COMPONENTS OF REPRODUCTIVE ISOLATION BETWEEN ORCHIS MASCULA AND ORCHIS PAUCIFLORA

Studies of the strength and nature of reproductive isolation (RI) between species can greatly contribute to our understanding of speciation. Although the role of RI in speciation is well recognized, there is a dearth of information on the contributions of different barriers between related plant species. Here, we estimated multiple components of RI between two Mediterranean orchid sister species (Orchis mascula and Orchis pauciflora), disentangling the strength and absolute contributions of seven different isolating mechanisms. Our survey includes one prepollination, two postpollination prezygotic (pollen–stigma incompatibility, conspecific pollen precedence), two intrinsic postzygotic (embryo mortality and hybrid sterility) and two extrinsic postzygotic (hybrid habitat differentiation and hybrid pollination) isolating mechanisms. We found strong RI between the investigated species, although none of the barriers were able to completely impede gene flow. Five isolating mechanisms contributed positively to the maintenance of species boundaries. Contrary to most surveys of isolating mechanisms, our data speak against a clear predominance of prepollination or of prezygotic barriers but confirm the emerging pattern of multiple barriers contributing to the maintenance of species integrity. These findings suggest an allopatric condition during early phases of species divergence. We discuss our data in the wider context of previous studies carried out in this orchid group by using a comparative approach.     

Drivers of population genetic differentiation in the wild: isolation by dispersal limitation,isolation by adaptation and isolation by colonization

Empirical population genetic studies have been dominated by a neutralist view, according to which gene flow and drift are the main forces driving population genetic structure in nature. The neutralist view in essence describes a process of isolation by dispersal limitation (IBDL) that generally leads to a pattern of isolation by distance (IBD). Recently, however, conceptual frameworks have been put forward that view local genetic adaptation as an important driver of population genetic structure. Isolation by adaptation (IBA) and monopolization (M) posit that gene flow among natural populations is reduced as a consequence of local genetic adaptation. IBA stresses that effective gene flow is reduced among habitats that show dissimilar ecological characteristics, leading to a pattern of isolation by environment. In monopolization, local genetic adaptation of initial colonizing genotypes results in a reduction in gene flow that fosters the persistence of founder effects. Here, we relate these different processes driving landscape genetic structure to patterns of IBD and isolation by environment (IBE). We propose a method to detect whether IBDL, IBA and M shape genetic differentiation in natural landscapes by studying patterns of variation at neutral and non‐neutral markers as well as at ecologically relevant traits. Finally, we reinterpret a representative number of studies from the recent literature by associating patterns to processes and identify patterns associated with local genetic adaptation to be as common as IBDL in structuring regional genetic variation of populations in the wild. Our results point to the importance of quantifying environmental gradients and incorporating ecology in the analysis of population genetics.     

Isolation of single cell suspensions from the rat mammary gland: Separation,characterization, and primary culture of various cell populations

Summary Isolation and characterization of a single cell suspension from the rat mammary gland was achieved by combining selective enzymatic digestion and the mechanical agitation of a Stomacher laboratory blender with immunohistological identification of cell-specific markers. Utilizing this procedure we were able to isolate single cell suspensions of high yield (10 to 15×10⁶ cells/rat) and viability (>98%) with a concurrent decrease in isolation time and the amount of proteolytic enzymes required. Five distinct cell fractions were isolated from the mammary gland cell suspension after banding on discontinuous Percoll gradients. These populations were characterized both before and after primary cell culture by a combination of histological, immunohistological, and autoradiographic techniques. Fractions two and three were found to be enriched for mammary epithelial cells, as identified by their high binding of antikeratin antibodies. These populations also exhibited a minimal degree of binding to actin, myosin, and fibronectin antibodies. Fraction three also exhibited a high labeling index as measured by autoradiography following in vivo administration of [methyl-³H]thymidine. The remaining fractions were found to contain higher percentages of myoepithelial cells or other mammary cell types. Inasmuch as there is a direct correlation between mammary gland cell types and susceptibility to mammary gland carcinomas, further studies of these cell populations may provide new insights into the mechanisms underlying mammary gland carcinogenesis. This work was supported by grant R809580 from the U. S. Environmental Protection Agency, Office of Research Grants and Centers, Washington, D. C.     

Evolution of reproductive isolation in stickleback fish

To understand how new species form and what causes their collapse, we examined how reproductive isolation evolves during the speciation process, considering species pairs with little to extensive divergence, including a recently collapsed pair. We estimated many reproductive barriers in each of five sets of stickleback fish species pairs using our own data and decades of previous work. We found that the types of barriers important early in the speciation process differ from those important late. Two premating barriers—habitat and sexual isolation—evolve early in divergence and remain two of the strongest barriers throughout speciation. Premating isolation evolves before postmating isolation, and extrinsic isolation is far stronger than intrinsic. Completing speciation, however, may require postmating intrinsic incompatibilities. Reverse speciation in one species pair was characterized by significant loss of sexual isolation. We present estimates of barrier strengths before and after collapse of a species pair; such detail regarding the loss of isolation has never before been documented. Additionally, despite significant asymmetries in individual barriers, which can limit speciation, total isolation was essentially symmetric between species. Our study provides important insight into the order of barrier evolution and the relative importance of isolating barriers during speciation and tests fundamental predictions of ecological speciation.     

Sexual isolation evolves faster than hybrid inviability in a diverse and sexually dimorphic genus of fish (Percidae: Etheostoma)

Abstract .Theory predicts that sexual (or behavioral) isolation will be the first form of reproductive isolation to evolve in lineages characterized by sexual selection. Here I directly compare the rate of evolution of sexual isolation with that of hybrid inviability in a diverse and sexually dimorphic genus of freshwater fish. The magnitude of both sexual isolation and hybrid inviability were quantified for multiple pairs of allopatric species. Rates of evolution were inferred by comparing genetic distances of these species pairs with the magnitude of each form of reproductive isolation: the slope of the regression of genetic distance on the magnitude of reproductive isolation represents the rate of evolution. Of the two forms of isolation, the magnitude of sexual isolation exhibited the steeper slope of regression, indicating that sexual isolation will tend to evolve to completion earlier than hybrid inviability, strictly as a by-product of evolution in geographically isolated populations. Additional evidence from the literature is used to qualitatively compare rates of evolution of sexual isolation with that of other forms of reproductive isolation. Preliminary comparisons support the prediction that sexual isolation will evolve more rapidly than other forms. Because Etheostoma is characterized by striking sexual dimorphism, these results are consistent with the hypothesis that sexual selection for exaggerated mate-recognition characters causes the relatively rapid evolution of sexual isolation.     

Reproductive isolation among allopatric Drosophila montana populations

An outstanding goal in speciation research is to trace the mode and tempo of the evolution of barriers to gene flow. Such research benefits from studying incipient speciation, in which speciation between populations has not yet occurred, but where multiple potential mechanisms of reproductive isolation (RI: i.e., premating, postmating‐prezygotic (PMPZ), and postzygotic barriers) may act. We used such a system to investigate these barriers among allopatric populations of Drosophila montana. In all heteropopulation crosses we found premating (sexual) isolation, which was either symmetric or asymmetric depending on the population pair compared. Postmating isolation was particularly strong in crosses involving males from one of the study populations, and while sperm were successfully transferred, stored, and motile, we experimentally demonstrated that the majority of eggs produced were unfertilized. Thus, we identified the nature of a PMPZ incompatibility. There was no evidence of intrinsic postzygotic effects. Measures of absolute and relative strengths of pre‐ and postmating barriers showed that populations differed in the mode and magnitude of RI barriers. Our results indicate that incipient RI among populations can be driven by different contributions of both premating and PMPZ barriers occurring between different population pairs and without the evolution of postzygotic barriers.     

Impact of ancestral populations on postzygotic isolation in allopatric speciation

Postzygotic isolation evolves due to an accumulation of substitutions (potentially deleterious alleles in hybrids) in populations that have become geographically isolated. These potentially deleterious alleles might also be maintained in ancestral populations before geographic isolation. We used an individual-based model to examine the effect of the genetic state of an ancestral population on the evolution of postzygotic isolation after geographic isolation of a population. The results showed that the number of loci at which degenerative alleles are fixed in an ancestral population at equilibrium significantly affects the evolutionary rates of postzygotic isolation between descendant allopatric populations. Our results suggest that: (1) a severe decrease in population size (e.g., less than ten individuals) is not necessary for the rapid evolution of postzygotic isolation (e.g., <10,000 generation); (2) rapid speciation can occur when there is a large difference in the equilibrium number of accumulated degenerative alleles between ancestral and descendant populations; and (3) in an ancestral population maintained at a small effective population size for a long period of time, postzygotic isolation rarely evolves if back mutations that restore the function of degenerative alleles are limited.     

Evolution of postmating isolation: comparison of three models based on possible genetic mechanisms

In this study, we simulated the process of the evolution of postmating isolation using three models in which postmating isolation is caused by (1) genetic divergence through collaborative coevolution, (2) genetic divergence through antagonistic coevolution resulting from sexual conflict, and (3) genetic divergence through combinational incompatibility. The collaborative coevolution model and the combinational incompatibility model showed a similar decreasing pattern of hybrid compatibility over generations depending on population size and mutation rates. The antagonistic coevolution model showed that reproductive isolation can evolve rapidly depending on the intensity of selection. In the combinational incompatibility model, the increasing number of loci that interact and result in incompatibility would have both promoting and inhibiting effects on the formation of hybrid incompatibility in the earlier stage of isolation. Mutation rates for genes causing incompatibility significantly affect the number of generations required for postmating isolation, which indicates that models assuming high mutation rates (e.g., μ = 10⁻⁴) might predict much faster evolution for reproductive isolation than those observed in real populations. Received: January 29, 2001 / Accepted: July 4, 2001     

COMPONENTS OF REPRODUCTIVE ISOLATION BETWEEN NORTH AMERICAN PHEROMONE STRAINS OF THE EUROPEAN CORN BORER

Of 12 potential reproductive isolating barriers between closely related Z‐ and E‐pheromone strains of the European corn borer moth (Ostrinia nubilalis), seven significantly reduced gene flow but none were complete, suggesting that speciation in this lineage is a gradual process in which multiple barriers of intermediate strength accumulate. Estimation of the cumulative effect of all barriers resulted in nearly complete isolation (>99%), but geographic variation in seasonal isolation allowed as much as ～10% gene flow. With the strongest barriers arising from mate‐selection behavior or ecologically relevant traits, sexual and natural selection are the most likely evolutionary processes driving population divergence. A recent multilocus genealogical study corroborates the roles of selection and gene flow ( Dopman et al. 2005 ), because introgression is supported at all loci besides Tpi, a sex‐linked gene. Tpi reveals strains as exclusive groups, possesses signatures of selection, and is tightly linked to a QTL that contributes to seasonal isolation. With more than 98% of total cumulative isolation consisting of prezygotic barriers, Z and E strains of ECB join a growing list of taxa in which species boundaries are primarily maintained by the prevention of hybridization, possibly because premating barriers evolve during early stages of population divergence.     

Isolation of an iridovirus from two terrestrial isopods,the pill bug, Armadillidium vulgare, and the sow bug, Porcellio dilatatus

An iridovirus was isolated from two terrestrial isopods (class Crustacea, order Isopoda), the pill bug, Armadillidium vulgare, and the sow bug, Porcellio dilatatus, collected in southern California. The isolates have been designated Type 31 (from A. vulgare) and Type 32 (from P. dilatatus). Diseased isopods were recognized by a characteristic blue discoloration of the normally gray cuticle. Based on the relative number of virions observed in diseased cells, viral replication was most extensive in epidermal, muscle, and adipose tissue. Additionally, small clusters of midgut epithelial cells were heavily infected in many specimens, although replication throughout this tissue was never observed. Nerve and reproductive tissues were lightly infected. Infection was not observed in hemocytes or the hepatopancreatic caeca. Virions of both isolates measured ca. 125 nm in diameter in ultrathin sections and 141 nm in negatively stained preparations, and formed paracrystalline arrays in heavily infected cells. The isolation of a typical iridovirus from isopods further demonstrates that the natural host range of this virus group extends beyond the class Insecta.     

Components of reproductive isolation between the monkeyflowers Mimulus lewisii and M. cardinalis (Phrymaceae)

Evolutionists have long recognized the role of reproductive isolation in speciation, but the relative contributions of different reproductive barriers are poorly understood. We examined the nature of isolation between Mimulus lewisii and M. cardinalis, sister species of monkeyflowers. Studied reproductive barriers include: ecogeographic isolation; pollinator isolation (pollinator fidelity in a natural mixed population); pollen competition (seed set and hybrid production from experimental interspecific, intraspecific, and mixed pollinations in the greenhouse); and relative hybrid fitness (germination, survivorship, percent flowering, biomass, pollen viability, and seed mass in the greenhouse). Additionally, the rate of hybridization in nature was estimated from seed collections in a sympatric population. We found substantial reproductive barriers at multiple stages in the life history of M. lewisii and M. cardinalis. Using range maps constructed from herbarium collections, we estimated that the different ecogeographic distributions of the species result in 58.7% reproductive isolation. Mimulus lewisii and M. cardinalis are visited by different pollinators, and in a region of sympatry 97.6% of pollinator foraging bouts were specific to one species or the other. In the greenhouse, interspecific pollinations generated nearly 50% fewer seeds than intraspecific controls. Mixed pollinations of M. cardinalis flowers yielded >75% parentals even when only one-quarter of the pollen treatment consisted of M. cardinalis pollen. In contrast, both species had similar siring success on M. lewisii flowers. The observed 99.915% occurrence of parental M. lewisii and M. cardinalis in seeds collected from a sympatric population is nearly identical to that expected, based upon our field observations of pollinator behavior and our laboratory experiments of pollen competition. F1 hybrids exhibited reduced germination rates, high survivorship and reproduction, and low pollen and ovule fertility. In aggregate, the studied reproductive barriers prevent, on average, 99.87% of gene flow, with most reproductive isolation occurring prior to hybrid formation. Our results suggest that ecological factors resulting from adaptive divergence are the primary isolating barriers in this system. Additional studies of taxa at varying degrees of evolutionary divergence are needed to identify the relative importance of pre- and postzygotic isolating mechanisms in speciation.     

POLLINATOR SPECIFICITY DRIVES STRONG PREPOLLINATION REPRODUCTIVE ISOLATION IN SYMPATRIC SEXUALLY DECEPTIVE ORCHIDS

Few studies have quantified the full range of pre‐ and postzygotic barriers that limit introgression between closely related plant species. Here, we assess the strength of four isolating mechanisms operating between two morphologically similar and very closely related sympatric orchid taxa, Chiloglottis valida and C. aff. jeanesii. Each taxon sexually attracts its specific wasp pollinator via distinct floral volatile chemistry. Behavioral experiments with flowers and synthetic versions of their floral volatiles confirmed that very strong pollinator isolation is mediated by floral odor chemistry. However, artificially placing flowers of the two taxa in contact proximity revealed the potential for rare interspecific pollination. Although we found hybrid vigor in F1 hybrids produced by hand‐crossing, genetic analysis at both nuclear and chloroplast loci showed significant and moderate‐to‐strong genetic differentiation between taxa. A Bayesian clustering method for the detection of introgression at nuclear loci failed to find any evidence for hybridization across 571 unique genotypes at one site of sympatry. Rather than inhibiting gene flow, postpollination barriers surveyed here show no contribution to overall reproductive isolation. This demonstrates the primacy of pollinators in maintaining species boundaries in these orchids, which display one of the strongest known examples of prepollination floral isolation.     

Differences in offspring size predict the direction of isolation asymmetry between populations of a placental fish

Crosses between populations or species often display an asymmetry in the fitness of reciprocal F₁ hybrids. This pattern, referred to as isolation asymmetry or Darwin''s Corollary to Haldane''s Rule, has been observed in taxa from plants to vertebrates, yet we still know little about which factors determine its magnitude and direction. Here, we show that differences in offspring size predict the direction of isolation asymmetry observed in crosses between populations of a placental fish, Heterandria formosa. In crosses between populations with differences in offspring size, high rates of hybrid inviability occur only when the mother is from a population characterized by small offspring. Crosses between populations that display similarly sized offspring, whether large or small, do not result in high levels of hybrid inviability in either direction. We suggest this asymmetric pattern of reproductive isolation is due to a disruption of parent–offspring coadaptation that emerges from selection for differently sized offspring in different populations.     

Rapid evolution of prezygotic barriers in non‐territorial damselflies

A central question in evolutionary biology concerns the accumulation of reproductive barriers during speciation. However, separating the reproductive barriers that have led to speciation from those that have secondarily accumulated (i.e. after initial divergence) is a widely recognized problem. Ideal candidate species for overcoming this problem are young species, where time for additional barriers to accrue has been limited. In the present study, we add to previous studies investigating the strength of reproductive barriers between the parapatric damselflies Ischnura elegans and Ischnura graellsii by quantifying seven prezygotic barriers between the allopatric pairs of I. elegans and Ischnura genei, as well as I. graellsii and I. genei. Specifically, we measured four premating (temporal, sexual, mechanical _I, and mechanical _II) and three postmating (oviposition success, fecundity, and fertility) barriers using experimental approaches and, for first time, we investigated the mechanisms causing mechanical isolation, which is the strongest reproductive barrier in ischnurans. The findings of the present study support the notion that premating barriers are generally strong and contribute significantly to total reproductive isolation in young lineages (65–98%), although they never solely lead to complete isolation. Asymmetry was generally stronger in premating than in postmating barriers, and was driven mostly through asymmetry in mechanical isolation, which is caused by morphological divergence of secondary sexual appendages. We found that barriers act multiplicatively in all species combinations tested, with the exception of sexual isolation, which was not detected. Our results are consistent with a recent allopatric speciation scenario driven by differences in male anal appendages, either impeding copulation or affecting female preferences. Taken together, the results from this and previous studies in diverse odonate genera suggest that premating barriers have evolved rapidly in ischnuran damselflies and, although reproductive isolation in ischnurans is more commonly the result of several barriers acting together, morphological divergence of secondary sexual appendages appears to be a common factor facilitating premating isolation in this group. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 485–496.     

Speciation with gene flow could be common

The likelihood of speciation in the face of homogenizing gene flow (i.e. without complete geographical isolation) is one of the most debated topics in evolutionary biology. Demonstrating this phenonemon is hampered by the difficulty of isolating the effects of time since population divergence vs. gene flow on levels of molecular genetic differentiation. For example, weak genetic differentiation between taxa could be due to recent divergence, gene flow, or a combination of these factors. Nonetheless, a number of convincing examples of speciation with gene flow have recently emerged, owing in part to the development of new analytical methods designed to estimate gene flow specifically. A recent example of speciation with gene flow in salamanders (Niemiller et al. 2008) further advances our understanding of this phenonemon, by showing that gene flow between cave and spring salamanders was ongoing during speciation, rather than having occurred after a long period of allopatric divergence. Future work on the ecological and genetic factors reducing gene flow will likely increase our understanding of the conditions that faciliate divergence in the face of gene flow.     

Divergence of premating behaviors in the closely related species Drosophila subquinaria and D. recens

Most animal species use distinctive courship patterns to choose among potential mates. Over time, the sensory signaling and preferences used during courtship can diverge among groups that are reproductively isolated. This divergence of signal traits and preferences is thought to be an important cause of behavioral isolation during the speciation process. Here, we examine the sensory modalities used in courtship by two closely related species, Drosophila subquinaria and Drosophila recens, which overlap in geographic range and are incompletely reproductively isolated. We use observational studies of courtship patterns and manipulation of male and female sensory modalities to determine the relative roles of visual, olfactory, gustatory, and auditory signals during conspecific mate choice. We find that sex‐specific, species‐specific, and population‐specific cues are used during mate acquisition within populations of D. subquinaria and D. recens. We identify shifts in both male and female sensory modalities between species, and also between populations of D. subquinaria. Our results indicate that divergence in mating signals and preferences have occurred on a relatively short timescale within and between these species. Finally, we suggest that because olfactory cues are essential for D. subquinaria females to mate within species, they may also underlie variation in behavioral discrimination across populations and species.     

TEMPERATURE-BASED EXTRINSIC REPRODUCTIVE ISOLATION IN TWO SPECIES OF DROSOPHILA

Drosophila santomea and D. yakuba are sister species that live on the African volcanic island of São Tomé, where they are ecologically isolated: D. yakuba inhabits low-altitude open and semiopen habitats while D. santomea lives in higher-elevation rain and mist forest. To determine whether this spatial isolation reflected differential preference for and tolerance of temperature, we estimated fitness components of both species at different temperatures as well as their behavioral preference for certain temperatures. At higher temperatures, especially 28°C, D. santomea was markedly inferior to D. yakuba in larval survival, egg hatchability, and longevity. Moreover, D. santomea females, unlike those of D. yakuba , become almost completely sterile after exposure to a temperature of 28°C, and conspecific males become semisterile. Drosophila santomea adults prefer temperatures 2–3°C lower than do adults of D. yakuba . Drosophila santomea , then, is poorly adapted to high temperature, partially explaining its restriction to cool, high habitats, which leads to extrinsic premating isolation and immigrant inviability. Rudimentary genetic analysis of the interspecific difference in egg hatchability and larval survival showed that these differences are due largely to cytoplasmic effects and to autosomal genes, with sex chromosomes playing little or no role.     

阿克苏高盐咸水滩放线菌分离新策略及系统发育多样性

[目的]探讨高盐咸水滩放线菌的分离新策略,为高盐地区放线菌资源的分离提供理论依据.[方法]以甘油-精氨酸培养基、海藻糖-肌酸培养基、甘油-天冬氨酸培养基、甘露醇-酸水解酪蛋白培养基、干酪素-甘露醇、甘露醇-丙氨酸培养基、壳聚糖-天冬酰胺培养基和高氏一号琼脂培养基8种培养基为基础培养基,采用营养成分十倍稀释法、据土样理化性质模拟原始生态环境、免培养分子技术检测菌株的分离培养基及培养条件指导放线菌可培养以及借鉴半咸水海洋环境放线菌分离培养基等4种策略来优化设计培养基,进行阿克苏高盐咸水滩土样放线菌的分离,并采用细菌通用引物进行16S rRNA基因扩增和序列测定,并构建系统发育树.[结果]共分离到403株,分属于放线菌的8个亚目10个科,Streptomyces、Streptomonospora、Saccharomonospora、Plantactinospora、Nocardia、Amycolatopsis、Glycomyces、Micromonospora、Nocardiopsis、Isoptericola、Nonomuraea、Thermobifida、Actinopolyspora和Actinomadura等14个属.69.96%菌株属于链霉菌亚目(Streptomycineae),9.68%菌株属于链孢囊菌亚目(Streptosporangineae),9株为潜在新种.[结论]4种分离新策略显著地提高了高盐地区放线菌的可培养性,还发现了许多新物种,为放线菌的分离提供了新思路与途径.