Insights into the Witheringia solanacea (Solanaceae) Complex in Costa Rica. I. Breeding Systems and Crossing Studies1

The Witheringia solanacea complex consists of three species, W. asterotricha, W. meiantha, and W, solanacea, native to Central and South America. The three taxa are morphologically similar, and their distinctions and relationships have been the subject of taxonomic controversy. To investigate breeding systems and potential for hybridization among the taxa of the complex, two Costa Rican accessions per species were used in a crossing program. All plants were self‐incompatible except for one accession of W. solanacea. Hybrid plants resulted from all crosses among accessions of W. asterotricha and W. solanacea. Most crosses were unsuccessful using W. meiantha in combination with either of the other two taxa. It is suggested that W. meiantha and W. solanacea be recognized as separate taxa, but that W. asterotricha be considered a synonym of W. solanacea.     

The effectiveness of pre‐ and post‐zygotic barriers in avoiding hybridization between two snapdragons (Antirrhinum L.: Plantaginaceae)

Reproductive barriers play an important role in the maintenance of species boundaries. However, to date, few studies have provided a detailed analysis of reproductive isolation barriers between species or examined their importance in maintaining species identity. This is the first detailed study into pre‐ and post‐zygotic reproductive isolation barriers in Antirrhinum, based on a mixed population with two species that rarely co‐occur. The study revealed that pollinator constancy and preference and poor hybrid seed viability were the most important reproductive isolating mechanisms. Reproductive isolation was practically complete by both pre‐ and post‐zygotic barriers. Average pre‐zygotic isolation was greater than post‐zygotic isolation, in accordance with the trend observed in flowering plants in which reproductive isolation is principally caused by pre‐zygotic mechanisms. However, average post‐zygotic isolation was also high, in contrast to what was expected among Antirrhinum spp. This case highlights the importance of quantifying the reproductive isolation barriers thoroughly to understand how and why species boundaries are maintained. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 159–172.     

Floral scent and species divergence in a pair of sexually deceptive orchids

Speciation is typically accompanied by the formation of isolation barriers between lineages. Commonly, reproductive barriers are separated into pre‐ and post‐zygotic mechanisms that can evolve with different speed. In this study, we measured the strength of different reproductive barriers in two closely related, sympatric orchids of the Ophrys insectifera group, namely Ophrys insectifera and Ophrys aymoninii to infer possible mechanisms of speciation. We quantified pre‐ and post‐pollination barriers through observation of pollen flow, by performing artificial inter‐ and intraspecific crosses and analyzing scent bouquets. Additionally, we investigated differences in mycorrhizal fungi as a potential extrinsic factor of post‐zygotic isolation. Our results show that floral isolation mediated by the attraction of different pollinators acts apparently as the sole reproductive barrier between the two orchid species, with later‐acting intrinsic barriers seemingly absent. Also, the two orchids share most of their fungal mycorrhizal partners in sympatry, suggesting little or no importance of mycorrhizal symbiosis in reproductive isolation. Key traits underlying floral isolation were two alkenes and wax ester, present predominantly in the floral scent of O. aymoninii. These compounds, when applied to flowers of O. insectifera, triggered attraction and a copulation attempt of the bee pollinator of O. aymoninii and thus led to the (partial) breakdown of floral isolation. Based on our results, we suggest that adaptation to different pollinators, mediated by floral scent, underlies species isolation in this plant group. Pollinator switches may be promoted by low pollination success of individuals in dense patches of plants, an assumption that we also confirmed in our study.     

Evaluation of reproductive barriers and realisation of interspecific hybridisations depending on genetic distances between species in the genus Helleborus

The genus Helleborus comprises 22 species, which are allocated to six sections. H. x hybridus and H. niger, which belong to different Helleborus sections, are economically important ornamentals. Several other species with minor impact exhibit interesting features, e.g. flower size, flower colour, foliage, scent and disease resistance, which should be introgressed into H. x hybridus or H. niger through interspecific hybridisation. The aims of this study were to investigate whether and which kind of hybridisation barriers occur in crosses between Helleborus species and if they differ in their manifestations, depending on the genetic distance of the respective partners. In order to obtain interspecific hybrids despite crossing barriers, a method to overcome these barriers should be developed. Crossing barriers in Helleborus were localised as predominantly post‐zygotic according to in situ pollen tube staining with aniline blue. For certain crosses, pre‐zygotic barriers could also be assumed, but pollen tube growth was not totally inhibited. Therefore, embryo rescue techniques via ovule culture were established to overcome the post‐zygotic barriers. Ovules were isolated from maternal plants 5–7 weeks after pollination in most cases and then cultured in vitro. Overall, 219 hybrids were successfully obtained, of which 16 were derived from inter‐sectional crosses. Hybrids were verified by flow cytometry and/or by molecular DNA markers.     

Crossability Barriers in Neotropical Heliconia

Artificial hybridization among species of neotropical Heliconiawas studied at two sites in Costa Rica, centralAmerica. At LasCruces Tropical Botanical Garden individuals in cultivationwere used as parents in crosses primarily between species withpendent inflorescences that normally are distributed allopatrically.At Finca La Selva normally sympatric species with either pendentor erect inflorescences were crossed in their natural habitats.Observation of pollen tube growth by means of fluorescence microscopyand seed set were used to determine the extent of crossability.Crossability barriers between the majority of species are strongand foreign pollen tubes are inhibited at the stigmatic surface,within the stylar tissue or within the ovary. The site of inhibitionis consistent for each pair of species, and is dependent onthe parentage and the direction of the cross. Although additionalisolating mechanisms, such as pollinator specificity and phenologicalseparation, are present in Heliconia, pre-fertilization crossabilitybarriers act as the ultimate mechanism to prevent hybridization.The type of barrier (stigmatic, stylar or ovarian) that existsbetween two species is not dependent upon the geographical distributionof the parental species or the specific types of pollinatorsthat visit them, but in some cases may indicate taxonomic relationships. Heliconia spp, isolating mechanisms, crossability barriers, progamic phase, hybridization, Costa Rica, hummingbirds, taxonomy, pollinator sharing     

Multiple sources of reproductive isolation in a bimodal butterfly hybrid zone

An important evolutionary question concerns whether one or many barriers are involved in the early stages of speciation. We examine pre‐ and post‐zygotic reproductive barriers between two species of butterflies (Heliconius erato chestertonii and H. e. venus) separated by a bimodal hybrid zone in the Cauca Valley, Colombia. We show that there is both strong pre‐ and post‐mating reproductive isolation, together leading to a 98% reduction in gene flow between the species. Pre‐mating isolation plays a primary role, contributing strongly to this isolation (87%), similar to previous examples in Heliconius. Post‐mating isolation was also strong, with absence of Haldane’s rule, but an asymmetric reduction in fertility (< 11%) in inter‐specific crosses depending on maternal genotype. In summary, this is one of the first examples of post‐zygotic reproductive isolation playing a significant role in early stages of parapatric speciation in Heliconius and demonstrates the importance of multiple barriers to gene flow in the speciation process.     

Incipient speciation in the Anastrepha fraterculus cryptic species complex: reproductive compatibility between A. sp.1 aff. fraterculus and A. sp.3 aff. fraterculus

Reproductive isolation among biological strains can be detected by analyzing reproductive barriers between populations. The Anastrepha fraterculus Wiedemann (Diptera: Tephritidae) cryptic species complex comprises eight morphotypes defined by morphometric characteristics. The present study analyzed the existence of pre‐ and post‐zygotic isolation among populations of A. sp.1 aff. fraterculus (A. sp.1) and A. sp.3 aff. fraterculus (A. sp.3) determined by tests of sexual compatibility, analyses of the temporal pattern of calling behavior, and the reproductive performance of the offspring of homotypic and heterotypic crosses. Pre‐zygotic tests indicated asymmetric matings with preferences for homotypic crosses. The A. sp.3 population demonstrated distinct characteristics, including low copulation percentages and a high proportion of homotypic copulations under crowded conditions. The females of the A. sp.1 population demonstrated lower copulation latency. The analyzed morphotypes demonstrated two peaks in pheromone emission activity, at the beginning and at the end of the morning and were differentiated in the abundance of males. The calling behavior of the hybrids between A. sp.1 male and A. sp.3 female crosses had an intermediate pattern compared to either parental type. Post‐zygotic compatibility tests indicated low viability of the cross between A. sp.3 males and A. sp.1 females. Pronounced asymmetries were found in the sex ratios of the offspring of heterotypic crosses, and only hybrids from A. sp.1 male vs. A. sp.3 female crosses produced descendants. The observed pre‐ and post‐zygotic incompatibilities reflect the probable occurrence of incipient speciation between A. sp.1 and A. sp.3. A more adequate taxonomic classification of this species complex that considers the distinct characteristics of each morphotype will be needed to improve environmentally wise control methods against this insect pest.     

Pre‐zygotic reproductive isolation between two synchronopatric Opuntia (Cactaceae) species in the Brazilian Chaco

• Opuntia (Cactaceae) is known for high rates of hybridization and ploidisation, resulting in the formation of new species. The occurrence of two sympatric and closely related species of Opuntia, O. elata and O. retrorsa, in Brazilian Chaco enabled us to test the hypothesis that pre‐zygotic reproductive isolation mechanisms operate in both species.
• We monitored the flowering period, as well as floral biology, and compared the morphological variation of floral structures through measurements, performed intra‐ and interspecific cross‐pollination tests, and recorded the guild of floral visitors and pollinators.
• Flowering was seasonal and highly synchronous. Floral biology exhibits similar strategies, and although floral morphology differs significantly in many of the compared structures, such morphological variation does not result in the selection of exclusive pollinators. Floral visitors and pollinators are oligolectic bees shared by both species. Opuntia elata and O. retrorsa are self‐compatible. While interspecific cross‐pollination (bidirectional) resulted in germination, the pollen tube did not penetrate the stigma.
• Opuntia elata and O. retrorsa are closely related; however, they are isolated and do not hybridise in Brazilian Chaco. We found that both have weak pre‐pollination barriers, but that they are strongly isolated by pollen–pistil incompatibility, i.e. post‐pollination barrier.

     

Quantification of the zygotic barrier between interbreeding taxa using gene flow data

Hybridization and introgression via interspecific gene flow are common processes in the plant kingdom. The effectiveness of these processes is governed by the strengths of multiple zygotic barriers. These barriers have often been quantified in artificial settings using laborious and time‐consuming hand‐pollination experiments, but their quantification is nonexistent in the landscape. In this study, we utilized gene flow data within a spatially explicit simulation to assess the strengths of zygotic barriers. Our model system consisted of Populus nigra and its hybrid, P. × canadensis, which interbreed under natural conditions. The study population was located in the floodplain of the Eder River in central Germany. Pollen‐mediated introgression rates from hybrid males into the seeds of individual female trees were used as the target pattern using an inverse modeling approach. Simulations that treated pollen from both taxa equally revealed a large discrepancy between the observed and modeled rates of introgression for both taxa. The discrepancy was reduced by introducing a zygotic barrier against the pollen from the hybrid males. The best model outcome values indicated comparably strong zygotic barriers acting against pollen‐mediated introgressive gene flow into the two parental taxa, P. nigra and P. × canadensis. The sensitivity of our model was tested by applying different dispersal functions. Four common probability density functions were used along with a pollen dispersal function that had previously been fitted to gene flow data from the same dataset. The best barrier value was almost independent of the dispersal functions used here. Moreover, it was within the range previously determined in hand‐pollination‐based investigations, validating our model. These data indicate that the inverse modeling approach is a powerful method for quantifying hidden processes, and we discuss its use as a valuable tool for generating new insights into plant mating systems that are relevant to evolutionary biology and risk analyses in conservation efforts.     

Asymmetric Assortative Mating Behaviour Reflects Incomplete Pre‐zygotic Isolation in the Nasonia Species Complex

Preference of con‐ over heterospecific mates leading to assortative mating can substantially contribute to pre‐zygotic reproductive isolation and prevent fitness losses if post‐zygotic hybridization barriers already exist. The jewel wasp genus Nasonia displays quite strong and well‐studied post‐zygotic reproductive isolation due to a ubiquitous Wolbachia infection causing cytoplasmic incompatibility between different species. Pre‐zygotic isolation, however, has received far less research attention in this model organism, especially concerning the mechanisms and criteria of mate choice. In the present study, we analysed mate rejection and mate acceptance rates in cross‐comparisons between all four Nasonia species. We put emphasis on observing which sex is more likely to interrupt interspecific matings and how discriminatory behaviour varies across the different species in all possible combinations. We found an asymmetric distribution of assortative mating among the four Nasonia species that appears to be highly influenced by the respective combinations of sex and species. Females appeared to be the main discriminators against heterospecific mating partners, but interestingly, we could also detect mate discrimination and rejection behaviour in males, a widely neglected factor in research on mating behaviour in general and on Nasonia in particular. Moreover, the asymmetry in the assortative mating behaviour was partially reflective of sym‐ or allopatric distributions of natural Nasonia populations.     

Barriers to hybridisation and their conservation implications for a highly threatened Australian fish species

Hybridisation and introgression are natural phenomena that may lead to the transfer of adaptive alleles from one species to another and increased species diversity. At the same time, hybridisation and subsequent introgression threaten many species world‐wide through the loss of genetic and species diversity. In Australia, introgressive hybridisation between native and alien species has not typically been considered a significant threat to native biodiversity because of the taxonomic distance between native and alien biota. However, many native fish have been introduced outside their natural range. Recently, four taxa in the genus Melanotaenia have been nationally listed as threatened due to introgressive hybridisation with introduced Melanotaenia splendida. We examined pre‐ and post‐zygotic barriers to hybridisation between M. splendida and one of these threatened taxa—Running River rainbowfish (RRR)—to assess the potential for hybridisation to occur. We used dichotomous mate choice experiments to examine pre‐zygotic barriers and mating experiments to examine post‐zygotic barriers. Size was not a significant predictor of the proportion of time subjects spent with a potential mate, nor was there any significant difference in the amount of time subjects spent with potential mates of their own or the opposite species. Eggs from hybrid pairings with female RRR had a slightly higher hatching rate than those from hybrid pairings with female M. splendida, but neither were significantly different from intraspecies crosses. We could not identify any definite barriers to hybridisation, demonstrating that the introduction of “native” fish species outside their natural range poses a higher risk of hybridisation than previously thought. We call for better education around the consequences of moving “native” fish and the development of rapid response plans to deal with recently established alien populations of Australian fish species in order to prevent future extinctions due to introgressive hybridisation.     

Evolution of pre‐zygotic and post‐zygotic barriers to gene flow among three cryptic species within the Anastrepha fraterculus complex

Tropical tephritids are ideally suited for studies on population divergence and speciation because they include species groups undergoing rapid radiation, in which morphologically cryptic species and sister species are abundant. The fraterculus species group in the Neotropical genus Anastrepha is a case in point, as it is composed of a complex of up to seven A. fraterculus morphotypes proposed to be cryptic species. Here, we document pre‐ and post‐zygotic barriers to gene flow among adults of the Mexican A. fraterculus morphotype and three populations (Argentina, Brazil, and Peru) belonging to two separate morphotypes (Brazilian 1 and Peruvian). We unveiled three forms of pre‐zygotic reproductive isolation resulting in strong assortative mating. In field cages, free‐ranging male and female A. fraterculus displayed a strong tendency to form couples with members of the opposite sex belonging to their own morphotype, suggesting that male pheromone emission, courtship displays, or both intervene in shaping female choice before actual contact and coupling. In addition, males and females of the Peruvian morphotype became receptive and mated significantly later than adults of the Mexican and Brazilian 1 morphotypes. After contact, Mexican females exhibited greater mating discrimination than males when facing adults of the opposite sex belonging to either the Peruvian or the Brazilian 1 morphotype as evidenced by vigorous resistance to penetration once they had been forcefully mounted by heterotypic males. Forced copulations resulted in production of F1 hybrids that were either less viable (and partially fertile) than parental crosses or even sterile. Our results suggest that the Mexican morphotype is a distinct biological entity and that pre‐zygotic reproductive isolation through divergence in courtship or male‐produced pheromone and other mechanisms appear to evolve faster than post‐zygotic isolation in the fraterculus species group.     

Impact of pre‐ and post‐pollination barriers on pollen transfer and reproductive isolation among three sympatric Pedicularis (Orobanchaceae) species

• Pedicularis is the largest genus in the Orobanchaceae (>300) with many species co‐occurring and co‐blooming in subalpine to alpine meadows in the Himalayas. Although it is well known that different Pedicularis species place pollen on different parts of the same bumblebee's body, thus reducing interspecific pollen transfer, it is not known whether post‐pollination components also contribute to reproductive isolation (RI).
• In this study, we quantified the individual strengths and absolute contributions of six pre‐ and post‐pollination components of RI between three sympatric species in two pairs; Pedicularis gruina × Pedicularis tenuisecta (gru × ten) and Pedicularis comptoniifolia × Pedicularis tenuisecta (com × ten).
• All three Pedicularis species shared the same Bombus species. Individual foragers showed a high, but incomplete, floral constancy for each species. Therefore, pre‐pollination barriers were potentially ‘leaky’ as Bombus species showed a low but consistent frequency of interspecific visitation. The RI strength of pre‐pollination was lower in com × ten than in gru × ten. In contrast, post‐pollination barriers completely blocked gene flow between both sets of species pairs. Two post‐pollination recognition sites were identified. Late acting rejection of interspecific pollen tube growth occurred in com♀ × ten♂, while seeds produced in bi‐directional crosses of gru × ten failed to germinate.
• We propose that, although floral isolation based on pollen placement on pollinators in the genus Pedicularis is crucial to avoid interspecific pollen transfer, the importance of this mode of interspecific isolation may be exaggerated. Post‐pollination barriers may play even larger roles for currently established populations of co‐blooming and sympatric species in this huge genus in the Himalayas.

     

Comparative phylogeography of three common Neotropical tree species

Aim To determine spatial and temporal commonalities in patterns of chloroplast DNA (cpDNA) variation in three widespread Neotropical tree species. We examine whether patterns of genetic variation are more consistent with Pliocene or Pleistocene divergence. Location Central American forests, located in El Salvador, Nicaragua, Costa Rica and Panama. Methods We collected sequences from two cpDNA loci from c. 30 locations for each of three species –Bursera simaruba (Burseraceae; n = 278), Brosimum alicastrum (Moraceae; n = 210) and Ficus insipida (Moraceae; n = 222) – and additionally sequenced one nuclear locus for Bursera simaruba (n = 45). We used Monmonier’s algorithm to detect genetic barriers between regions. Divergence times between these regions were estimated using coalescent analyses. Results Spatial genetic boundaries were found in similar areas for these species, namely between Costa Rica and Nicaragua for all three species, and between El Salvador and Nicaragua for two species. These boundaries visually coincide with the spatial delimitations of Pliocene islands and previously hypothesized Pleistocene refugia. Divergence time estimates between regions are more consistent with Pleistocene divergence in two of the three species. Main conclusions Our results point to strong commonalities in the spatial locations of genetic boundaries in these three species, despite the complex geological and climatological history of this region, and ecological differences between the species. While spatial genetic boundaries coincide conspicuously with possible Pliocene and Pleistocene barriers to gene flow, we cannot distinguish between the two scenarios because of the strong spatial overlap of both barriers. However, the temporal data tentatively suggest that some of this divergence occurred in the Pleistocene, although limitations in the analysis cannot confirm Pleistocene divergence without external, corroborating data. While we cannot definitively implicate a single historical process as driving patterns of genetic differentiation in all three species, our results represent an initial step towards identifying a common history of Central American tree species.     

Ecological speciation in sympatric palms: 2. Pre‐ and post‐zygotic isolation

We evaluated reproductive isolation in two species of palms (Howea) that have evolved sympatrically on Lord Howe Island (LHI, Australia). We estimated the strength of some pre‐ and post‐zygotic mechanisms in maintaining current species boundaries. We found that flowering time displacement between species is consistent across in and ex situ common gardens and is thus partly genetically determined. On LHI, pre‐zygotic isolation due solely to flowering displacement was 97% for Howea belmoreana and 80% for H. forsteriana; this asymmetry results from H. forsteriana flowering earlier than H. belmoreana and being protandrous. As expected, only a few hybrids (here confirmed by genotyping) at both juvenile and adult stages could be detected in two sites on LHI, in which the two species grow intermingled (the Far Flats) or adjacently (Transit Hill). Yet, the distribution of hybrids was different between sites. At Transit Hill, we found no hybrid adult trees, but 13.5% of younger palms examined there were of late hybrid classes. In contrast, we found four hybrid adult trees, mostly of late hybrid classes, and only one juvenile F1 hybrid in the Far Flats. This pattern indicates that selection acts against hybrids between the juvenile and adult stages. An in situ reciprocal seed transplant between volcanic and calcareous soils also shows that early fitness components (up to 36 months) were affected by species and soil. These results are indicative of divergent selection in reproductive isolation, although it does not solely explain the current distribution of the two species on LHI.     

Airborne‐pollen pool and mating pattern in a hybrid zone between Pinus pumila and P. parviflora var. pentaphylla

The reproductive isolation barriers and the mating patterns among Pinus pumila, P. parviflora var. pentaphylla and their hybrids were examined by flowering phenology and genetic assays of three life stages: airborne‐pollen grains, adults and seeds, in a hybrid zone on Mount Apoi, Hokkaido, Japan. Chloroplast DNA composition of the airborne‐pollen was determined by single‐pollen polymerase chain reaction. Mating patterns were analysed by estimating the molecular hybrid index of the seed parent, their seed embryos and pollen parents. The observation of flowering phenology showed that the flowering of P. pumila precedes that of P. parviflora var. pentaphylla by about 6 to 10 days within the same altitudinal ranges. Although this prezygotic isolation barrier is effective, the genetic assay of airborne‐pollen showed that the two pine species, particularly P. pumila, still have chances to form F₁ hybrid seeds. Both parental species showed a strong assortative mating pattern; F₁ seeds were found in only 1.4% of seeds from P. pumila mother trees and not at all in P. parviflora var. pentaphylla. The assortative mating was concluded as the combined result of flowering time differentiation and cross‐incompatibility. In contrast to the parental species, hybrids were fertilized evenly by the two parental species and themselves. The breakdown of prezygotic barriers (intermediate flowering phenology) and cross‐incompatibility may account for the unselective mating. It is suggested that introgression is ongoing on Mount Apoi through backcrossing between hybrids and parental species, despite strong isolation barriers between the parental species.     

Species distribution models predict the geographic expansion of an enzootic amphibian pathogen

Globally, numerous amphibian species have declined due to the introduction of the chytrid fungus Batrachochytrium dendrobatidis (Bd). However, the understanding of the spatiotemporal dynamics remains incomplete. Therefore, estimating the current geographic distribution of Bd is urgently needed, especially in countries like Costa Rica, where susceptible species are still recovering from Bd‐driven declines. We conducted model tuning and spatial analysis to compare the habitat suitability for epizootic and enzootic Bd in Costa Rica and to identify data‐deficient regions, opportunistic sampling, and Bd hotspots. Our dataset combined two methods of detection (histology and PCR methods) for a total of 451 Bd‐positive records from 34 localities. We found that the distribution of enzootic Bd in Costa Rica increased 60% since previous estimates in the early 2000s and extended to highlands and dry lowlands that were considered unsuitable for Bd. We also found that Bd is common across protected lands (80%) and within the herpetological provinces containing the highest amphibian richness and endemism in Costa Rica. Opportunistic sampling of Bd has focused on sites where epizootics occurred with the strongest intensity, leading to deficient or absent sampling across the Talamanca Range, the Nicoya Peninsula, and the northern lowlands. Our results showed that PCR increased the power of Bd detection in lowlands and favored the identification of Bd hotspots across the Caribbean side of Costa Rica. Our results add to the understanding of disease spread during enzootics and can be used to identify new hotspots for disease to mitigate future outbreaks of this pathogen. Abstract in Spanish is available with online material     

Some Entolomataceae (Agaricales) from Costa Rica

A preliminary account of Entolomataceae from Costa Rica is presented. Three new taxa are described, two inClitopilus, one inRhodocybe, and three new combinations are proposed inInocephalus. Five taxa ofClitopilus are reported for the first time from Costa Rica and a key to species is provided. Four species ofRhodocybe are discussed and a key to the six species known from Costa Rica is also provided.Alboleptonia earlei, Inocephalus murraii, Inocephalus quadratum, Rhodocybe incarnata andRhodocybe pseudonitellina are now known to occur in Costa Rica.     

Widespread co‐occurrence of divergent mitochondrial haplotype lineages in a Central American species of poison frog (Oophaga pumilio)

Aim To analyse the phylogeographic structure of the strawberry poison frog, Oophaga pumilio (Dendrobatidae), across a large part of its range using a combination of mitochondrial and nuclear markers. Location Costa Rica and Panama. Methods Sequence analyses of a mitochondrial (cytochrome b) and a nuclear (RAG‐1) gene fragment as well as analyses of seven microsatellite loci were carried out on 269 individuals of O. pumilio sampled from 24 localities and on two individuals of O. vicentei. Results Two main mitochondrial haplotype lineages, corresponding to a northern (north Costa Rica) and a southern (south Costa Rica and eastern Panama) lineage, were identified. They differed by up to 7% uncorrected distance. We observed co‐occurrence of both lineages in seven populations in Costa Rica and Panama, indicating a pattern of extensive admixture. The two main mitochondrial lineages of O. pumilio roughly corresponded to a previously described phylogeographic pattern. Microsatellites indicate admixture spanning over a wide geographic area, but significant variation between the northern and southern groups was also found with microsatellite data. While microsatellite data reconstructed a separation south of an assumed Caribbean valley barrier, mitochondrial haplotypes of the ‘southern lineage’ shifted this barrier towards the north. Main conclusions Despite admixture, all three markers showed significant variation between the northern and southern groups. Phylogeographical breaks known from other anuran species in the study region could not be verified for O. pumilio. The unexpected clustering of the population from Escudo de Veraguas and the individuals of O. vincentei with the northern O. pumilio lineage indicates the need for a fundamental and careful taxonomic revision, including an interspecific phylogeography of the entire genus.     

Post‐zygotic hybrid viability in sympatric species pairs: a case study from European lampreys

Ecological speciation mechanisms are widely assumed to play an important role in the early stages of divergence between incipient species, and this especially true of fishes. In the present study, we tested explicitly for post‐zygotic barriers to gene flow between a sympatric, recently diverged lamprey species pair that likely arose through ecological divergence. Experimental in vitro hybridization between anadromous parasitic Lampetra fluviatilis and resident nonparasitic Lampetra planeri resulted in a high proportion of embryos capable of attaining the burrowing pro‐larval stage, strongly indicating no post‐zygotic barriers to gene flow between these species. A sympatric, locally‐adapted resident parasitic form of L. fluviatilis was also found to successfully hybridize with both members of this species pair. The consequences of these findings are discussed in the context of lamprey speciation. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 108 , 378–383.