Heterospecific courtship,minority effects and niche separation between cryptic butterfly species

Species interacting in varied ecological conditions often evolve in different directions in different local populations. The butterflies of the cryptic Leptidea complex are sympatrically distributed in different combinations across their Eurasian range. Interestingly, the same species is a habitat generalist in some regions and a habitat specialist in others, where a sibling species has the habitat generalist role. Previous studies suggest that this geographically variable niche divergence is generated by local processes in different contact zones. By varying the absolute and relative densities of Leptidea sinapis and Leptidea juvernica in large outdoor cages, we show that female mating success is unaffected by conspecific density, but strongly negatively affected by the density of the other species. Whereas 80% of the females mated when a conspecific couple was alone in a cage, less than 10% mated when the single couple shared the cage with five pairs of the other species. The heterospecific courtships can thus affect the population fitness, and for the species in the local minority, the suitability of a habitat is likely to depend on the presence or absence of the locally interacting species. If the local relative abundance of the different species depends on the colonization order, priority effects might determine the ecological roles of interacting species in this system.     

A striking lack of genetic diversity across the wide-ranging amphibian <Emphasis Type="Italic">Gastrophryne carolinensis</Emphasis> (Anura: Microhylidae)

We examine phylogeographic structure across a wide-ranging microhylid frog (Gastrophryne carolinensis) using both mitochondrial (mtDNA) and nuclear (AFLP) data. Species with similar ecological characteristics such as large range size, low vagility, or existence across known biogeographic barriers, often are comprised of multiple, cryptic lineages. Surprisingly, our analyses of both portions of the genome show very little phylogeographic or population genetic structure. The family Microhylidae is one of the largest families of anurans with over 60 genera and around 400 species distributed across much of the world (Americas, Asia, Africa, and Madagascar), but very few phylogeographic studies have assessed intraspecific genetic diversity across the mitochondrial and nuclear genomes. Our results suggest that G. carolinensis, one of only three species of microhylid native to the US, has experienced a severe population bottleneck with subsequent range expansion. Comparable molecular data from closely related microhylids, in addition to demographic and ecological analyses, will provide valuable insight into patterns of genetic diversity and the processes driving phylogeographic diversity in these wide-ranging frogs.     

Ecological Differentiation of Cryptic Species within an Asexual Protist Morphospecies: A Case Study of Filamentous Green Alga Klebsormidium (Streptophyta)

Taxa of microbial eukaryotes defined on morphological basis display a large degree of genetic diversity, implying the existence of numerous cryptic species. However, it has been postulated that genetic diversity merely mirrors accumulation of neutral mutations. As a case taxon to study cryptic diversity in protists, we used a widely distributed filamentous genus, Klebsormidium, specifically the lineage E (K. flaccidum/K. nitens complex) containing a number of morphologically similar strains. Fourteen clades were recognized in the phylogenetic analysis based on a concatenated ITS rDNA + rbcL data set of more than 70 strains. The results of inferred character evolution indicated the existence of phylogenetic signal in at least two phenotypic characters (production of hydro‐repellent filaments and morphology of zoosporangia). Moreover, the lineages recovered exhibited strong ecological preferences to one of the three habitat types: natural subaerial substrata, artificial subaerial substrata, and aquatic habitats. We interpret these results as evidence of existence of a high number of cryptic species within the single morphospecies. We consider that the permanent existence of genetically and ecologically well‐defined cryptic species is enabled by the mechanism of selective sweep.     

Cryptic diversity in the genus <Emphasis Type="Italic">Adineta</Emphasis> Hudson &; Gosse, 1886 (Rotifera: Bdelloidea: Adinetidae): a DNA taxonomy approach

Cryptic species are continuously discovered in rotifers using different methods to delineate these units of diversity. DNA taxonomy is the most effective method taxonomists have to untie potential cryptic taxa. Here, we estimate hidden diversity in a genus of bdelloid rotifers, Adineta. We analyse cryptic diversity using a coalescent approach to infer evolutionarily significant units from a phylogenetic tree obtained from cytochrome oxidase I sequences. Cryptic diversity was measured for eight traditional species and from several additional undetermined populations. Taxonomic inflation of up to 36 taxa was found in A. vaga from DNA taxonomy. As observed in other microscopic organisms, cryptic taxa within each traditional species were not geographically isolated, but had significantly narrower ecological niches than expected by chance alone.     

Hidden diversity in the freshwater planktonic diatom Asterionella formosa

Many freshwater and marine algal species are described as having cosmopolitan distributions. Whether these widely distributed morphologically similar algae also share a similar gene pool remains often unclear. In the context of island biogeography theory, stronger spatial isolation deemed typical of freshwater lakes should restrict gene flow and lead to higher genetic differentiation among lakes. Using nine microsatellite loci, we investigate the genetic diversity of a widely distributed freshwater planktonic diatom, Asterionella formosa, across different lakes in Switzerland and the Netherlands. We applied a hierarchical spatial sampling design to determine the geographical scale at which populations are structured. A subset of the isolates was additionally analysed using amplified fragment length polymorphism (AFLP) markers. Our results revealed complex and unexpected population structure in A. formosa with evidence for both restricted and moderate to high gene flow at the same time. Different genetic markers (microsatellites and AFLPs) analysed with a variety of multivariate methods consistently revealed that genetic differentiation within lakes was much stronger than among lakes, indicating the presence of cryptic species within A. formosa. We conclude that the hidden diversity found in this study is expected to have implications for the further use of A. formosa in biogeographical, conservation and ecological studies. Further research using species‐level phylogenetic markers is necessary to place the observed differentiation in an evolutionary context of speciation.     

Hidden in the Arabian Mountains: Multilocus phylogeny reveals cryptic diversity in the endemic Omanosaura lizards

An increase in studies in the Hajar Mountains from the southeastern Arabian Peninsula has revealed a high richness of endemic evolutionary lineages with many cryptic taxa. Omanosaura is the only lacertid lizard genus endemic to the Hajar Mountains, with two species O. cyanura and O. jayakari distributed throughout this mountain range. The phylogenetic relationships and genetic diversity between and within these species have been poorly studied. In this study, we collected mitochondrial (12S, cytb, and nd4) and nuclear (cmos and mc1r) sequences for 25 specimens of Omanosaura, including 15 individuals of O. jayakari and 10 of O. cyanura. We performed phylogenetic analyses based on network reconstruction, maximum likelihood and Bayesian inference to estimate the relationships and intraspecific genetic diversity of these species. We estimated the time of divergence between the two species in the Miocene, around 8.5 million years ago. Omanosaura jayakari shows little genetic diversity, while O. cyanura presents two differentiated lineages. These are reciprocally monophyletic at mitochondrial and nuclear genes and present a high genetic distance between them. These two lineages are associated with the geographic features of the Hajar Mountains, with one lineage distributed in the northernmost part of the Hajar Mountains and the other in the rest of the western Hajars, the Jebel Akhdar, and the eastern Hajars. This geographic relationship has been recovered previously in other reptile taxa and is generally associated with high levels of local genetic diversity. Our results suggest the existence of cryptic diversity within O. cyanura and support a general biogeographic pattern of high diversity and endemism in the northern Hajar Mountains that certainly deserves additional research in the future.     

Extreme levels of hidden diversity in microscopic animals (Rotifera) revealed by DNA taxonomy

Knowledge and estimates of species richness at all scales are biased both by our understanding of the evolutionary processes shaping diversity and by the methods used to delineate the basic units of diversity. DNA taxonomy shows that diversity may be underestimated by traditional taxonomy, especially for microscopic animals. The effects of such hidden diversity are usually overlooked in ecological studies. Here, we estimate hidden diversity in bdelloid rotifers, a group of microscopic animals. We analyse cryptic diversity using a coalescent approach to infer taxonomical units from phylogenetic trees. Cryptic diversity was measured for eight traditional species of bdelloid rotifers and the results compared to that of the monogonont rotifer Brachionus plicatilis species complex, which is well studied and for which cross-mating experiments have been performed to explicitly define some of the species boundaries. A taxonomic inflation of up to 34 potential cryptic taxa was found in bdelloids. Cryptic taxa within each traditional species may be spatially isolated, but do not have narrower ecological niches. The species deemed as generalists exhibit the highest cryptic diversity. Cryptic diversity based on molecular characterization is commonly found in animals; nevertheless, the amount of cryptic diversity in bdelloids is much higher than in other groups analysed so far, maybe because of their peculiar parthenogenetic reproduction, other than microscopic size. We discuss this hypothesis in the light of the available empirical evidence from other groups of microscopic invertebrates, such as tardigrades and mites, which share size, habitat heterogeneity, potential for dispersal, and/or parthenogenetic reproduction.     

Diurnal butterflies (Lepidoptera, Rhopalocera) of the Solovetskie Islands (Northwestern Russia, the White Sea)

The species composition and diversity of Lepidoptera Rhopalocera were studied on the Solovetskie Islands during five years (2001–2005). Twenty-nine species of five families were recorded. The most abundant species are Leptidea sinapis, Boloria aquilonaris, Heodes virgaureae, Plebejus argus, Arcia eumedon, and Cyaniris semiargus. The fauna of these islands is poorer than local faunas of the continental northern taiga, forest-tundra, and southern tundra of Europe. The species diversity of insular assemblages of diurnal lepidopterans is lower in comparison with analogous continental assemblages. They are characterized by the mixed species composition, when species with different ecological preference occur within the same habitat.     

The interplay of colour and bioacoustic traits in the differentiation of a Southeast Asian songbird complex

Morphological traits have served generations of biologists as a taxonomic indicator, and have been the main basis for defining and classifying species diversity for centuries. A quantitative integration of behavioural characters, such as vocalizations, in studies on biotic differentiation has arisen more recently, and the relative importance of these different traits in the diversification process remains poorly understood. To provide a framework within which to interpret the evolutionary interplay between morphological and behavioural traits, we generated a draft genome of a cryptic Southeast Asian songbird, the limestone wren‐babbler Napothera crispifrons. We resequenced whole genomes of multiple individuals of all three traditional subspecies and of a distinct leucistic population. We demonstrate strong genomic and mitochondrial divergence among all three taxa, pointing to the existence of three species‐level lineages. Despite its great phenotypic distinctness, the leucistic population was characterized by shallow genomic differentiation from its neighbour, with only a few localized regions emerging as highly diverged. Quantitative bioacoustic analysis across multiple traits revealed deep differences especially between the two taxa characterized by limited plumage differentiation. Our study demonstrates that differentiation in these furtive songbirds has resulted in a complex mosaic of colour‐based and bioacoustic differences among populations. Extreme colour differences can be anchored in few genomic loci and may therefore arise and subside rapidly.     

Integrated species delimitation and conservation implications of an endangered weevil Pachyrhynchus sonani (Coleoptera: Curculionidae) in Green and Orchid Islands of Taiwan

Oceanic islands are productive habitats for generating new species and high endemism, which is primarily due to their geographical isolation, smaller population sizes and local adaptation. However, the short divergence times and subtle morphological or ecological divergence of insular organisms may obscure species identity, so the cryptic endemism on islands may be underestimated. The endangered weevil Pachyrhynchus sonani Kôno (Coleoptera: Curculionidae: Entiminae: Pachyrhynchini) is endemic to Green Island and Orchid Island of the Taiwan‐Luzon Archipelago and displays widespread variation in coloration and host range, thus raising questions regarding its species boundaries and degree of cryptic diversity. We tested the species boundaries of P. sonani using an integrated approach that combined morphological (body size and shape, genital shape, coloration and cuticular scale), genetic (four genes and restriction site‐associated DNA sequencing, RAD‐seq) and ecological (host range and distribution) diversity. The results indicated that all the morphological datasets for male P. sonani, except for the colour spectrum, reveal overlapping but statistically significant differences between islands. In contrast, the morphology of the female P. sonani showed minimum divergence between island populations. The populations of P. sonani on the two islands were significantly different in their host ranges, and the genetic clustering and phylogenies of P. sonani established two valid evolutionary species. Integrated species delimitation combining morphological, molecular and ecological characters supported two distinct species of P. sonani from Green Island and Orchid Island. The Green Island population was described as P. jitanasaius sp.n. Chen & Lin, and it is recommended that its threatened conservation status be recognized. Our findings suggest that the inter‐island speciation of endemic organisms inhabiting both islands may be more common than previously thought, and they highlight the possibility that the cryptic diversity of small oceanic islands may still be largely underestimated.     

Cryptic species diversity in a widespread bumble bee complex revealed using mitochondrial DNA RFLPs

Cryptic species diversity is thought to be common within the class Insecta, posing problems for basic ecological and population genetic studies and conservation management. Within the temperate bumble bee (Bombus spp.) fauna, members of the subgenus Bombus sensu stricto are amongst the most abundant and widespread. However, their species diversity is controversial due to the extreme difficulty or inability morphologically to identify the majority of individuals to species. Our character-based phylogenetic analyses of partial CO1 (700 bp) from 39 individuals spread across their sympatric European ranges provided unequivocal support for five taxa (3–22 diagnostic DNA base pair sites per species). Inclusion of 20 Irish specimens to the dataset revealed ≥2.3% sequence divergence between taxa and ≤1.3% within taxa. We developed a PCR-RFLP based method for unequivocally distinguishing amongst the four cryptic European taxa of this subgenus, B. cryptarum, B. lucorum, B. magnus and B. terrestris, and used it to analyse 391 females of the former three species collected across Ireland, all of which could be unambiguously assigned to species. Bombus lucorum was the most widely distributed and abundant of the cryptarum–lucorum–magnus species complex, comprising 56% of individuals, though it was significantly less abundant at higher altitudes (>200 m) whilst B. cryptarum was relatively more abundant at higher altitudes. Bombus magnus was rarely encountered at urban sites. Both B. lucorum and B. terrestris are nowadays reared commercially for pollination and transported globally. Our RFLP approach to identify native fauna can underpin ecological studies of these important cryptic species as well as the impact of commercial bumble bees on them.     

Cryptic diversity in ant-mimic Micaria spiders (Araneae,Gnaphosidae) and a tribute to early naturalists

Spiders of the genus Micaria are ground-living mimics of ants. Species delineation in these spiders is challenging, mainly because of exceptional high levels of intraspecific variation masking species boundaries. As implied by preliminary DNA barcode data from Central Europe, the Holarctic and very widely distributed glossy ant-spider M. pulicaria shows cryptic diversity. Here, we disentangle the hidden diversity by means of an integrative taxonomy approach, using mitochondrial DNA, morphometrics, traditional genitalic characters and ecology. Our data suggest the clear delineation of two distinct species, which supports the conception of 19th century taxonomists. These early naturalists distinguished M. pulicaria and a second closely related species based on morphology and natural history, which were synonymized in subsequent taxonomic studies. Therefore, we re-circumscribe M. pulicaria and revalidate the long forgotten M. micans. These two Micaria species co-occur sympatrically in vast areas of the western Palearctic, while the Nearctic region is populated by M. pulicaria alone. Male genitalic traits are more dissimilar in the area of sympatry than in allopatry, suggesting a decisive role of reproductive character displacement in species diversification. Our study emphasizes the value of the early taxonomic literature in integrative taxonomic studies, as it may contain crucial information on natural history that are not regularly recorded by modern taxonomists.     

Geographic isolation facilitates the evolution of reproductive isolation and morphological divergence

Geographic isolation is known to contribute to divergent evolution, resulting in unique phenotypes. Oftentimes morphologically distinct populations are found to be interfertile while reproductive isolation is found to exist within nominal morphological species revealing the existence of cryptic species. These disparities can be difficult to predict or explain especially when they do not reflect an inferred history of common ancestry which suggests that environmental factors affect the nature of ecological divergence. A series of laboratory experiments and observational studies were used to address what role biogeographic factors may play in the ecological divergence of Hyalella amphipods. It was found that geographic isolation plays a key role in the evolution of reproductive isolation and divergent morphology and that divergence cannot be explained by molecular genetic variation.     

Partitioning of herbivore hosts across time and food plants promotes diversification in the Megastigmus dorsalis oak gall parasitoid complex

Communities of insect herbivores and their natural enemies are rich and ecologically crucial components of terrestrial biodiversity. Understanding the processes that promote their origin and maintenance is thus of considerable interest. One major proposed mechanism is ecological speciation through host‐associated differentiation (HAD), the divergence of a polyphagous species first into ecological host races and eventually into more specialized daughter species. The rich chalcid parasitoid communities attacking cynipid oak gall wasp hosts are structured by multiple host traits, including food plant taxon, host gall phenology, and gall structure. Here, we ask whether the same traits structure genetic diversity within supposedly generalist parasitoid morphospecies. We use mitochondrial DNA sequences and microsatellite genotypes to quantify HAD for Megastigmus (Bootanomyia) dorsalis, a complex of two apparently generalist cryptic parasitoid species attacking oak galls. Ancient Balkan refugial populations showed phenological separation between the cryptic species, one primarily attacking spring galls, and the other mainly attacking autumn galls. The spring species also contained host races specializing on galls developing on different host‐plant lineages (sections Cerris vs. Quercus) within the oak genus Quercus. These results indicate more significant host‐associated structuring within oak gall parasitoid communities than previously thought and support ecological theory predicting the evolution of specialist lineages within generalist parasitoids. In contrast, UK populations of the autumn cryptic species associated with both native and recently invading oak gall wasps showed no evidence of population differentiation, implying rapid recruitment of native parasitoid populations onto invading hosts, and hence potential for natural biological control. This is of significance given recent rapid range expansion of the economically damaging chestnut gall wasp, Dryocosmus kuriphilus, in Europe.     

Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads (Pelobates)

Cryptic phylogeographic diversifications provide unique models to examine the role of phylogenetic divergence on the evolution of reproductive isolation, without extrinsic factors such as ecological and behavioural differentiation. Yet, to date very few comparative studies have been attempted within such radiations. Here, we characterize a new speciation continuum in a group of widespread Eurasian amphibians, the Pelobates spadefoot toads, by conducting multilocus (restriction site associated DNA sequencing and mitochondrial DNA) phylogenetic, phylogeographic and hybrid zone analyses. Within the P. syriacus complex, we discovered species‐level cryptic divergences (>5 million years ago [My]) between populations distributed in the Near‐East (hereafter P. syriacus sensu stricto [s.s.]) and southeastern Europe (hereafter P. balcanicus), each featuring deep intraspecific lineages. Altogether, we could scale hybridizability to divergence time along six different stages, spanning from sympatry without gene flow (P. fuscus and P. balcanicus, >10 My), parapatry with highly restricted hybridization (P. balcanicus and P. syriacus s.s., >5 My), narrow hybrid zones (~15 km) consistent with partial reproductive isolation (P. fuscus and P. vespertinus, ~3 My), to extensive admixture between Pleistocene and refugial lineages (≤2 My). This full spectrum empirically supports a gradual build up of reproductive barriers through time, reversible up until a threshold that we estimate at ~3 My. Hence, cryptic phylogeographic lineages may fade away or become reproductively isolated species simply depending on the time they persist in allopatry, and without definite ecomorphological divergence.     

Contrasting patterns of population structure and demographic history in cryptic species of Bostrychia intricata (Rhodomelaceae,Rhodophyta) from New Zealand

Spatial patterns of genetic diversity provide insight into the demography and history of species. Morphologically similar but genetically distinct “cryptic” species are increasingly being recognized in marine organisms through molecular analyses. Such species are, on closer inspection, often discovered to display contrasting life histories or occasionally minor morphological differences; molecular tools can thus be useful indicators of diversity. Bostrychia intricata, a marine red alga, is widely distributed throughout the Southern Hemisphere and comprises many cryptic species. We used mitochondrial cytochrome c oxidase I gene sequences to assess the genetic variation, population genetic structure, and demographic history of B. intricata in New Zealand. Our results supported the existence of three cryptic species of B. intricata (N2, N4, and N5) in New Zealand. Cryptic species N4, which was found throughout New Zealand, showed a higher genetic diversity and wider distribution than the other two species, which were only found in the North Island and northern South Island. Our analyses showed low to moderate genetic differentiation among eastern North Island populations for cryptic species N2, but high differentiation among North and South Island populations for N4, suggesting different population structure between these cryptic species. Data also indicated that N2 has recently undergone population expansion, probably since the Last Glacial Maximum (LGM), while the higher genetic diversity in N4 populations suggests persistence in situ through the LGM. The contrasting population structures and inferred demographic histories of these species highlight that life history can vary greatly even among morphologically indistinguishable taxa.     

The southwestern Carpathians as an ancient centre of diversity of freshwater gammarid amphipods: insights from the Gammarus fossarum species complex

Gammarus fossarum is a diverse species complex of epigean freshwater amphipods throughout Europe. Due to their poor dispersal capabilities and ubiquity, these crustaceans may serve as a model for investigating the influence of historical factors on the contemporary distribution and diversity patterns of freshwater macrozoobenthos. Here, we investigate the fine‐scale phylogeographic structure of this complex across its range in the southwestern Carpathian Mountains, which comprises two areas that are geographically isolated from its main European distribution area as well as from each other. Given the Tertiary age of many freshwater Gammarus species, we hypothesize that the southwestern Carpathian populations reflect a relict distribution pattern. We used two mitochondrial and three nuclear markers from 32 localities to reconstruct phylogenetic relationships and estimate the timings of divergence among southwestern Carpathian and non‐Carpathian lineages. Cryptic diversity was evaluated from mitochondrial markers by employing phylogenetic and distance‐based methods. We distinguished at least 16 cryptic microendemic taxa, some of them coexisting, distributed in the southwestern Carpathians in a mosaic‐like pattern. These lineages form a monophyletic group together with several lineages from southeastern Europe. Estimated divergence times indicate a Middle Miocene origin of this clade, with many deep splits dating back to more than 10 Ma. This time frame corresponds with a period of intense geological subsidence in the region that gave birth to the Pannonian Basin. We conclude that subsidence could have been an important driver of diversification in freshwater Gammarus and that the southwestern Carpathians represent an ancient centre of diversity for these crustaceans.     

Testing the ecological relevance of Daphnia species designations

1. Molecular approaches have increasingly revealed hidden genetic structure within ecologically important species, leading to the creation of sibling species whose ecological relevance is often unclear. A prime example is Daphnia galeata mendotae, which was split into D. dentifera and D. mendotae based on differences at two allozyme loci. 2. In a set of lake populations in Michigan USA, we test the geographical and temporal consistency of the genetic structure underlying this species split. We also test the morphological relevance of this molecular variation and its ecological significance in lakes. In essence, we ask: does recognition of these new species provide valuable information for plankton ecologists? 3. We found that D. dentifera and D. mendotae represent morphologically and ecologically distinct forms that are distributed among lakes in non‐random fashion, which were remarkably stable over 6 years. Key differences between the species concern their body and head shape, vertical habitat use within lakes and distribution among lakes of different size. We hypothesise that these differences represent specialisation to habitats that differ in risk of invertebrate predation. 4. Reproductive barriers alone are insufficient to explain the pattern of genetic structure; in some lakes complete introgression is apparent. However, parent species and hybrids exhibit a stable co‐existence in many lakes, which suggests that ecological specialisation reinforces divergence within this taxon.     

Molecular patterns of differentiation in canyon treefrogs (Hyla arenicolor): evidence for introgressive hybridization with the Arizona treefrog (H. wrightorum) and correlations with advertisement call differences

Detection of genetic and behavioural diversity within morphologically similar species has led to the discovery of cryptic species complexes. We tested the hypothesis that US populations of the canyon treefrog (Hyla arenicolor) may consist of cryptic species by examining mate‐attraction signals among three divergent clades defined by mtDNA. Using a multi‐locus approach, we re‐analysed phylogenetic relationships among the three clades and a closely related, but morphologically and behaviourally dissimilar species, the Arizona treefrog (H. wrightorum). We found evidence for introgression of H. wrightorum’s mitochondrial genome into H. arenicolor. Additionally, the two‐clade topology based on nuclear data is more congruent with patterns of call variation than the three‐clade topology from the mitochondrial dataset. The magnitude of the call divergence is probably insufficient to promote isolation of the nuclear DNA‐defined clades should they become sympatric, but further divergence in call properties significant in species identification could promote speciation in the future.