Genetic structure and signature of population decrease in the critically endangered freshwater cyprinid <Emphasis Type="Italic">Chondrostoma lusitanicum</Emphasis>

The endemic and critically endangered cyprinid Chondrostoma lusitanicum has a very restricted distribution range. In order to estimate genetic diversity, characterize population structure and infer the demographic history, we examined six microsatellite loci and cytochrome b (mtDNA) sequences from samples taken throughout C. lusitanicum’s geographical range. Estimates of genetic diversity were low in all samples (average He < 0.35). The microsatellite data pointed to a major difference between northern (Samarra and Tejo drainages) and southern (Sado and Sines drainages) samples. This separation was not so clear with mtDNA, since one sample from the Tejo drainage grouped with the southern samples. This could be related with ancestral polymorphism or with admixture events between northern and southern sites during the late Pleistocene. Nevertheless, both markers indicate high levels of population differentiation in the north (for microsatellites F _ST >  0.23; and for mtDNA Φ_ST > 0.74) and lower levels in the south (F _ST < 0.05; Φ_ST < 0.40). With microsatellites we detected strong signals of a recent population decrease in effective size, by more than one order of magnitude, starting in the last centuries. This is consistent with field observations reporting a severe anthropogenic-driven population decline in the last decades. On the contrary mtDNA suggested a much older expansion. Overall, these results suggest that the distribution of genetic diversity in C. lusitanicum is the result of both ancient events related with drainage system formation, and recent human activities. The potential effect of population substructure generating genetic patterns similar to a population decrease is discussed, as well as the implications of these results for the conservation of C. lusitanicum. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sequence Polymorphism and Geographical Variation at a Positively Selected MHC-DRB Gene in the Finless Porpoise (Neophocaena phocaenoides): Implication for Recent Differentiation of the Yangtze Finless Porpoise?

Sequence polymorphism at the MHC class II DRB locus was investigated in three finless porpoise (Neophocaena phocaenoides) populations in Chinese waters. Intragenic recombination and strong positive selection were the main forces in generating sequence diversity in the DRB gene. MHC sequence diversity changed significantly along the study period. Significant decrease in heterozygosity and lost alleles have been detected in the Yangtze River population and South China Sea population since 1990. Furthermore, there is a trend of increasing population differentiation over time. Especially, the genetic differentiation between the Yangtze River population and the Yellow Sea population was very low prior to 1990 (F _ST = 0.036, P = 0.009), but became very significant after 1990 (F _ST = 0.134, P < 0.001), suggesting a recent augmentation of genetic differentiation between both populations probably in a relatively short-term period. Porpoises from the Yangtze River displayed divergent frequencies of shared and private alleles from those displayed by two marine populations, which suggest that the former riverine population has been under a different selection regime (characteristic of a fresh water environment) than that of its marine counterparts.     

Genetic Diversity of Chinese Water Deer (Hydropotes inermis inermis): Implications for Conservation

The Chinese water deer (Hydropotes inermis inermis) is endemic to China. Historically, the species was widely distributed, but now, habitat loss and poaching have reduced its range and number drastically. In order to provide useful information for its conservation, we have investigated the genetic diversity and population structure of the Chinese water deer by analyzing the 403 bp fragment of the mitochondrial DNA (mtDNA) control region (D-loop). Eighteen different haplotypes were detected in 40 samples. Overall, Chinese water deer have a relatively high-genetic diversity compared to other rare cervid species, with a haplotype diversity of 0.923±0.025 and nucleotide diversity of 1.318 ± 0.146%. No obvious phylogenetic structure among haplotypes was found for samples of different origin. An analysis of molecular variance (AMOVA) showed significant differentiation between the Zhoushan and the mainland population (F_ST= 0.088, P < 0.001; Φ _ST = 0.075, P = 0.043), which suggests that exchanges of individuals between Zhoushan and the mainland should be avoided. We also recommend that a breeding center be set up for the mainland population.     

Conservation genetics of Houbara Bustard (Chlamydotis undulata undulata): population structure and its implications for the reinforcement of wild populations

The major decline of Houbara Bustards—sensu lato—throughout their range has led to their classification as ‘vulnerable’ on the IUCN Red List. Within this context, a captive breeding program was established in Morocco to restock North African wild populations of Houbara Bustard (Chlamydotis undulata undulata). At present, founders originate from locations that are currently being reinforced through the addition of captive bred individuals. However, it is planned to restock others areas of the species’ range. Before this could be considered, an accurate knowledge of the genetic structure of the wild recipient population is essential to delineate potential conservation units. We therefore sampled populations throughout most of the remaining range, and used several methods to evaluate their genetic differentiation by means of both mtDNA and microsatellite markers. Very little genetic differentiation was found for both genetic markers (Φ_st ranged from −0.181 to 0.118 and F _st ranged from −0.004 to 0.019). This suggests that C. u. undulata populations could be managed as a single Conservation Unit.     

长江安庆段航道整治对长江江豚数量和分布的时空影响

栖息地丧失和破碎化是威胁长江江豚(Neophocaena asiaeorientalis)种群长期发展的主要因素之一。航道整治工程是导致江豚栖息地丧失和破碎化的一个主要原因,研究工程施工和运行对江豚数量和分布的影响可为江豚栖息地管护提供有益信息。通过对湖口-吉阳矶水域江豚数量和分布的持续调查,分析了安庆段航道整治工程施工和运行对江豚的影响。12次目视调查共在研究水域发现江豚258群次613头次,工程施工前、施工期和初步运行期单次考察分别平均观察到江豚(67.0±24.3)头次、(35.6±22.2)头次和(50.0±2.8)头次。在主要施工区共发现江豚79群次185头次,施工前、施工期和初步运行期单次考察分别平均观察到江豚(18.60±12.03)头次、(10.4±7.3)头次和(20.0±4.2)头次。工程不同时期主要施工区江豚的数量及占比在统计学上没有显著差异。主要施工区S2和S3工程段施工期江豚平均分布密度((0.18±0.17)头/km、(0.04±0.08)头/km)较施工前((0.62±0.16)头/km、(0.40±0.29)头/km)显著下降,S5和S6工程段施工期平均...     

Development and characterization of polymorphic microsatellite loci in the endangered Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis)

The finless porpoise (Neophocaena phocaenoides) is one of the smallest cetacean species widely distributed in the shallow coastal waters of the Indo-Pacific Oceans. The population size of the Yangtze subspecies (N. p. asiaeorientalis) has sharply decreased in the last two decades and access to objective data on its population structure and genetic diversity would be of great assistance for their proper management. Here we report on the isolation of nine polymorphic microsatellite using the “Fast Isolation by AFLP of Sequences Containing repeats” (FIASCO) protocol. Polymorphism was assessed using 30 porpoise individuals randomly sampled in the Yangtze River. The number of alleles per locus varies from 2 to 9, with an average value of 5.56, whereas the ranges of observed and expected heterozygosities were 0.300–0.633 (mean 0.496) and 0.473–0.804 (mean 0.659), respectively.     

Mitochondrial DNA reveals low population differentiation in elongate loach, <Emphasis Type="Italic">Leptobotia elongata</Emphasis> (Bleeker): implications for conservation

Elongate loach (Leptobotia elongata (Bleeker)), an endemic fish species to China, is a famous ornamental freshwater fish. Here, a comparative study of mtDNA control region (D-loop) (835 bp) sequences was performed to analyze its wild population structure and evaluate the genetic diversity for 110 individuals from five locations in the upper reaches of the Yangtze River, China. A total of 49 polymorphic sites and 45 haplotypes yielded high haplotype diversity (h = 0.952), but low nucleotide diversity (π = 0.00454) as that of many fish species. Sequence divergences between haplotypes ranged from 0.0033 ± 0.0011 to 0.0050 ± 0.0012 in intra-groups, and from 0.0037 ± 0.0.0011 to 0.0050 ± 0.0012 between groups. Significant values of Tajima’s D (−1.86383, P < 0.01) and Fu’s F _S (−25.93, P < 0.01), together with uni-modal mismatch distribution, indicated a recent genetic bottleneck or population expansion of the species. Analysis of molecular variance (AMOVA) indicated a small amount of differentiation among groups (1.7%); most of the total variation occurred within groups (98.3%). Also, there was no significant population structure (F _ST = 0.017, P > 0.05), and estimates of gene flows among groups were extremely high (Nm = 28.88), suggesting low genetic divergence between populations in the species. The lack of genetic differentiation among groups is most likely due to the combined gene flow from the downstream movement of eggs and larvae with currents and the upstream or downstream migration of adults throughout the distribution. These groups of L. elongata distributed in upper reaches of the Yangtze River should be considered as a single management unit.     

Mitochondrial DNA diversity of the vulnerable Chinese Egret (Egretta eulophotes) from China

The genetic diversity and population structure of the vulnerable Chinese Egret (Egretta eulophotes) were surveyed in the present study from three archipelagoes that cover the most southerly to the very northerly parts of the Chinese distribution range of this species, using a 433-bp fragment of the mitochondrial control region (CR). Among 90 individual samples, 31 different haplotypes were defined by 30 polymorphic sites. Overall haplotype diversity, nucleotide diversity and mean sequence divergence (p-distance) of this egret were 0.920, 0.0088 and 1.11%, respectively. NJ tree and parsimony network for the CR haplotypes of the Chinese Egret showed little genetic structure, and analysis of molecular variance indicated low but significant genetic differentiation (haplotype-based Φ_ST = 0.03267, P < 0.05 and distance-based Φ_ST = 0.04194, P < 0.05) among populations. The significant Fu’s F _S tests (Fu’s F _S  = −16.946, P < 0.01) and mismatch distribution analysis (τ = 4.463, SSD = 0.0081, P = 0.12) suggested that the low genetic differentiation and little geographical structure of the genetic differentiation might be explained by the population expansion. The Mantel test (haplotype-based F _ST, r = 0.639, P = 0.34 and distance-based F _ST, r = 0.947, P = 0.15) suggest that the significant genetic differentiation among populations was likely due to isolation by distance.     

Mitochondrial phylogeography and population history of finless porpoises in Sino‐Japanese waters

Phylogeographical patterns of intraspecific variation can provide insights into the population‐level processes responsible for speciation and yield information useful for conservation purposes. In the present study, three hundred and forty‐five base pairs of the mitochondrial DNA control region were sequenced to analyse the genetic diversity, population structure and history, and phylogeography of finless porpoises (Neophocaena phocaenoides) in Chinese and Japanese waters of the North Pacific. Nucleotide and haplotype diversities were 0.44% and 0.79 ± 0.01 for Chinese and Japanese waters, respectively, but varied significantly among populations. Analysis of molecular variance showed a high level of genetic structure between populations (Φ_ST = 0.61, P < 0.001; F_ST = 0.52, P < 0.001). Eleven of 18 haplotypes were restricted to a single population, common haplotypes were found in two to four populations, but no haplotype was found throughout Sino‐Japanese waters, suggesting multiple colonization events followed by limited gene flow. The inferred age of demographic expansion was from the end stage of the last ice age to the Holocene. No obvious phylogeographical pattern was revealed, including between saline and fresh water populations. A low level of genetic diversity for each population and high among‐population differentiation in haplotype frequency were revealed, which suggest a role for random genetic drift, recent demographic bottlenecks, and reduced or limited gene flow in these populations. Some conservation considerations, with special reference to the unique Yangtze population, are discussed. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 193–204.     

Population genetics of the freshwater mussel, <Emphasis Type="Italic">Amblema plicata</Emphasis> (Say 1817) (Bivalvia: Unionidae): Evidence of high dispersal and post-glacial colonization

Over 70% of North American freshwater mussel species (families Unionidae and Margaritiferidae) are listed as threatened or endangered. Knowledge of the genetic structure of target species is essential for the development of effective conservation plans. Because Ambelma plicata is a common species, its population genetic structure is likely to be relatively intact, making it a logical model species for investigations of freshwater mussel population genetics. Using mtDNA and allozymes, we determined the genotypes of 170+ individuals in each of three distinct drainages: Lake Erie, Ohio River, and the Lower Mississippi River. Overall, within-population variation increased significantly from north to south, with unique haplotypes and allele frequencies in the Kiamichi River (Lower Mississippi River drainage). Genetic diversity was relatively low in the Strawberry River (Lower Mississippi River drainage), and in the Lake Erie drainage. We calculated significant among-population structure using both molecular markers (A.p. Φ_st = 0.15, θ_st = 0.12). Using a hierarchical approach, we found low genetic structure among rivers and drainages separated by large geographic distances, indicating high effective population size and/or highly vagile fish hosts for this species. Genetic structure in the Lake Erie drainage was similar to that in the Ohio River, and indicates that northern populations were founded from at least two glacial refugia following the Pleistocene. Conservation of genetic diversity in Amblema plicata and other mussel species with similar genetic structure should focus on protection of a number of individual populations, especially those in southern rivers.     

High MHC DQB Variation and Asymmetric Allelic Distribution in the Endangered Yangtze Finless Porpoise, Neophocaena phocaenoides asiaeorientalis

The endangered Yangtze finless porpoise is found in the middle and lower reaches of the Yangtze River and its adjoining big lakes. To explore the major histocompatibility complex (MHC) genetic diversity and allelic distribution patterns across its range, we investigated variation at DQB exon 2. From 76 porpoises, we identified 18 DQB sequences. The freshwater Yangtze populations had much higher allelic diversity than marine populations. Among these freshwater populations, the middle-reach population had higher allelic diversity than the lower-reach population. The high DQB diversity level, relative to that of a neutral mtDNA locus, suggests that balancing selection is acting at the DQB gene and that rapid evolution and local positive selection play critical roles in generating and retaining high MHC diversity in the freshwater population. As the balancing selection might be driven by environmental pathogens, we suggest that maintaining MHC variation should be a high priority in the conservation and management of this endangered population, especially as an ex situ conservation strategy.     

Conservation genetics of Butte County meadowfoam (Limnanthes floccosa ssp. californica Arroyo), an endangered vernal pool endemic

The endangered annual plant Limnanthes floccosa ssp. californica Arroyo is restricted to vernal pools in Butte County, California. To identify populations with unique genetic resources, guide reintroduction efforts, and design seed collection scenarios for long-term ex situ seed storage we determined extant genetic diversity and structure by surveying 457 individuals from 21 distinct populations using nine polymorphic microsatellite markers. We found low within population genetic diversity: low allelic diversity (1.9 [0.06 SE] alleles/locus); low heterozygosity (H _obs = 0.10 ± 0.018, H _exp = 0.19 ± 0.015), and a high fixation index (0.556 ± 0.044). The number of polymorphic loci ranged between 11 and 89%. Bayesian ordination determined 20 distinct populations and we found high genetic structure among these (F _st = 0.65, P < 0.0001). We identified notable gene flow barriers across populations, confirming regional structuring between three previously defined population density centers and two outlying populations (F _st = 0.21, P < 0.0001). Population size estimates ranged between ~50 and >5,000 extant plants per site. Our study confirms previous isozyme-based results and suggests that the loss of any population would represent a significant loss in the species’ genetic diversity. Recovery requires active restoration of existing populations and permanent habitat protection. We recommend close comparison of microhabitats of declining populations with genetically similar populations, to determine the potential for human assisted gene flow via seed movement to recover declining populations.     

固化河岸对长江江豚栖息活动的影响

长江江豚主要栖居在近岸浅水水域,喜好泥沙质类型的河岸。但在长江干流,有很多自然河岸被固化,河岸固化给长江江豚分布和栖息活动造的影响仍不甚清楚。2016年3月—2017年1月,对长江干流2个自然河段江豚的数量和分布做了12次考察,并收集了这2个河段岸型的相关数据,来分析固化河岸对江豚栖息活动的影响。12次考察累计发现江豚215头次,平均每次考察观察到江豚(17.92±7.09)头次。研究区域的固化河岸约占岸线总长的59%,分析发现,仅约13.9%的江豚分布在固化河岸水域,86.1%的江豚均分布在自然河岸水域。江豚在单位河岸长度的分布数量与该段固化河岸长度所占的比例呈显著负相关(r=-0.639,P0.01)。在自然河岸,分布在近岸50 m水域的江豚占31.8%,而在固化河岸,仅观察到2头江豚活动在近岸50 m水域内。由此可见,固化河岸对江豚的近岸分布有明显影响。调查还发现,安庆城区建设带约10 km江段12次考察均未发现江豚分布,城区建设带可能对江豚栖息已造成严重影响。长江干流的固化河岸所占比例非常高,研究结果提示这可能导致长江干流江豚栖息地的丧失和破碎化加剧,在制定长江江豚保护措施必需慎重考虑此因素的影响,并据此提出相应的栖息地保护和恢复方案。     

Genetic structure of the Far Eastern population of Eurasian wigeon <Emphasis Type="Italic">Anas penelope</Emphasis> inferred from sequencing of the mitochondrial DNA control region

Sequence variation of the 5′ end of the mitochondrial DNA control region (600 bp) was examined in the population samples of Eurasian wigeon Anas penelope from Anadyr’ and Primorye. A total of 11 different mtDNA haplotypes were identified, with one of these belonging to American wigeon Anas americana. The presence of the mtDNA haplotype from the species closely relative to A. penelope in the Anadyr’ sample can be considered as the genetic evidence in favor of interspecific hybridization. This suggestion is in the good agreement with ornithological data. Genetic differentiation of the Primorye and Anadyr’ populations was low (Φ_st = 0.096). The phylogeographic structure was not pronounced.     

Risso’s dolphins (<Emphasis Type="Italic">Grampus griseus</Emphasis>) in UK waters are differentiated from a population in the Mediterranean Sea and genetically less diverse

The Risso’s dolphin (Grampus griseus) has a worldwide distribution, but little is known about their population genetic structure. Local coastal populations are never known to be abundant, and are sometimes under anthropogenic impact. Therefore the question of regional differentiation by genetic drift and overall diversity levels is of conservation interest. Here we present preliminary data that clearly indicate genetic differentiation and lower genetic diversity of a population sampled in UK waters (primarily from the Western Isles, Scotland) compared to a Mediterranean sample. Significant differentiation was found in comparison with a sample from the Mediterranean for both microsatellite DNA markers (F _ST = 0.0296) and mtDNA sequence data (F _ST = 0.260; ϕ _ST = 0.542). Allelic diversity was lower in the UK for nearly all loci.     

Genetic diversity within and between seedstock populations of several German autochthonous provenances and conventionally propagated nursery material of blackthorn (<Emphasis Type="Italic">Prunus spinosa</Emphasis> L.)

Genetic diversity within and between seedstocks of autochthonous provenances of common blackthorn (Prunus spinosa L.) from several locations in Germany was determined and compared with the diversity in conventionally propagated (German and Hungarian) seedstocks using a highly reproducible high-annealing-temperature random amplified polymorphic DNA (HAT-RAPD) protocol. Based on the distribution of 359 markers obtained with 11 primers we found relatively low genetic diversity in the studied autochthonous blackthorn populations (H ₀ 0.1182–0.1333), with the majority distributed within the populations (92.22%) and only 7.78% among them. Similar levels of diversity were also found in the conventional seedstocks. Accordingly, genetic differentiation among these populations is rather low (pairwise F _st 0.0284–0.1266). In one case, we were not able to differentiate between an autochthonous population and conventional (F _st 0.0284) one. We discuss the results with respect to German conservation laws and their practical implementation.     

The genetic structure of the loggerhead sea turtle (Caretta caretta) in the Mediterranean as revealed by nuclear and mitochondrial DNA and its conservation implications

The population genetic structure of the loggerhead sea turtle (Caretta caretta) nesting in the eastern Mediterranean was assessed by sequencing a fragment of the control region of the mitochondrial DNA (n = 190) and seven microsatellites (n = 112). The two types of markers revealed genetic structuring (mtDNA: γ_st = 0.212, P < 0.001; nDNA F _st = 0.006, P < 0.001), thus indicating that both females and males are philopatric and that gene flow between populations is restricted. Mitochondrial DNA data indicate that the female populations nesting on the islands of Crete and Cyprus have suffered a recent bottleneck or colonization event. However, no bottleneck or founder effect was revealed by nuclear markers, thus indicating male-mediated gene flow from other populations that would increase nuclear genetic variability. Crete, and to a lower extent Cyprus, are thought to play a central role in such male-mediated gene flow that may reduce the negative effect of genetic drift or inbreeding on the small populations of Lebanon and Israel. This population structure indicates that assessing population relevance only on the basis of genetic variability and size would be misleading, as some populations not fulfilling those requirements may play a relevant role in genetic exchange and hence contribute to the overall genetic variability.     

Establishment and characterization of fibroblast cell lines from the skin of the Yangtze finless porpoise

The Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), as the sole freshwater subspecies of N. phocaenoides, is endemic to the Yangtze River and its adjacent lakes. Its population has declined significantly over recent decades. In this study, we established a skin-derived finite fibroblast cell line of the Yangtze finless porpoise, named YFP-SF1, using primary cell culture methods, and an immortalized cell line, T-YFP-SF1, through co-transfection (GFP and SV40 T antigens) techniques. YFP-SF1 proliferated continuously with a minimum population doubling time of 31 h and exhibited age-dependent changes in growth rate. T-YFP-SF1 cells exhibited fibroblast morphology and were characterized by a shorter doubling time, higher attachment efficiencies, colony formation at a low seeding density, and growth in low serum concentrations. Anchorage independence and foci formation in the cell monolayer were observed from passage 36. The chromosome number of YFP-SF1 and T-YFP-SF1 remained stable at 2n = 44 in the early passages, and the viability of thawed cells remained above 90% after cryopreservation in liquid nitrogen. Taken together, we have established fibroblast cell lines of Yangtze finless porpoise for the first time, which might assist as an in vitro model for this endangered mammal.     

The influence of spatial scale on the genetic structure of a widespread tropical wetland tree, Pterocarpus officinalis (Fabaceae)

Identifying factors that cause genetic differentiation in plant populations and the spatial scale at which genetic structuring can be detected will help to understand plant population dynamics and identify conservation units. In this study, we determined the genetic structure and diversity of Pterocarpus officinalis, a widespread tropical wetland tree, at three spatial scales: (1) drainage basin “watershed” (<10 km), (2) within Puerto Rico (<100 km), and (3) Caribbean-wide (>1000 km) using AFLP. At all three spatial scales, most of the genetic variation occurred within populations, but as the spatial scale increased from the watershed to the Caribbean region, there was an increase in the among population variation (Φ_ST=0.19 to Φ_ST=0.53). At the watershed scale, there was no significant differentiation (P=0.77) among populations in the different watersheds, although there was some evidence that montane and coastal populations differed (P<0.01). At the island scale, there was significant differentiation (P<0.001) among four populations in Puerto Rico. At the regional scale (>1000 km), we found significant differentiation (P<0.001) between island and continental populations in the Caribbean region, which we attributed to factors associated with the colonization history of P. officinalis in the Neotropics. Given that genetic structure can occur from local to regional spatial scales, it is critical that conservation recommendations be based on genetic information collected at the appropriate spatial scale.     

Conservation genetics of the alligator snapping turtle: cytonuclear evidence of range-wide bottleneck effects and unusually pronounced geographic structure

A previous mtDNA study indicated that female-mediated gene flow was extremely rare among alligator snapping turtle populations in different drainages of the Gulf of Mexico. In this study, we used variation at seven microsatellite DNA loci to assess the possibility of male-mediated gene flow, we augmented the mtDNA survey with additional sampling of the large Mississippi River System, and we evaluated the hypothesis that the consistently low within-population mtDNA diversity reflects past population bottlenecks. The results show that dispersal between drainages of the Gulf of Mexico is rare (F _STmsat  = 0.43, Φ_STmtDNA = 0.98). Past range-wide bottlenecks are indicated by several genetic signals, including low diversity for microsatellites (1.1–3.9 alleles/locus; H _e = 0.06–0.53) and mtDNA (h = 0.00 for most drainages; π = 0.000–0.001). Microsatellite data reinforce the conclusion from mtDNA that the Suwannee River population might eventually be recognized as a distinct taxonomic unit. It was the only population showing fixation or near fixation for otherwise rare microsatellite alleles. Six evolutionarily significant units are recommended on the basis of reciprocal mtDNA monophyly and high levels of microsatellite DNA divergence.