Cryptic species of a water hyacinth biological control agent revealed in South Africa: host specificity,impact, and thermal tolerance

The discovery that cryptic species are more abundant than previously thought has implications for weed biological control, as there is a risk that cryptic species may be inadvertently released with consequences for the safety of the practice. A cryptic species of a biological control agent released for the control of the invasive alien macrophyte, water hyacinth, Eichhornia crassipes (C. Mart.) Solms. (Pontederiaceae), was recently discovered in South Africa. The two species were considered a single species prior to genetic analysis and interbreeding experiments. The original biological control agent retains the name Eccritotarsus catarinensis (Carvalho) (Heteroptera: Miridae) whereas the new species has been described as Eccritotarsus eichhorniae Henry. In this study, we compared the host specificity, efficacy, and thermal physiologies of the two species. The host specificity of the two species within the Pontederiaceae was very similar and both are safe for release in South Africa. Comparison of the per capita impact of the two species indicated that E. eichhorniae was the more damaging species but this is likely to be influenced by temperature, with E. catarinensis being more effective under lower temperatures and E. eichhorniae being more effective under higher temperatures. Releasing the correct species for the thermal environment of each release site will improve the level of control of water hyacinth in South Africa. This example highlights the need to keep populations of biological control agents from different native range collection localities separate, and to screen for host specificity and efficacy.     

High levels of interspecific gene flow in an endemic cichlid fish adaptive radiation from an extreme lake environment

Studying recent adaptive radiations in isolated insular systems avoids complicating causal events and thus may offer clearer insight into mechanisms generating biological diversity. Here, we investigate evolutionary relationships and genomic differentiation within the recent radiation of Alcolapia cichlid fish that exhibit extensive phenotypic diversification, and which are confined to the extreme soda lakes Magadi and Natron in East Africa. We generated an extensive RAD data set of 96 individuals from multiple sampling sites and found evidence for genetic admixture between species within Lake Natron, with the highest levels of admixture between sympatric populations of the most recently diverged species. Despite considerable environmental separation, populations within Lake Natron do not exhibit isolation by distance, indicating panmixia within the lake, although individuals within lineages clustered by population in phylogenomic analysis. Our results indicate exceptionally low genetic differentiation across the radiation despite considerable phenotypic trophic variation, supporting previous findings from smaller data sets; however, with the increased power of densely sampled SNPs, we identify genomic peaks of differentiation (F_ST outliers) between Alcolapia species. While evidence of ongoing gene flow and interspecies hybridization in certain populations suggests that Alcolapia species are incompletely reproductively isolated, the identification of outlier SNPs under diversifying selection indicates the radiation is undergoing adaptive divergence.     

Speciation in the marine crop Pyropia yezoensis (Bangiales,Rhodophyta)

In the marine red alga Pyropia yezoensis, commonly known in Japan as nori, sympatric occurrence of two cryptic species Pyropia sp. 2 and Pyropia sp. 3 on the same rock in a natural habitat has been confirmed by molecular analysis and detailed morphological observations. To confirm whether Pyropia sp. 2 and Pyropia sp. 3 were reproductively isolated in the sympatric population, 170 blades that had previously been studied using a maternally inherited plastid marker were examined with a nuclear gene marker. The results suggested that Pyropia sp. 2 and Pyropia sp. 3 with identical morphological features were reproductively isolated in the sympatric population and that they were different species based on the biological species concept. Although gametophytic blades of Pyropia were usually assumed to be haploid, 18 of 170 blades possessed both of the two genotypes derived from Pyropia sp. 2 and from Pyropia sp. 3. These results inferred that allodiploid blades were generated from the interspecific hybridization between these two cryptic species. The present findings provide insights for future studies on the speciation mechanism in seaweeds, particularly for genera that contain numerous species.     

Cryptic diversity,reproductive isolation and cytoplasmic incompatibility in a classic biological control success story

Molecular genetics and symbiont diagnostics have revolutionized our understanding of insect species diversity, and the transformative effects of bacterial symbionts on host life history. Encarsia inaron is a parasitoid wasp that has been shown to harbour two bacterial endosymbionts, Wolbachia and Cardinium. Known then as E. partenopea, it was introduced to the USA in the late 1980s from populations collected in Italy and Israel for the biological control of an ornamental tree pest, the ash whitefly, Siphoninus phillyreae. We studied natural populations from sites in the USA, the Mediterranean and the Middle East as well as from a Cardinium‐infected laboratory culture established from Italy, with the aims of characterizing these populations genetically, testing reproductive isolation, determining symbiont infection status in their native and introduced range, and determining symbiont role. The results showed that the two Encarsia populations introduced to the USA are genetically distinct, reproductively isolated, have different symbionts and different host–symbiont interactions, and can be considered different biological species. One (‘E. inaron’) is doubly infected by Wolbachia and Cardinium, while only Cardinium is present in the other (‘E. partenopea’). The Cardinium strains in the two species are distinct, although closely related, and crossing tests indicate that the Cardinium infecting ‘E. partenopea’ induces cytoplasmic incompatibility. The frequency of symbiont infection found in the native and introduced range of these wasps was similar, unlike the pattern seen in some other systems. These results also lead to a retelling of a successful biological control story, with several more characters than had been initially described.     

The importance of cryptic species and subspecific populations in classic biological control of weeds: a North American perspective

Classical biological control of weeds depends on finding agents that are highly host-specific. This requires not only correctly understanding the identity of the target plant, sometimes to subspecific levels, in order to find suitable agents, but also identifying agents that are sufficiently specific to be safe and effective. Behavioral experiments and molecular genetic tools have revealed that some arthropod species previously thought to be polyphagous really consist of multiple cryptic species, host races or biotypes, some of which are more host-specific than others. Whereas true species are reproductively isolated, individuals from subspecific populations may potentially interbreed with those of other populations if they should encounter them. Furthermore, biotypes may consist of individuals sharing a genotype that is not fixed within a monophyletic group, and thus may not be evolutionarily stable. This raises the question of how such populations should be classified, and how to confirm the identity of live arthropods before releasing them as classical biological control agents. The existence of host races or cryptic species may greatly increase the number of prospective biological control agents available. However, it may also create new challenges for governmental regulation. These issues are discussed using pertinent examples, mainly from North America.     

Glacial cycles drive rapid divergence of cryptic field vole species

Understanding the factors that contribute to the generation of reproductively isolated forms is a fundamental goal of evolutionary biology. Cryptic species are an especially interesting challenge to study in this context since they lack obvious morphological differentiation that provides clues to adaptive divergence that may drive reproductive isolation. Geographical isolation in refugial areas during glacial cycling is known to be important for generating genetically divergent populations, but its role in the origination of new species is still not fully understood and likely to be situation dependent. We combine analysis of 35,434 single‐nucleotide polymorphisms (SNPs) with environmental niche modeling (ENM) to investigate genomic and ecological divergence in three cryptic species formerly classified as the field vole (Microtus agrestis). The SNPs demonstrate high genomic divergence (pairwise F_ST values of 0.45–0.72) and little evidence of gene flow among the three field vole cryptic species, and we argue that genetic drift may have been a particularly important mechanism for divergence in the group. The ENM reveals three areas as potential glacial refugia for the cryptic species and differing climatic niches, although with spatial overlap between species pairs. This evidence underscores the role that glacial cycling has in promoting genetic differentiation and reproductive isolation by subdivision into disjunct distributions at glacial maxima in areas relatively close to ice sheets. Future investigation of the intrinsic barriers to gene flow between the field vole cryptic species is required to fully assess the mechanisms that contribute to reproductive isolation. In addition, the Portuguese field vole (M. rozianus) shows a high inbreeding coefficient and a restricted climatic niche, and warrants investigation into its conservation status.     

Genetic rescue in a severely inbred wolf population

Natural populations are becoming increasingly fragmented which is expected to affect their viability due to inbreeding depression, reduced genetic diversity and increased sensitivity to demographic and environmental stochasticity. In small and highly inbred populations, the introduction of only a few immigrants may increase vital rates significantly. However, very few studies have quantified the long‐term success of immigrants and inbred individuals in natural populations. Following an episode of natural immigration to the isolated, severely inbred Scandinavian wolf (Canis lupus) population, we demonstrate significantly higher pairing and breeding success for offspring to immigrants compared to offspring from native, inbred pairs. We argue that inbreeding depression is the underlying mechanism for the profound difference in breeding success. Highly inbred wolves may have lower survival during natal dispersal as well as competitive disadvantage to find a partner. Our study is one of the first to quantify and compare the reproductive success of first‐generation offspring from migrants vs. native, inbred individuals in a natural population. Indeed, our data demonstrate the profound impact single immigrants can have in small, inbred populations, and represent one of very few documented cases of genetic rescue in a population of large carnivores.     

Male mating preference of two cryptic species of the herbivorous insect Eccritotarsus catarinensis

When using biological control against pest populations, more than one biocontrol agent might be introduced simultaneously. This could be counterproductive in the event of negative interactions between the biocontrol agents. Within and between species interactions have a strong impact on mating behaviour and reproduction, and can have an impact on the effectiveness of biological control. We studied the reproductive compatibility between two geographically isolated strains (Brazil and Peru) of Eccritotarsus catarinensis (Heteroptera: Miridae), a biocontrol agent against the invasive aquatic weed, Eichhornia crassipes. By performing inter- and intra-species mating experiments, we investigated whether or not males from each of the cryptic species would be able to distinguish between partners from either species, and if they would mate with partners from the opposite species. Our results showed the decrease in lifetime fecundity, and most importantly, the lack of production of offspring from eggs resulting from forced hybridisation. We showed that Peruvian males mated only with females from their own species, and did not mate with females from the Brazilian species. In contrast, Brazilian males mated equally with females from both species, but needed significantly more time in order to commence a mating, and no offspring were produced from eggs resulting from hybridisation. Although future studies demand more rigorous controls, our results indicate asymmetrical sexual isolation between the two species. We speculate on mechanisms involved in reproductive isolation in the two cryptic species, and the possible implications for effective biocontrol, and include some morphological measurements that might support our assumptions.     

The effects of diadromy and its loss on genomic divergence: The case of amphidromous Galaxias maculatus populations

Understanding the evolutionary mechanisms that affect the genetic divergence between diadromous and resident populations across heterogeneous environments is a challenging task. While diadromy may promote gene flow leading to a lack of genetic differentiation among populations, resident populations tend to be affected by local adaptation and/or plasticity. Studies on these effects on genomic divergence in nonmodel amphidromous species are scarce. Galaxias maculatus, one of the most widespread fish species in the Southern Hemisphere, exhibits two life histories, an ancestral diadromous, specifically, amphidromous form, and a derived freshwater resident form. We examined the genetic diversity and divergence among 20 estuarine and resident populations across the Chilean distribution of G. maculatus and assessed the extent to which selection is involved in the differentiation among resident populations. We obtained nearly 4,400 SNP markers using a RADcap approach for 224 individuals. As expected, collections from estuarine locations typically consist of diadromous individuals. Diadromous populations are highly differentiated from their resident counterparts by both neutral and putative adaptive markers. While diadromous populations exhibit high gene flow and lack site fidelity, resident populations appear to be the product of different colonization events with relatively low genetic diversity and varying levels of gene flow. In particular, the northernmost resident populations were clearly genetically distinct and reproductively isolated from each other suggesting local adaptation. Our study provides insights into the role of life history differences in the maintenance of genetic diversity and the importance of genetic divergence in species evolution.     

Detecting cryptic species in sympatry and allopatry: analysis of hidden diversity in Polyommatus (Agrodiaetus) butterflies (Lepidoptera: Lycaenidae)

Modern multilocus molecular techniques are a powerful tool in the detection and analysis of cryptic taxa. However, its shortcoming is that with allopatric populations it reveals phylogenetic lineages, not biological species. The increasing power of coalescent multilocus analysis leads to the situation in which nearly every geographically isolated or semi‐isolated population can be identified as a lineage and therefore raised to species rank. It leads to artificial taxonomic inflation and as a consequence creates an unnecessary burden on the conservation of biodiversity. To solve this problem, we suggest combining modern lineage delimitation techniques with the biological species concept. We discuss several explicit principles on how genetic markers can be used to detect cryptic entities that have properties of biological species (i.e. of actually or potentially reproductively isolated taxa). Using these principles we rearranged the taxonomy of the butterfly species close to Polyommatus (Agrodiaetus) ripartii. The subgenus Agrodiaetus is a model system in evolutionary research, but its taxonomy is poorly elaborated because, as a rule, most of its species are morphologically poorly differentiated. The taxon P. (A.) valiabadi has been supposed to be one of the few exceptions from this rule due to its accurately distinguishable wing pattern. We discovered that in fact traditionally recognized P. valiabadi is a triplet of cryptic species, strongly differentiated by their karyotypes and mitochondrial haplotypes.     

Biodiversity and Molecular Phylogeny of Australian Clevelandella Species (Class Armophorea,Order Clevelandellida,Family Clevelandellidae), Intestinal Endosymbiotic Ciliates in the Wood‐Feeding Roach Panesthia cribrata Saussure, 1864

There are over 100 species in the Order Clevelandellida distributed in many hosts. The majority is assigned to one of the five families, the Nyctotheridae. Our knowledge of clevelandellid genetic diversity is limited to species of Nyctotherus and Nyctotheroides. To increase our understanding of clevelandellid genetic diversity, species were isolated from intestines of the Australian wood‐feeding roach Panesthia cribrata Saussure, 1864 from August to October, 2008. Four morphospecies, similar to those reported in Java and Japan by Kidder [Parasitologica, 29 :163–205], were identified: Clevelandella constricta, Clevelandella nipponensis, Clevelandella parapanesthiae, and Clevelandella panesthiae. Small subunit rRNA gene sequences assigned all species to a “family” clade that was sister to the clade of species assigned to the Family Nyctotheridae in the Order Clevelandellida. Genetics and morphology were consistent for the first three Clevelandella species, but isolates assigned to C. panesthiae were assignable to three different genotypes, suggesting that this may be a cryptic species complex.     

Effects of floral resources on the efficacy of a primary parasitoid and a facultative hyperparasitoid

Resources added to agroecosystems to enhance biological control are potentially available to multiple members of the resident insect community—not only the biological control agents for which the resources are intended. Many studies have examined the effects of sugar feeding on the efficacy of biological control agents. However, such information is lacking for other, interacting species such as facultative hyperparasitoids, which may contribute to pest suppression but can also interfere with introduced biological control agents. Under greenhouse conditions, we tested the direct effects of sugar and nectar provisioning on the longevity, host‐killing impact and offspring production of two pupal parasitoids associated with leek moth, Acrolepiopsis assectella: the introduced biological control agent, Diadromus pulchellus, and the native facultative hyperparasitoid, Conura albifrons. Adding sucrose, buckwheat or a combination of buckwheat and common vetch to a sugar‐deprived system (potted leek plants in cages) increased parasitoid longevity and resulted in higher leek moth parasitism and mortality compared to water or common vetch treatments. However, the two parasitoid species exhibited a distinct temporal response to the treatments, likely influenced by differences in their life histories. This study provides insight into how integrating conservation biological control techniques could affect the success of a classical biological control programme.     

Reproductive isolation betweenTetranychus lintearius and two related mites,T. urticae andT. turkestani (Acarina: Tetranychidae)

Gorse,Ulex europaeus L. (Leguminosae), is a serious weed in New Zealand and some other temperate parts of the world.Tetranychus lintearius Dufour (Tetranychidae) has been released in New Zealand as a biological control agent for the weed. Before it could be introduced from Europe, it was necessary to show thatT. lintearius is reproductively isolated from closely related pest mites.Crosses and reciprocal crosses were made betweenT. lintearius and four populations ofT. urticae Koch, and betweenT. lintearius and one population ofT. turkestani (Ugarov and Nikolski). In a second experiment, males of a second population ofT. lintearius were crossed with females of fiveT. urticae populations. The sex-ratio of F₁ progeny in both experiments suggested thatT. lintearius was reproductively isolated from the other species, and could therefore be used as a biological control agent for gorse.     

No inbreeding depression in laboratory‐reared individuals of the parasitoid wasp Allotropa burrelli

Inbreeding depression is a major concern in almost all human activities relating to plant and animal breeding. The biological control of pests with natural enemies is no exception, because populations of biocontrol agents experience a series of bottlenecks during importation, rearing, and introduction. A classical biological control program for the Comstock mealybug Pseudococcus comstocki (Hemiptera: Pseudococcidae) was initiated in France in 2008, based on the introduction of an exotic parasitoid, Allotropa burrelli Mues. (Hymenoptera: Platygastridae), a haplodiploid parasitoid imported from Japan. We evaluated the sensitivity of A. burrelli to inbreeding, to optimize rearing and release strategies. We compared several morphological and life‐history traits between the offspring of siblings and the offspring of unrelated parents. We took into account the low level of genetic variability due to the relatively small size of laboratory‐reared populations by contrasting two types of pedigree: one for individuals from a strain founded from a single field population, and the other generated by hybridizing individuals from two strains founded from two highly differentiated populations. Despite this careful design, we obtained no evidence for a negative impact of inbreeding on laboratory‐reared A. burrelli. We discussed the results in light of haplodiploid sex determination and parasitoid mating systems, and classical biological control practices.     

Responses of different geographic populations of two potato tuber moth species to genetic variants of Phthorimaea operculella granulovirus

Phthorimaea operculella granulovirus (PhopGV) belongs to the genus Betabaculovirus of the arthropod‐infecting Baculoviridae. PhopGV is able to infect several gelechiid species. Among them are the potato tuber moths Phthorimaea operculella Zeller and Tecia solanivora Povolny (both Lepidoptera: Gelechiidae). In various South American countries, PhopGV‐based biopesticides are used to control either P. operculella or T. solanivora. Many trials have indicated that a particular viral isolate can exhibit very distinct pathogenicity when infecting different host species or different populations of one host species. In this study, we compared host–pathogen interactions using various PhopGV isolates and various populations of P. operculella and T. solanivora. Virus isolates from P. operculella were more pathogenic against their original host species than against T. solanivora. A PhopGV isolated from T. solanivora was less efficient against P. operculella. In addition, virus isolates differed in pathogenicity toward their hosts (i.e., lethal concentrations of isolates ranged from low to high). Unexpectedly, we also found that host populations of one species from distinct geographic origins did not differ significantly in susceptibility to the same PhopGV isolate. This was the case for both host species and for five PhopGV isolates. Comparative restriction fragment length polymorphism (RFLP) analyses of 11 isolates including those used in bio‐assays indicated three main regions of variation in the genome of PhopGV, corresponding to the regions of open reading frame PhopGV046, gene PhopGV129 (egt), and repeat 9 (located between open reading frames PhopGV083 and PhopGV084). Comparison of the nucleotide sequences of the insertions/deletions present in these regions were carried out for the most variable isolate, JLZ9f. The results are discussed in the context of the production and use of PhopGV as a biological agent against these two pest species.     

Population genetics of Anopheles koliensis through Papua New Guinea: New cryptic species and landscape topography effects on genetic connectivity

New Guinea is a topographically and biogeographically complex region that supports unique endemic fauna. Studies describing the population connectivity of species through this region are scarce. We present a population and landscape genetic study on the endemic malaria‐transmitting mosquito, Anopheles koliensis (Owen). Using mitochondrial and nuclear sequence data, as well as microsatellites, we show the evidence of geographically discrete population structure within Papua New Guinea (PNG). We also confirm the existence of three rDNA ITS2 genotypes within this mosquito and assess reproductive isolation between individuals carrying different genotypes. Microsatellites reveal the clearest population structure and show four clear population units. Microsatellite markers also reveal probable reproductive isolation between sympatric populations in northern PNG with different ITS2 genotypes, suggesting that these populations may represent distinct cryptic species. Excluding individuals belonging to the newly identified putative cryptic species (ITS2 genotype 3), we modeled the genetic differences between A. koliensis populations through PNG as a function of terrain and find that dispersal is most likely along routes with low topographic relief. Overall, these results show that A. koliensis is made up of geographically and genetically discrete populations in Papua New Guinea with landscape topography being important in restricting dispersal.     

Self‐fertilization,long‐distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at the worldwide scale

Population genetic studies are efficient for inferring the invasion history based on a comparison of native and invasive populations, especially when conducted at species scale. An expected outcome in invasive populations is variability loss, and this is especially true in self‐fertilizing species. We here focus on the self‐fertilizing Pseudosuccinea columella, an invasive hermaphroditic freshwater snail that has greatly expanded its geographic distribution and that acts as intermediate host of Fasciola hepatica, the causative agent of human and veterinary fasciolosis. We evaluated the distribution of genetic diversity at the largest geographic scale analysed to date in this species by surveying 80 populations collected during 16 years from 14 countries, using eight nuclear microsatellites and two mitochondrial genes. As expected, populations from North America, the putative origin area, were strongly structured by selfing and history and harboured much more genetic variability than invasive populations. We found high selfing rates (when it was possible to infer it), none‐to‐low genetic variability and strong population structure in most invasive populations. Strikingly, we found a unique genotype/haplotype in populations from eight invaded regions sampled all over the world. Moreover, snail populations resistant to infection by the parasite are genetically distinct from susceptible populations. Our results are compatible with repeated introductions in South America and flash worldwide invasion by this unique genotype/haplotype. Our study illustrates the population genetic consequences of biological invasion in a highly selfing species at very large geographic scale. We discuss how such a large‐scale flash invasion may affect the spread of fasciolosis.     

Increased genetic divergence between two closely related fir species in areas of range overlap

Because of introgressive hybridization, closely related species can be more similar to each other in areas of range overlap (parapatry or sympatry) than in areas where they are geographically isolated from each other (allopatry). Here, we report the reverse situation based on nuclear genetic divergence between two fir species, Abies chensiensis and Abies fargesii, in China, at sites where they are parapatric relative to where they are allopatric. We examined genetic divergence across 126 amplified fragment length polymorphism (AFLP) markers in a set of 172 individuals sampled from both allopatric and parapatric populations of the two species. Our analyses demonstrated that AFLP divergence was much greater between the species when comparisons were made between parapatric populations than between allopatric populations. We suggest that selection in parapatry may have largely contributed to this increased divergence.     

Species identity of geographically distinct populations of the glassy-winged sharpshooter parasitoid Gonatocerus ashmeadi: morphology, DNA sequences, and reproductive compatibility

Gonatocerus ashmeadi is a common and seemingly widespread egg parasitoid of Homalodisca coagulata, the glassy-winged sharpshooter (GWSS). Location records for G. ashmeadi indicate its natural range to be the southeastern USA and northeastern Mexico (which coincides with the presumed native range of GWSS), and possibly southern and central California (CA) (the adventive range of GWSS). The purpose of our work was to determine whether G. ashmeadi in the USA and northeastern Mexico is one species or a complex of reproductively incompatible sibling species. We used three approaches to determine the species identity of different G. ashmeadi populations: (1) reassessment of key morphological features using scanning electron microscopy (SEM) to determine if subtle morphological differences exist between G. ashmeadi populations which could indicate species differences; (2) to determine if molecular differences exist between G. ashmeadi populations collected from different regions by comparing mitochondrial and ribosomal DNA sequences; (3) mating compatibility studies to determine if different populations of G. ashmeadi are reproductively isolated, or if mating occurs, whether offspring are viable thereby defining species groups on the basis of successful interbreeding. Results from these three areas (morphology, DNA sequences, and reproductive compatibility) have been evaluated collectively; leading us to the conclusion that G. ashmeadi as it is currently viewed is a valid species and not an aggregate of morphologically indistinguishable cryptic species.     

Multidisciplinary approaches for species delimitation in Sisyrinchium (Iridaceae)

A remarkable morphological similarity in natural populations of different groups of Sisyrinchium spp. (Iridaceae) makes classification of some species difficult. The present paper focused on two morphological categories of S. sellowianum (MC‐I and MC‐II) with distinct morphological traits. Morphological, cytogenetic, reproductive biology and genetic studies were performed, aiming to elucidate and better characterize such variation. The basic chromosome number established for the species was x = 9 and diploid and tetraploid populations were observed for MC‐I, whereas MC‐II showed only diploid populations. Different pollen morphologies were recognized in each morphological category. Based on pollination experiments, MC‐I is out‐crossing, whereas MC‐II is selfing. The populations were highly differentiated (F_ST = 0.46, θ^B = 0.62). In MC‐I, observed variation was greater within populations (69%) than among populations (31%), whereas in MC‐II, 61% of the variation was among populations and 39% within populations. This study evidenced that the two morphological categories correspond to distinct and reproductively isolated species and that floral traits reflect their breeding system, with allogamous plants in MC‐I and autogamous in MC‐II. Furthermore, MC‐II is unveiled as S. platycaule, a neglected taxon of Sisyrinchium in Brazil. The tools applied in this study were substantially able to check the identity of the two morphological categories as two different species and present the variation encountered in the taxa.