonomy of allopolyploid Dactylorhiza (Orchidaceae) and its implications for conservation

This review is based on recent molecular studies of Dactylorhiza (Orchidaceae). Most of the studies have focused on the allotetraploid members of the genus in general and on D. majalis ssp. lapponica in particular. It was concluded that most of the allotetraploid taxa have derived from hybridizations between the parental lineages D. maculata s.l. and D. incarnata s.l., with D. maculata s.l. serving as the seed parent. Evidence of multiple origins was found both among northern European allotetraploids as well as among Greek allotetraploids. Introgression from both parental lineages and hybridizations between independently derived polyploid lineages was also detected. The three morphologically similar taxa D. majalis ssp. traunsteineri, ssp. lapponica and ssp. russowii should be treated as one and most of the Greek allotetraploids should be regarded as regional variants of the southeastern European D. majalis ssp. cordigera. The Balkans and the Alps most probably served as refugia for the genus during the last glaciations and at least two waves of immigration reached Scandinavia. Finally, we suggest that the conservation of allotetraploid Dactylorhiza should emphasize important geographic areas and habitats and that the allopolyploids should have the same conservation status as the diploids.     

Population genetics and conservation priorities for the critically endangered island endemic Delphinium pentagynum subsp. formenteranum (Ranunculaceae)

Isozyme electrophoresis was used to evaluate levels of genetic diversity and population genetic structure of the critically endangered (CR) perennial larkspur, Delphinium pentagynum subsp. formenteranum (Ranunculaceae), endemic to the island of Formentera (Balearic Islands, Spain). There is only one known population for this taxon, containing only 480 individuals. Moderate values of diversity were detected (P = 40.7%, A = 1.6 and H _e = 0.180), within the range observed in other surveyed larkspurs, but higher than most island endemics. Moderate levels of inbreeding were detected, probably as a consequence of the population's genetic structuring (biparental inbreeding). Threats to this taxon are mainly anthropogenic (fires, grazing, pathway works, and building pressures), although given that only one population exists, stochastic risks cannot be ignored. Conservation of D. pentagynum subsp. formenteranum requires in situ strategies, such as implementing a monitoring program and establishing a botanical reserve, and ex situ strategies, such as collection and long-term storage of seeds.     

Molecular population genetics, phylogeography, and conservation biology of the mottled duck (Anas fulvigula)

The mottled duck (Anas fulvigula) is a year-round endemicresident of the Gulf Coast and one of two non-migratory dabbling ducksthat inhabit North America. To investigate population genetic structureof allopatric mottled duck populations, we collected 5' control regionsequences (bp 78–774) from the mitochondria of 219 mottled duckssampled at 11 widely spaced geographic localities in Texas, Louisiana,and Florida and compared them to each other and to homologous sequencesfrom 4 Mexican ducks (A. diazi), 13 American black ducks(A. rubripes), and 10 mallards (A. platyrhynchos). Weidentified 57 unique haplotypes composed of 665 or 666 nucleotides inthe 246 control region sequences. Of the 665 homologous positions,8.3% (n = 55) vary among haplotypes, and98.2% (n = 54) of these occur within the first351 nucleotides from the 5' end of the outgroup sequence.Neighbor-joining analysis shows a large distal clade (52.5% ofmottled ducks sampled in our study) composed of two reciprocallymonophyletic clades of mottled duck haplotypes, one of which is endemicto Texas and Louisiana and the other endemic to Florida. No mottledducks sampled in Florida occur in the clade composed of mottled ducksfrom Texas and Louisiana or vice versa, suggesting that (1) an enduringgeographic split has existed for many years between east and west, and(2) gene flow currently is non-existent (or at least undetectable)across the central Gulf Coast. The remaining 47.5% of mottledducks sampled in our study branch basally from this derived clade, showsubstantially less hierarchical structure, and fall into various lineagegroups of mixed species composition with no geographic orspecies-specific pattern. Pairwise F _ST valuescorroborate the pattern of strong differentiation observed betweenTexas/Louisiana and Florida. Our findings are consistent with apattern of partial lineage sorting from a polymorphic ancestral genepool reshuffled by hybridizing mallards. Control region data andpatterns of divergence in mallard-like species worldwide, furthermore,suggest that mottled ducks are close relatives of Mexican ducks, and inturn nested within black ducks. Genetic similarities to nominatemallards are less likely to be the product of common ancestry, but theresult of past hybridization with a dichromatic mallard ancestor thatinvaded North America from Asia many generations ago. Our findings haveseveral important consequences for the conservation biology of mottledducks across the Gulf Coast and our understanding of the phylogeographyof mallard-like species worldwide.