Phylogeny and phylogeography of critically endangered Gyps species based on nuclear and mitochondrial markers

Populations of Oriental White-backed Vulture (Gyps bengalensis) and Long-billed Vulture (Gyps indicus) dramatically declined by 95–100% on the Indian subcontinent in mid-1990s. The present study was conducted to discover the phylogeny and phylogeography of Gyps species based on nuclear (recombination activating gene, RAG-1) and mitochondrial (cytochrome b, cytb) markers. Gyps species showed monophyly and no geographic partition was observed within the three groups of Gyps species (G. bengalensis, G. indicus and G. fulvus) despite the large sample size available (n = 149). Our study supports the treatment of G. indicus and G. tenuirostris as separate species. In all analyses, the earliest divergence separated G. bengalensis from all other Gyps taxa while a sister relationship was supported between G. fulvus and G. rueppellii, and these two taxa together were sister group to a clade consisting of G. indicus, G. tenuirostris and G. coprotheres. Molecular clock estimates of both nuclear and mitochondrial (RAG-1, cytb) genes indicated a rapid and recent diversification within the Gyps species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The use of a triploid hybrid for introgression in Lolium species

Summary Triploid hybrids of Lolium multiflorum (4x) x L. perenne (2x) behaved cytologically as autotriploids but the segregation of isozyme variants did not always agree with the expected trisomic ratios. The overall effect of these deviations from expectation was a greater proportion than expected of diploid progeny from the cross L. multiflorum (2x) x triploid hybrid which did not include any of the L. perenne alleles at the three marker isozyme loci. The possible mechanisms for these aberrant segregation ratios are discussed together with the advantages of the crossing scheme to accelerate the recovery of the genotype of the recurrent parent in a backcrossing programme to transfer characters from one species to another.     

Thinopyrum bessarabicum: biochemical and cytological markers for the detection of genetic introgression in its hybrid derivatives with Triticum aestivum L.

Summary Thinopyrum bessarabicum (2n = 2x = 14, JJ) with its unique property of salt tolerance provides a potential means for the transfer of this important and complex trait into cultivated wheat through intergeneric hybridization. To accomplish this, diagnostic markers for detecting the presence of Th. bessarabicum chromosomes in a wheat background have to be established. The C-banded karyotype of Th. bessarabicum distinctly identifies individual Th. bessarabicum chromosomes and separates them from those of Triticum aestivum. Also, seven protein/isozymes, i.e., malate dehydrogenase, high-molecular-weight glutenin, Superoxide dismutase, grain esterase, glutamate oxaloacetate transaminase, -amylase and -amylase, were identified as being positive markers specific to Th. bessarabicum; these were also expressed in the T. aestivum/Th. bessarabicum amphiploid. These diagnostic biochemical markers could be useful in detecting and establishing homoeology of Th. bessarabicum chromosomes in T. aestivum/Th. bessarabicum intergeneric hybrid derivatives.     

Biogeography and conservation biology of Florida's Geolycosa wolf spiders: threatened spiders in endangered ecosystems

The state of Florida has a complex biogeography which is the product of past sea level changes and associated ocean currents creating numerous inland sand ridge systems which are home to a unique biota adapted to the seasonally dry conditions found on these sandy uplands. Among these xeric-adapted taxa is a suite of wolf spider species in the genus Geolycosa. Geolycosa wolf spiders are habitat specialists and poor dispersers, and many taxa have ranges restricted to specific ridge systems. We found that the six Geolycosa species typical of Florida's scrubs and sandhills (G. escambiensis Wallace, G. ornatipes (Bryant), G. micanopy Wallace, G. patellonigra Wallace, G. hubbelli Wallace and G. xera McCrone) have current distributions that correlate with the historical biogeography of the state. There are species typical of the Lake Wales Ridge and associated ridges (G. xera and G. hubbelli), species found on the more coastal ridges (G. patellonigra and G. micanopy) and species whose ranges are divided by the Apalachicola River drainage (G. escambiensis and G. ornatipes). We also found that Geolycosa wolf spiders tend to occur in species pairs which use different microhabitats: those which burrow in areas covered in leaf litter, and those which use only barren sites. These latter species require habitat management plans that include burns, that maintain the habitat in an open condition.     

The utility of nuclear introns for investigating hybridization and genetic introgression: a case study involving Brachyramphus murrelets

Interspecific hybridization can have importantconsequences for conservation, but can bedifficult to detect using morphologicalmarkers. To test the utility of nuclear intronsfor investigating hybridization and geneticintrogression, we analyzed variation in fivenuclear introns and the mitochondrialcytochrome b gene in two species ofseabirds that are declining and may behybridizing: marbled murrelets(Brachyramphus marmoratus) and Kittlitz'smurrelets (B. brevirostris). No alleleswere shared between samples of the two species,and intron alleles formed reciprocallymonophyletic groups in gene trees. Our resultssuggest that few murrelets in Alaska areF ₁, F ₂or back-cross hybrids,and that gene pools of these species have beenindependent for 1.8 to 5.7 million years. Weconclude that introns are a potentially richsource of markers for analyzing hybridizationand introgression in endangered or decliningspecies.     

11 S seed storage proteins fromHelianthus species (Compositae): Biochemical,size and charge heterogeneity

The seeds of 19 sunflower species were compared on the basis of their protein contents and the relative proportions of their protein fractions. The globulin content varied from 50% to about 70% and the albumin content from 18% to 35% according to the species. The level of intermediateMr polypeptides showed a great variability (9.6 to 24.3%). Comparative studies onMr polymorphism were carried out by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of non reduced and/or reduced samples using both mono- and bidimensional procedures. Polypeptide constituents of helianthinin were compared including both number and molecular size (cultivatedH. annuus was used as a standard). Studies focused on differences observed between the major two (Mr 38 000), (Mr 32 000) and (Mr 25 500), (21 000) polypeptides families constituting the main A, B, and C subunits. and polypeptides analyses permit to discriminate easilyH. petiolaris from the other species. Charge polymorphism was studied using isoelectric focusing (IEF) and IEF-PAGE in mono and bidimensional procedures in the presence or absence of 2-mercaptoethanol (2-ME). Only a specific ₄ polypeptide enables an easy discrimination betweenH. petiolaris and all the other species. Detailed nomenclature of the , and , polypeptides constituting the different helianthinin globulin subunits is given via the results of pI andMr analyses. Monodimensional IEF patterns of the more basic albumins (pI > 8.0) appear to provide a more valuable approach to identifying specific protein markers.     

Biochemical genetic markers in sugarcane

Summary Isozyme variation was used to identify biochemical markers of potential utility in sugarcane genetics and breeding. Electrophoretic polymorphism was surveyed for nine enzymes among 39 wild and noble sugarcane clones, belonging to the species most closely related to modern varieties. Up to 114 distinct bands showing presence versus absence type of variation were revealed and used for qualitative characterization of the materials. Multivariate analysis of the data isolated the Erianthus clone sampled and separated the Saccharum spontaneum clones from the S. robustum and S. officinarum clones; the latter two were not differentiated from one another. The analysis of self-progenies of a 2n=112 S. spontaneum and of a commercial variety showed examples of mono- and polyfactorial segregations. Within the progeny of the variety, co-segregation of two isozymes frequent in S. spontaneum led to them being assigned to a single chromosome initially contributed by a S. spontaneum donor. This illustrates how combined survey of ancestral species and segregation analysis in modern breeding materials should permit using the lack of interspecific cross-over to establish linkage groups in a sugarcane genome.     

Genetic relationships among Mediterranean Pistacia species evaluated by RAPD and AFLP markers

Polymorphisms among Mediterranean basin Pistacia species and accessions within species were assessed by random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses. Twenty-eight Pistacia accessions representing six species from geographically diverse locations in the Mediterranean area were analyzed. With RAPD, a total of 259 DNA fragments were amplified by 27 pre-selected primers, 254 were polymorphic fragments. AFLP analysis with 15 primer sets, produced 954 (93%) polymorphic bands out of a total of 1026. A Mantel test revealed an extremely high correlation (r=0.99) between similarity matrices generated from RAPD and AFLP data sets, indicating that similar results were obtained by the two techniques. Dendrograms constructed from the similarity matrices showed that Pistacia species could be clustered into two groups, one group containing all the #E5/E5#. lentiscus and the second group containing all other accessions. The latter group was divided into two subgroups, one consisting of #E5/E5#. palaestina and #E5/E5#. terebinthus; the other consisting of #E5/E5#. atlantica, #E5/E5#. khinjuk and #E5/E5#. vera. P. vera and P. khinjuk were highly similar, as were P. palaestina and P. terebinthus.     

A suite of molecular markers for identifying species, detecting introgression and describing population structure in spadefoot toads (Spea spp.)

Two congeneric species of spadefoot toad, Spea multiplicata and Spea bombifrons, have been the focus of hybridization studies since the 1970s. Because complex hybrids are not readily distinguished phenotypically, genetic markers are needed to identify introgressed individuals. We therefore developed a set of molecular markers (amplified fragment length polymorphism, polymerase chain reaction-restriction fragment length polymorphism and single nucleotide polymorphism) for identifying pure-species, F1 hybrids and more complex introgressed types. To do so, we tested a series of markers across both species and known hybrids using populations in both allopatry and sympatry. We retained those markers that differentiated the two pure-species and also consistently identified known species hybrids. These markers are well suited for identifying hybrids between these species. Moreover, those markers that show variation within each species can be used in conjunction with existing molecular markers in studies of population structure and gene flow.     

Modulation of a neuronal calmodulin mRNA species in the rat brain stem by reserpine

Reserpine evokes transsynaptic impulse activity by depleting catecholaminergic neurotransmitters in the rat brain. Previous studies suggest a relationship between catecholaminergic activity and calmodulin concentration. In this report we employ Northern blot analysis to examine the effect of a single subcutaneous injection of reserpine on levels of calmodulin mRNA species which are preferentially expressed in neurons of the rat brain. Regional differences in mRNA levels were also investigated byin situ hybridization and drug-induced changes were noted particularly in specific regions of the rat brain stem. The riboprobe used in thein situ hybridization study recognized a 4.0 kilobase neuronal calmodulin mRNA species (NGB1), which was derived from the rat CaM1 gene. A calmodulin radio-immunoassay was utilized to demonstrate a drug-induced increased in calmodulin protein levels in a region which included the brain stem.     

Allozymes,mtDNA and microsatellites study introgression in a stocked trout population in France

A comparative study of disturbed (downstream) and undisturbed (upstream) populations in a French Mediterranean river (the Sorgue) was conducted to assess the fate of introgressing hatchery-genes in the aboriginal gene pool. Variation was assessed at 27 protein-coding loci, three microsatellite loci and two mtDNA restriction sites. The results were compared to those from another Mediterranean drainage basin (that of the river Orb). The genetic pattern observed among markers and drainage basins was found to be noticeably different. A reduction in population size is hypothesized to explain the lower variability observed in the upper part of the river Sorgue. Estimations of hatchery gene flow were different between loci but in the same range of values between markers.     

Rapid identification of white-Engelmann spruce species by RAPD markers

Fragments of random amplified polymorphic DNA (RAPDs) were used as markers to distinguish Picea glauca (Moench) Voss (white spruce) and Picea engelmannii Parry (Engelmann spruce). These species and their putative hybrids are difficult to differentiate morphologically and are collectively known as interior spruce. Four oligodeoxynucleotide decamer primers showed species-specific amplification products between white spruce and Engelmann spruce. These fragments are highly conserved among seed lots and individual trees of each species from diverse geographic origins. The consistency and reproducibility of these species-specific amplification products were tested in more than two amplification reactions. Therefore, RAPD markers can provide genetic markers for easy and rapid identification of the specific genetic entry of these spruce species and their reported putative hybrids. According to the frequencies of the species-specific RAPD markers, it is possible to estimate the hybrid fraction, indicative of true introgression between the two species. These results are useful for quick identification of both species and their hybrid swarms at any stage in the sporophyte phase of the life cycle, for determining the occurrence and the magnitude of introgressive hybridization in an overlap zone between the two species, and for certification purposes in operational re-forestation and tree-improvement programs.     

The endangered Sonoran topminnow: Examination of species and ESUs using three mtDNA genes

There has been controversy over the species status of Sonoran topminnows and debate about the presence of ESUs in the Gila topminnow. From examination of sequence variation at 2626 base pairs over three mtDNA genes, we found a 29 (1.1%) nucleotide genetic difference between Gila and Yaqui topminnows. This provides strong support that these two taxa are separate species, Poeciliopsis occidentalis (Gila topminnow) and P. sonoriensis (Yaqui topminnow) and have been separated for approximately one million years. All the Gila topminnows within Arizona have the same sequence for the three mtDNA genes, that is, there is not reciprocal monophyly for mtDNA sequence data for the two previously designated ESUs. However, evidence of the unique habitat for Monkey Spring, its long-term isolation from other Gila topminnow habitats, and the presence of unique fish and invertebrate taxa in Monkey Spring support the designation of the Monkey Spring topminnows as an ESU. Finally, theoretical considerations using molecular data and estimates of heterozygosity and genetic distance for nuclear genes between populations of the Gila topminnow show that the lack of mtDNA variation is not inconsistent with the level and pattern of nuclear genetic variation observed.     

Host recognition and selection by Aphytis species: Response to California red, oleander, and cactus scale cover extracts

One or more chemicals associated with the host's cover appear to play a major role in host recognition by Aphytis species. Aphytis melinus DeBach, A. lingnanensis Compère, A. coheni DeBach and Comperiella bifasciata Howard, all parasitoids of diaspidid scale-insects, were found to respond to water extracts of California red scale covers, Aonidiella aurantii (Mask.); A. melinus and A. lingnanensis responded to water extracts of cactus scale covers, Diaspis echinocacti (Bouché); and A. chilensis Howard to water extracts of oleander scale covers, Aspidiotus nerii Bouché. A. lingnanensis responded to water extracts of cactus scale covers only if it had been reared on this scale. A. melinus also responded to ethanol extracts of California red scale covers while A. lingnanensis did not. A. melinus failed to respond to water extracts of California red scale covers after a single ovipositional experience on California red scale. Such kairomones might prove useful in screening natural enemies as potential biological control agents for specific hosts as well as in elucidating the mechanisms by which parasitoids of scale-insects differentiate among potential hosts.

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Résumé Aphytis lingnanensis, A. melinus, A. coheni et Comperiella bifasciata réagissent tous à des extraits aqueux d'Aonidiella aurantii, tandis qu'A. chiliensis réagit aux extraits aqueux d'Aspidiotus nérii. La réaction de A. coheni aux extraits aqueux d'A. aurantii confirme les écrits de Quednau et Hübsche (1964). Nous n'avons pas été capables d'observer une réaction de A. chrysomphali aux extraits aqueux d'A. aurantii. Comme nous n'avons pas essayé de traiter les boucliers avec d'autres solvants, nous ne pouvons pas affirmer l'absence de réponse de cet aphélinide aux kairomones de cette cochenille.Que A. melinus et A. lingnanensis aient été élevés sur Aspidiotus nerii ou sur Aonidiella aurantii, ils ne réagissent pas aux extraits aqueux d'A. nerii; qui est accepté comme hôte par ces deux aphélinides au laboratoire. Ces résultats suggèrent que d'autres éléments, comme la forme ou la texture, jouent aussi un rôle important dans la détermination des hôtes. A. melinus et A. lingnanensis réagissent tous deux aux extraits aqueux de boucliers de Diaspis echinocacti; cependant, après élevage sur D. echinocacti, seul A. lingnanensis réagit à l'extrait. La réaction de A. melinus à l'extrait de D. echinocacti suggère que cet aphélinide est préadapté chimiosensoriellement à déceler cette cochenille, dont la distribution est néotropicale tandis que celle de A. melinus orientale. A. melinus a été élevé à partir de D. echinocacti récoltés dans la nature. La réaction de A. lingnanensis aux extraits de D. echinocacti uniquement lorsqu'il a élévé dessus, suggère qu'un conditionnement préimaginal peut aussi jouer un rôle dans la détermination de l'hôte.

     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

Morphological and molecular evidence for hybridization and introgression in Central European Arctium (Asteraceae)

Three species of burdock (Arctium lappa, A. tomentosum, and A. minus; Asteraceae), which commonly occur in sympatry in Central Europe, are thought to hybridize frequently, hence raising questions about their integrity. We surveyed mixed, monotypic, and/or single plant stands of these species in Central Germany to assess their extent of hybridization and introgression, and also the likelihood of insect host shifts between them. Individual plants were characterized by multivariate analyses of morphological and RAPD variation in combination with RAPD-based character-index-scores. All analyses recognized the three species as distinct units, but also identified a limited number of first (or early) generation hybrids between sympatric A. lappa and either A. tomentosum or A. minus, respectively, as well as one introgressant of A. tomentosum into A. lappa. While pre-zygotic isolation may partly account for the rarity of first generation crosses (F₁'s), the near absence of later generation hybrid segregants or introgressants likely reflects post-zygotic isolation due to the disruption of co-adapted gene complexes and/or habitat-mediated superiority shared by the ruderal parentals. As the tephritid fly Tephritis bardanae oviposits on both A. tomentosum (T) and A. minus (M), but not on A. lappa (L), the occurrence of L × M and L × T hybrids and/or introgressants may have important evolutionary consequences regarding the acquisition of a new host, i.e. A. lappa, by these insects.     

Nuclear markers reveal a complex introgression pattern among marine turtle species on the Brazilian coast

Surprisingly, a high frequency of interspecific sea turtle hybrids has been previously recorded in a nesting site along a short stretch of the Brazilian coast. Mitochondrial DNA data indicated that as much as 43% of the females identified as Eretmochelys imbricata are hybrids in this area (Bahia State of Brazil). It is a remarkable find, because most of the nesting sites surveyed worldwide, including some in northern Brazil, presents no hybrids, and rare Caribbean sites present no more than 2% of hybrids. Thus, a detailed understanding of the hybridization process is needed to evaluate natural or anthropogenic causes of this regional phenomenon in Brazil, which could be an important factor affecting the conservation of this population. We analysed a set of 12 nuclear markers to investigate the pattern of hybridization involving three species of sea turtles: hawksbill (E. imbricata), loggerhead (Caretta caretta) and olive ridley (Lepidochelys olivacea). Our data indicate that most of the individuals in the crossings L. olivacea × E. imbricata and L. olivacea × C. caretta are F1 hybrids, whereas C. caretta × E. imbricata crossings present F1 and backcrosses with both parental species. In addition, the C. caretta × E. imbricata hybridization seems to be gender and species biased, and we also found one individual with evidence of multispecies hybridization among C. caretta × E. imbricata × Chelonia mydas. The overall results also indicate that hybridization in this area is a recent phenomenon, spanning at least two generations or ～40 years.     

The effects of mating design on introgression between chromosomally divergent sunflower species

Population genetic theory suggests that mating designs employing one or more generations of sib-crossing or selfing prior to backcrossing are more effective than backcrossing alone for moving alleles across linkage groups where effective recombination rates are low (e.g., chromosomally divergent linkages). To test this hypothesis, we analyzed the effects of chromosomal structural differences and mating designs on the frequency and genomic distribution of introgressed markers using the domesticated sunflower, Helianthus annuus, and one of its wild relatives, H. petiolaris, as the experimental system. We surveyed 170 progeny, representing the end products of three different mating designs (design I, P-F₁-BC₁-BC₂-F₂-F₃; design II, P-F₁-F₂-BC₁-BC₂-F₃; and design III, P-F₁-F₂-F₃-BC₁-BC₂), for 197 parental RAPD markers of known genomic location. Comparison of observed patterns of introgression with expectations based on simulations of unrestricted introgression revealed that much of the genome was protected from introgression regardless of mating design or chromosomal structural differences. Although the simulations indicated that all markers should introgress into multiple individuals in each of the three mating designs, 20 of 58 (34%) markers from collinear linkage groups, and 112 of 139 (81%) markers from rearranged linkage groups did not introgress. In addition, the average size of introgressed fragments (12.2 cM) was less than half that predicted by theoretical models (26–33 cM). Both of these observations are consistent with strong selection against introgressed linkage blocks, particularly in chromosomally divergent linkages. Nonetheless, mating designs II and III, which employed one and two generations of sib-mating, respectively, prior to backcrossing, were significantly more effective at moving alleles across both collinear and rearranged linkages than mating design I, in which the backcross generations preceded sib-mating. Thus, breeding strategies that include sib-crossing, in combination with backcrossing, should significantly increase the effectiveness of gene transfer across complex genic or chromosomal sterility barriers.     

Biochemical analysis of four species ofCassia L. pollen

The present paper describes the comparative biochemical studies in terms of quantitative analyses of carbohydrates, lipids, proteins, free amino acids, nucleic acids, minerals, ash and moisture as well as the identification of free amino acids of pollen of four species ofCassia L. (C. alata L,C. fistula L,C. occidentalis L andC. siamea Lam.). A significant variation in the chemical constituents was observed among the four species.C. occidentalis showed the highest levels of carbohydrate (15.15%) and protein (22.45%), andC. siamea had the lowest levels of carbohydrate (7.15%), lipid (6.2%) and protein (13.85%).C. alata andC. fistula showed intermediate results. However,C. alata showed the highest amount of free amino acids (3.8%) and the least of 1.42% was found inC. fistula. Thin layer chromatography (TLC) of free amino acids of the four species showed some homology in their amino acid content, of which proline, glutamic acid, methionine and phenyl-alanine were the most dominant. The level of nucleic acids and minerals was found to be comparatively low.C. siamea andC. alata showed an exceptionally high level of ash content (8.6 and 8.8%, respectively) while moisture content varied from 8 to 11%.