Identification of purebred Crocodylus siamensis for reintroduction in Vietnam

Crocodylus siamensis, the Siamese crocodile, is a critically endangered species of freshwater crocodile previously distributed throughout much of SE Asia. Recovery plans call for reintroductions to the wild using founder individuals currently in captivity, mostly in commercial crocodile farms. On many farms C. siamensis has been intentionally hybridised with either Cuban crocodiles, C. rhombifer, or the estuarine crocodile, C. porosus, and hybrids may be difficult to distinguish morphologically. We report on the combined use of microsatellite and mtDNA genetic markers to determine the species status of potential founder individuals for reintroduction of C. siamensis. Genetic markers were used to characterise 103 captive and wild-caught individuals of C. siamensis, C. rhombifer and C. porosus in Vietnam and to distinguish purebred versus hybrid individuals. Although the microsatellite loci used had some overlap of allele sizes among species, assignment tests allowed differentiation. Four hybrids were identified, two of which had not been recognised morphologically as hybrids, and one of these was thought to be a C. siamensis suitable for reintroduction. Ten of the identified purebred C. siamensis were subsequently released into Cat Tien National Park in southern Vietnam.     

Karyotypic study of four gibbon forms provisionally considered as subspecies of Hylobates (Nomascus) concolor (Primates, Hylobatidae)

We report here a karyotypic study of 6 individuals of Hylobates concolor leucogenys, 2H, concolor siki, 3H, concolor gabriellae, 1 hybrid H, concolor leucogenys x H. concolor ski, 3 hybrid H. concolor gabriellae x H. concolor siki and 2 hybrid H. concolor hainanus x H. concolor leucogenys. Difficulties raised by the morphological identification of subspecies are discussed, and a new morphological characteristic for recognising female H. concolor gabriellae is described. Each of the 4 subspecies appears to be distinguishable on the basis of its karyotype: H. concolor leucogenys differs from H. concolor siki by a reciprocal translocation, from H. concolor hainanus by a pericentric inversion and from H. concolor gabriellae by the presence of both of these two rearrangements. However, these data do not allows us to identify a phylogenetic relationship between the subspecies because, with respect to the karyotypes, none occupies an ancestral position in comparison with the others.     

A complete species-level phylogeny of the Hylobatidae based on mitochondrial ND3-ND4 gene sequences

The Hylobatidae (gibbons) are among the most endangered primates and their evolutionary history and systematics remain largely unresolved. We have investigated the species-level phylogenetic relationships among hylobatids using 1257 bases representing all species and an expanded data set of up to 2243 bases for select species from the mitochondrial ND3-ND4 region. Sequences were obtained from 34 individuals originating from all 12 recognized extant gibbon species. These data strongly support each of the four previously recognized clades or genera of gibbons, Nomascus, Bunopithecus, Symphalangus, and Hylobates, as monophyletic groups. Among these clades, there is some support for either Bunopithecus or Nomascus as the most basal, while in all analyses Hylobates appears to be the most recently derived. Within Nomascus, Nomascus sp. cf. nasutus is the most basal, followed by N. concolor, and then a clade of N. leucogenys and N. gabriellae. Within Hylobates, H. pileatus is the most basal, while H. moloch and H. klossii clearly, and H. agilis and H. muelleri likely form two more derived monophyletic clades. The segregation of H. klossii from other Hylobates species is not supported by this study. The present data are (1) consistent with the division of Hylobatidae into four distinct clades, (2) provide the first genetic evidence for all the species relationships within Nomascus, and (3) call for a revision of the current relationships among the species within Hylobates. We propose a phylogenetic tree as a working hypothesis against which intergeneric and interspecific relationships can be tested with additional genetic, morphological, and behavioral data.     

ECOLOGICAL SEGREGATION IN A SMALL MAMMAL HYBRID ZONE: HABITAT‐SPECIFIC MATING OPPORTUNITIES AND SELECTION AGAINST HYBRIDS RESTRICT GENE FLOW ON A FINE SPATIAL SCALE

The degree to which closely related species interbreed is determined by a complex interaction of ecological, behavioral, and genetic factors. We examine the degree of interbreeding between two woodrat species, Neotoma bryanti and N. lepida, at a sharp ecological transition. We identify the ecological association of each genotypic class, assess the opportunity for mating between these groups, and test whether they have similar patterns of year‐to‐year persistence on our study site. We find that 13% of individuals have a hybrid signature but that the two parental populations and backcrosses are highly segregated by habitat type and use. Also, we find that adult hybrids are comparable to parental types in terms of year‐to‐year persistence on our site but that, among juveniles, significantly fewer hybrids reach adulthood on site compared to their purebred counterparts. Our analyses show that this hybrid zone is maintained by occasional nonassortative mating coupled with hybrid fertility, but that these factors are balanced by lower apparent survival of juvenile hybrids and habitat‐based preference or selection that limits heterospecific mating while promoting backcrossing to habitat‐specific genotypes. This system presents a novel example of the role that sharp resource gradients play in reproductive isolation and the potential for genetic introgression.     

Relative performance of hybrid nestlings in Ficedula flycatchers: a translocation experiment

Ecological speciation predicts that hybrids should experience relatively low fitness in the local environments of their parental species. In this study, we performed a translocation experiment of nestling hybrids between collared and pied flycatchers into the nests of conspecific pairs of their parental species. Our aim was to compare the performance of hybrids with purebred nestlings. Nestling collared flycatchers are known to beg and grow faster than nestling pied flycatchers under favorable conditions, but to experience higher mortality than nestling pied flycatchers under food limitation. The experiment was performed relatively late in the breeding season when food is limited. If hybrid nestlings have an intermediate growth potential and begging intensity, we expected them to beg and grow faster, but also to experience lower survival than pied flycatchers. In comparison with nestling collared flycatchers, we expected them to beg and grow slower, but to survive better. We found that nestling collared flycatchers indeed begged significantly faster and experienced higher mortality than nestling hybrids. Moreover, nestling hybrids had higher weight and tended to beg faster than nestling pied flycatchers, but we did not detect a difference in survival between the latter two groups of nestlings. We conclude that hybrid Ficedula nestlings appear to have a better intrinsic adaptation to food limitation late in the breeding season compared with nestling collared flycatchers. We discuss possible implications for gene flow between the two species.     

The molecular and evolutionary basis of reproductive isolation in plants

Reproductive isolation is defined as processes that prevent individuals of different populations from mating, survival or producing fertile offspring. Reproductive isolation is critical for driving speciation and maintaining species identity, which has been a fundamental concern in evolutionary biology. In plants, reproductive isolation can be divided into prezygotic and postzygotic reproductive barriers, according to its occurrence at different developmental stages. Postzygotic reproductive isolation caused by reduced fitness in hybrids is frequently observed in plants, which hinders gene flow between divergent populations and has substantial effects on genetic differentiation and speciation, and thus is a major obstacle for utilization of heterosis in hybrid crops. During the past decade, China has made tremendous progress in molecular and evolutionary basis of prezygotic and postzygotic reproductive barriers in plants. Present understandings in reproductive isolation especially with new data in the last several years well support three evolutionary genetic models, which represent a general mechanism underlying genomic differentiation and speciation. The updated understanding will offer new approaches for the development of wide-compatibility or neutral varieties, which facilitate breeding of hybrid rice as well as other hybrid crops.     

Efficiency of model-based Bayesian methods for detecting hybrid individuals under different hybridization scenarios and with different numbers of loci

Accurate detection of offspring resulting from hybridization between individuals of distinct populations has a range of applications in conservation and population genetics. We assessed the hybrid identification efficiency of two methods (implemented in the STRUCTURE and NEWHYBRIDS programs) which are tailored to identifying hybrid individuals but use different approaches. Simulated first- and second-generation hybrids were used to assess the performance of these two methods in detecting recent hybridization under scenarios with different levels of genetic divergence and varying numbers of loci. Despite the different approaches of the methods, the hybrid detection efficiency was generally similar and neither of the two methods outperformed the other in all scenarios assessed. Interestingly, hybrid detection efficiency was only minimally affected by whether reference population allele frequency information was included or not. In terms of genotyping effort, efficient detection of F1 hybrid individuals requires the use of 12 or 24 loci with pairwise F(ST) between hybridizing parental populations of 0.21 or 0.12, respectively. While achievable, these locus numbers are nevertheless higher than the number of loci currently commonly applied in population genetic studies. The method of STRUCTURE seemed to be less sensitive to the proportion of hybrids included in the sample, while NEWHYBRIDS seemed to perform slightly better when individuals from both backcross and F1 hybrid classes were present in the sample. However, separating backcrosses from purebred parental individuals requires a considerable genotyping effort (at least 48 loci), even when divergence between parental populations is high.     

Hybrid asexuality as a primary postzygotic barrier between nascent species: On the interconnection between asexuality,hybridization and speciation

Although sexual reproduction is ubiquitous throughout nature, the molecular machinery behind it has been repeatedly disrupted during evolution, leading to the emergence of asexual lineages in all eukaryotic phyla. Despite intensive research, little is known about what causes the switch from sexual reproduction to asexuality. Interspecific hybridization is one of the candidate explanations, but the reasons for the apparent association between hybridization and asexuality remain unclear. In this study, we combined cross‐breeding experiments with population genetic and phylogenomic approaches to reveal the history of speciation and asexuality evolution in European spined loaches (Cobitis). Contemporary species readily hybridize in hybrid zones, but produce infertile males and fertile but clonally reproducing females that cannot mediate introgressions. However, our analysis of exome data indicates that intensive gene flow between species has occurred in the past. Crossings among species with various genetic distances showed that, while distantly related species produced asexual females and sterile males, closely related species produce sexually reproducing hybrids of both sexes. Our results suggest that hybridization leads to sexual hybrids at the initial stages of speciation, but as the species diverge further, the gradual accumulation of reproductive incompatibilities between species could distort their gametogenesis towards asexuality. Interestingly, comparative analysis of published data revealed that hybrid asexuality generally evolves at lower genetic divergences than hybrid sterility or inviability. Given that hybrid asexuality effectively restricts gene flow, it may establish a primary reproductive barrier earlier during diversification than other “classical” forms of postzygotic incompatibilities. Hybrid asexuality may thus indirectly contribute to the speciation process.     

Multigenerational outbreeding effects in Chinook salmon (Oncorhynchus tshawytscha)

Outbreeding, mating between genetically divergent individuals, may result in negative fitness consequences for offspring via outbreeding depression. Outbreeding effects are of notable concern in salmonid research as outbreeding can have major implications for salmon aquaculture and conservation management. We therefore quantified outbreeding effects in two generations (F₁ hybrids and F₂ backcrossed hybrids) of Chinook salmon (Oncorhynchus tshawytscha) derived from captively-reared purebred lines that had been selectively bred for differential performance based on disease resistance and growth rate. Parental lines were crossed in 2009 to create purebred and reciprocal hybrid crosses (n = 53 families), and in 2010 parental and hybrid crosses were crossed to create purebred and backcrossed hybrid crosses (n = 66 families). Although we found significant genetic divergence between the parental lines (F_ST = 0.130), reciprocal F₁ hybrids showed no evidence of outbreeding depression (hybrid breakdown) or favorable heterosis for weight, length, condition or survival. The F₂ backcrossed hybrids showed no outbreeding depression for a suite of fitness related traits measured from egg to sexually mature adult life stages. Our study contributes to the current knowledge of outbreeding effects in salmonids and supports the need for more research to better comprehend the mechanisms driving outbreeding depression.     

Behavioural isolation may facilitate homoploid hybrid speciation in cichlid fish

Hybrid speciation is constrained by the homogenizing effects of gene flow from the parental species. In the absence of post‐mating isolation due to structural changes in the genome, or temporal or spatial premating isolation, another form of reproductive isolation would be needed for homoploid hybrid speciation to occur. Here, we investigate the potential of behavioural mate choice to generate assortative mating among hybrids and parental species. We made three‐first‐generation hybrid crosses between different species of African cichlid fish. In three‐way mate‐choice experiments, we allowed hybrid and nonhybrid females to mate with either hybrid or nonhybrid males. We found that hybrids generally mated nonrandomly and that hybridization can lead to the expression of new combinations of traits and preferences that behaviourally isolate hybrids from both parental species. Specifically, we find that the phenotypic distinctiveness of hybrids predicts the symmetry and extent of their reproductive isolation. Our data suggest that behavioural mate choice among hybrids may facilitate the establishment of isolated hybrid populations, even in proximity to one or both parental species.     

Genetic monitoring of two decades of hybridization between allis shad (Alosa alosa) and twaite shad (Alosa fallax)

Habitat alteration has been implicated in driving hybridization between the sympatric migratory shads Alosa alosa and Alosa fallax. Morphological and molecular evidence is consistent with hybridization across the overlapping range of these species, but the temporal extent of hybrid occurrence and genetic consequences for populations have not been explored. Using eight nuclear microsatellite loci and samples collected between 1989 and 2008 in the Solway Firth (UK), we genetically identified hybrids, studied temporal changes in their frequency, and explored changes in allele frequencies of parental populations. These molecular data confirmed the hybrid status of individuals identified using morphology (number of rakers on the outer gill arch), and enabled separation of hybrids from purebred individuals. Mitochondrial cytochrome-b sequencing revealed the presence of two haplogroups, each predominantly occurring in one species. Heterospecific haplotypes were found in 22.3 and 12.8% of A. alosa and A. fallax individuals, respectively, consistent with backcrossing and suggesting that hybrids are fertile. On average, microsatellite-identified hybrids comprised 12.7% of all samples, but when individuals with cytonuclear discordance were also considered introgressed on average 25.4% of individuals were of hybrid ancestry. Overall, allelic richness remained largely unchanged within species, but there were declines in the inbreeding coefficient (F _IS) of both species and episodes of significant temporal allelic frequency change. Hybrids sampled between 2004 and 2008 showed no evidence of lower fecundity relative to purebred individuals. Together, results suggest that hybridization between shad species in northern Europe is prevalent, and has been ongoing over at least two decades. The challenge is now to understand the extent to which observed patterns are linked to immigration from other populations, and the mechanisms that have prevented species collapse despite apparent hybrid fertility and longstanding introgression of neutral markers.     

鲟鱼分子鉴定方法的整合应用

鲟形目物种是国家重点保护水生野生动物和CITES附录物种。其人工养殖种群数量众多, 种类丰富, 产品贸易量大, 但种类鉴定困难。本文在厘清当前鲟鱼商业类群的基础上, 通过分析现有种类鉴定方法, 整合了线粒体DNA遗传分析、SNP分析和微卫星DNA分析的鉴定方法, 探讨其鉴定国际贸易所涉鲟鱼的可行性。结果表明: 上述3种方法整合应用可在11种纯种鲟鱼及其正反杂交产生的杂交鲟范围内进行盲检。当前共有贸易鲟鱼36种, 其中杂交鲟14种, 杂交鲟的亲本共涉及9种鲟鱼。整合方法可准确鉴定小体鲟(Acipenser ruthenus)、达氏鳇(Huso dauricus)、施氏鲟(Acipenser schrenckii)、欧洲鳇(Huso huso)、闪光鲟(Acipenser stellatus)、高首鲟(A. transmontanus)两两杂交所产生的杂交鲟, 小体鲟为母本与纳氏鲟(Acipenser naccarii)或富氏鲟(A. fulvescens)或中华鲟(A. sinensis)产生的杂交鲟, 纯种的达氏鳇、高首鲟、富氏鲟和中华鲟, 但无法准确鉴定纯种的西伯利亚鲟(Acipenser baerii)和俄罗斯鲟(A. gueldenstaedti)以及父母本涉及此两种鲟鱼的杂交鲟。由于已开发的分子标记仍有限, 上述结果是对当前CITES贸易所涉鲟鱼鉴定的最大范围, 可以满足一些鲟鱼野生种群保护、贸易产品检测、种质资源管理等情景下的鉴定需求。     

Methodological approaches for studying the european water frog <Emphasis Type="Italic">Pelophylax esculentus</Emphasis> complex

The European water frog (Pelophylax esculentus) complex represents a unique and adequate model system for the study of interspecific hybridization and the mechanisms enabling interspecific hybrids to overcome the reproductive barriers. The difficulties in the study of individuals from the P. esculentus complex are associated with high polymorphism of morphological characters in parental species and interspecific hybrids, as well as with the presence of polyploid hybrid forms. From the discovery of the phenomenon of interspecific hybridization and the demonstration of successful reproduction of interspecific hybrids, researchers constantly searched for the methods necessary for the most accurate identification of parental species and various hybrid forms. This review describes biochemical, cytogenetic, and molecular methods and approaches used to identify individuals from the European water frog complex, as well as to analyze the genomes transferred with the gametes of hybrids. The advantages and disadvantages of these approaches are discussed. The presented methods can be used for studying other hybrid complexes of fish, amphibians, and reptiles.     

Incomplete reproductive isolation between Rhododendron taxa enables hybrid formation and persistence

The evolutionary consequences of hybridization ultimately depend on the magnitude of reproductive isolation between hybrids and their parents. We evaluated the relative contributions of pre-and post-zygotic barriers to reproduction for hybrid formation, hybrid persistence and potential for reproductive isolation of hybrids formed between two Rhododendron species,R. spiciferum and R. spinuliferum. Our study established that incomplete reproductive isolation promotes hybrid formation and persistence and delays hybrid speciation.All pre-zygotic barriers to reproduction leading to hybrid formation are incomplete: parental species have overlapping flowering; they share the same pollinators;reciprocal assessments of pollen tube germination and growth do not differ among parents. The absence of post-zygotic barriers between parental taxa indicates that the persistence of hybrids is likely. Reproductive isolation was incomplete between hybrids and parents in all cases studied, although asymmetric differences in reproductive fitness were prevalent and possibly explain the genetic structure of natural hybrid swarms where hybridization is known to be bidirectional but asymmetric. Introgression, rather than speciation, is a probable evolutionary outcome of hybridization between the two Rhododendron taxa. Our study provides insights into understanding the evolutionary implications of natural hybridization in woody plants.     

Chromosomal phylogeny and evolution of gibbons (Hylobatidae)

Although human and gibbons are classified in the same primate superfamily (Hominoidae), their karyotypes differ by extensive chromosome reshuffling. To date, there is still limited understanding of the events that shaped extant gibbon karyotypes. Further, the phylogeny and evolution of the twelve or more extant gibbon species (lesser apes, Hylobatidae) is poorly understood, and conflicting phylogenies have been published. We present a comprehensive analysis of gibbon chromosome rearrangements and a phylogenetic reconstruction of the four recognized subgenera based on molecular cytogenetics data. We have used two different approaches to interpret our data: (1) a cladistic reconstruction based on the identification of ancestral versus derived chromosome forms observed in extant gibbon species; (2) an approach in which adjacent homologous segments that have been changed by translocations and intra-chromosomal rearrangements are treated as discrete characters in a parsimony analysis (PAUP). The orangutan serves as an "outgroup", since it has a karyotype that is supposed to be most similar to the ancestral form of all humans and apes. Both approaches place the subgenus Bunopithecus as the most basal group of the Hylobatidae, followed by Hylobates, with Symphalangus and Nomascus as the last to diverge. Since most chromosome rearrangements observed in gibbons are either ancestral to all four subgenera or specific for individual species and only a few common derived rearrangements at subsequent branching points have been recorded, all extant gibbons may have diverged within relatively short evolutionary time. In general, chromosomal rearrangements produce changes that should be considered as unique landmarks at the divergence nodes. Thus, molecular cytogenetics could be an important tool to elucidate phylogenies in other species in which speciation may have occurred over very short evolutionary time with not enough genetic (DNA sequence) and other biological divergence to be picked up.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Scent variation and hybridization cause the displacement of a sexually deceptive orchid species

In the sexually deceptive orchid genus Ophrys, reproductive isolation is based on the specific attraction of males of a single pollinator species, mostly bees, by mimicking the female sex pheromone of this species. Changes in the floral odor can lead to hybridization, introgression, and possibly speciation. We investigated hybrid swarms of O. lupercalis and O. iricolor on Sardinia using behavioral, electrophysiological (GC-EAD), chemical, morphological, and genetic methods (AFLPs). In behavioral experiments, approximately 20% of the flowers from both species and hybrids were attractive to the "wrong" or both pollinator species. Analysis of the EAD-active hydrocarbons in the floral odor showed an overlap in the two species, whereby hybrid individuals could not be separated from O. iricolor. The genetic analysis confirmed the hybridization of the species. Plants of O. iricolor and hybrids are genetically indistinguishable and form an O. iricolor × lupercalis hybrid population. Remaining plants of O. lupercalis will possibly be displaced by the O. iricolor × lupercalis hybrid population in the future. Our study showed that in deceptive orchids, variation in the pollinator attracting cues, in this case, scent, can be the first step for speciation and at the same time cause the displacement of a species.     

Confirmation of natural hybrids between Gentiana straminea and G. siphonantha (Gentianaceae) based on molecular evidence

A few individuals with intermediate morpho-logy always appeared in the sympatric distributions of Gentiana straminea and G. siphonantha. These intermedi-ate individuals were hypothesized to be the hybrids of two species after a careful evaluation of their morphological characteristics. To test this hypothesis, sequence compar-ison of the internal transcribed spacer (ITS) regions of the nuclear ribosomal and trnS (GCU)-trnG (UCC) inter-genic spacer region of the chloroplast DNA from Gentiana straminea, G. siphonantha and the putative hybrids was performed. The results suggest that most intermediate individuals were the natural hybrids between G. straminea and G. siphonantha. In addition, we exam-ined the sequence variation among the individuals of both parent species and analyzed the possibility leading to the incongruent identification in some individuals based on morphologic and molecular evidences, respectively. The intraspecific diversification of DNA fragments within both parent species and their high variability in hybrid swarms probably resulted from chloroplast genome recombination and incomplete lineage sorting during the early stages of speciation origin of the parent species.     

麻花艽和管花秦艽(龙胆科)之间自然杂交类型的分子验证

野外考察发现麻花艽(Gentiana stramineaMaxim.)和管花秦艽(G.siphonantha Maxim.ex Kusn.)同域分布时存在大量形态位于二者之间的个体。经形态变异研究后发现它们可能是这两个物种之间的杂交后代。对两个亲本种以及假设杂交群体共55个个体的核糖体ITS序列和叶绿体trnS-G序列的分析结果表明:中间形态个体是麻花艽和管花秦艽的自然杂交后代。此外分析了两个亲本种以及杂交群内个体间trnS-G和ITS序列的变异状况以及分子标记结果与形态鉴定不一致的可能原因;指出可能是杂交诱导的叶绿体基因组重组以及早期物种分化中的谱系筛选不彻底等原因造成了亲本种群体内序列变异的多样化。     

Microsatellites and single nucleotide polymorphisms in avian hybrid identification: a comparative case study

The correct identification of hybrids is essential in avian hybridisation studies, but selection of the appropriate set of genetic markers for this purpose is at times complicated. Microsatellites and single nucleotide polymorphisms (SNPs) are currently the most commonly used markers in this field. We compare the efficiency of these two marker types, and their combination, in the identification of the threatened avian species, the greater spotted eagle and the lesser spotted eagle, as well as hybrids between the two species. We developed novel SNP markers from genome-wide distributed 122 candidate introns using only sympatric samples, and tested these markers successfully in 60 sympatric and allopatric spotted eagles using Bayesian model-based approaches. Comparatively, only one out of twelve previously described avian nuclear intron markers showed significant species-specific allele frequency difference, thus stressing the importance of selecting the proper markers. Twenty microsatellites outperformed selected nine SNPs in species identification, but were poorer in hybrid detection, whereas the resolution power of ten microsatellites remained too low for correct assignment. A combination of SNPs and microsatellites resulted in the most efficient and accurate identification of all individuals. Our study shows that the use of various sets of markers could lead to strikingly different assignment results, hybridisation studies may have been affected by too low a resolution power of used markers, and that an appropriate set of markers is essential for successful hybrid identification.