Cryptic speciation in the field vole: a multilocus approach confirms three highly divergent lineages in Eurasia

Species are generally described from morphological features, but there is growing recognition of sister forms that show substantial genetic differentiation without obvious morphological variation and may therefore be considered ‘cryptic species’. Here, we investigate the field vole (Microtus agrestis), a Eurasian mammal with little apparent morphological differentiation but which, on the basis of previous sex‐linked nuclear and mitochondrial DNA (mtDNA) analyses, is subdivided into a Northern and a Southern lineage, sufficiently divergent that they may represent two cryptic species. These earlier studies also provided limited evidence for two major mtDNA lineages within Iberia. In our present study, we extend these findings through a multilocus approach. We sampled 163 individuals from 46 localities, mainly in Iberia, and sequenced seven loci, maternally, paternally and biparentally inherited. Our results show that the mtDNA lineage identified in Portugal is indeed a distinct third lineage on the basis of other markers as well. In fact, multilocus coalescent‐based methods clearly support three separate evolutionary units that may represent cryptic species: Northern, Southern and Portuguese. Divergence among these units was inferred to have occurred during the last glacial period; the Portuguese lineage split occurred first (estimated at c. 70 000 bp ), and the Northern and Southern lineages separated at around the last glacial maximum (estimated at c. 18 500 bp ). Such recent formation of evolutionary units that might be considered species has repercussions in terms of understanding evolutionary processes and the diversity of small mammals in a European context.     

Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis, a disease confined to Latin America and of marked importance in the endemic areas due to its frequency and severity. This species is considered to be clonal according to mycological criteria and has been shown to vary in virulence. To characterize natural genetic variation and reproductive mode in this fungus, we analyzed P. brasiliensis phylogenetically in search of cryptic species and possible recombination using concordance and nondiscordance of gene genealogies with respect to phylogenies of eight regions in five nuclear loci. Our data indicate that this fungus consists of at least three distinct, previously unrecognized species: S1 (species 1 with 38 isolates), PS2 (phylogenetic species 2 with six isolates), and PS3 (phylogenetic species 3 with 21 isolates). Genealogies of four of the regions studied strongly supported the PS2 clade, composed of five Brazilian and one Venezuelan isolate. The second clade, PS3, composed solely of 21 Colombian isolates, was strongly supported by the alpha-tubulin genealogy. The remaining 38 individuals formed S1. Two of the three lineages of P. brasiliensis, S1 and PS2, are sympatric across their range, suggesting barriers to gene flow other than geographic isolation. Our study provides the first evidence for possible sexual reproduction in P. brasiliensis S1, but does not rule it out in the other two species.     

Reproductive interference by male Drosophila subobscura on female D. persimilis: A laboratory experiment

While females often reject courtship attempts by heterospecific males, reproductive interference by harassment from such males can nonetheless occur, potentially reducing female fitness. Such effects may be profound following a range expansion, when males from a new species may suddenly encounter (and perhaps even become abundant relative to) females of related native species. Drosophila subobscura recently invaded North America and may impact native species through reproductive interference and other processes. We test for the potential for reproductive interference by D. subobscura males on D. persimilis females in the laboratory. D. subobscura males aggressively copulated with D. persimilis females, including many females that exhibit rejection behaviors. Despite females attempting to dismount the males, the heterospecific copulations are on average longer than conspecific copulations, and females exhibit some reluctance to remate with conspecific males following this harassment. Females confined with both conspecific and heterospecific males produce fewer adult progeny than those with either conspecific males only or with conspecific males and distantly related D. simulans males that do not engage in female harassment. Overall, our results illustrate how reproductive interference by an invasive species can have negative effects on resident natural populations.     

Adaptation,ancestral variation and gene flow in a ‘Sky Island’ Drosophila species

Over time, populations of species can expand, contract, fragment and become isolated, creating subpopulations that must adapt to local conditions. Understanding how species maintain variation after divergence as well as adapt to these changes in the face of gene flow is of great interest, especially as the current climate crisis has caused range shifts and frequent migrations for many species. Here, we characterize how a mycophageous fly species, Drosophila innubila, came to inhabit and adapt to its current range which includes mountain forests in south‐western USA separated by large expanses of desert. Using population genomic data from more than 300 wild‐caught individuals, we examine four populations to determine their population history in these mountain forests, looking for signatures of local adaptation. In this first extensive study, establishing D. innubila as a key genomic "Sky Island" model, we find D. innubila spread northwards during the previous glaciation period (30–100 KYA) and have recently expanded even further (0.2–2 KYA). D. innubila shows little evidence of population structure, consistent with a recent establishment and genetic variation maintained since before geographic stratification. We also find some signatures of recent selective sweeps in chorion proteins and population differentiation in antifungal immune genes suggesting differences in the environments to which flies are adapting. However, we find little support for long‐term recurrent selection in these genes. In contrast, we find evidence of long‐term recurrent positive selection in immune pathways such as the Toll signalling system and the Toll‐regulated antimicrobial peptides.     

Variation of within‐crop microhabitat use by Drosophila suzukii (Diptera: Drosophilidae) in blackberry

Drosophila suzukii Matsumara (Diptera: Drosophilidae) is an invasive vinegar fly that infests ripe and ripening soft skinned fruits. In the south‐eastern United States, blackberry (Rubus spp.) crops are heavily impacted by D. suzukii, and current management tactics rely on the use of broad‐spectrum insecticides targeted to adult populations. An improved understanding of D. suzukii biology and ecology are necessary to create sustainable management options. Knowledge of how D. suzukii interacts with resources will enable targeted management actions in the future. In this present study, we monitored larval infestation throughout the blackberry canopy and found that infestation was highest in the inner portion of the canopy and lower in more exposed locations. We also documented higher humidity within the cane canopy relative to the edge of the field. A difference in abiotic conditions may create within‐crop microhabitats that D. suzukii is able to exploit. Future research will explore how to take advantage of these microhabitats in pest management programs.     

Hidden diversity and cryptic speciation refute cosmopolitan distribution in Caprella penantis (Crustacea: Amphipoda: Caprellidae)

Caprella penantis is considered a cosmopolitan species and one of the most challenging caprellids in taxonomic terms because of its remarkable intraspecific morphological variation. This study examined DNA sequences from mitochondrial (COI) and nuclear (18S) markers together with morphological data from 25 localities of C. penantis, and closely related species Caprella dilatata and Caprella andreae, all traditionally considered part of the old ‘acutifrons’ complex. The large genetic divergence and reciprocally allopatric distributions point to the existence of a species complex of at least four species, of which one is reported as a cryptic species. This study provides the first evidence of cryptic speciation in the family Caprellidae, and questions the validity of some traditional morphological characters used to delimit species in the genus Caprella. Our results are consistent with the idea that main factors were probably isolation by distance and ecological traits, promoting diversification in C. penantis. The strong genetic structure reported for this species in the Iberian Peninsula and Moroccan coasts also suggests restriction to dispersal as well as the presence of refugial areas. These results highlight the utility of the COI and 18S genes in combination with morphological characters for shedding light on systematic questions in caprellids, and patterns of genetic connectivity.     

Species delineation in the Capitella species complex (Annelida: Capitellidae): geographic and genetic variation in the northern Gulf of Mexico

Capitella capitata was traditionally used as a biological indicator species due to its ubiquitous distribution and high densities in disturbed and polluted marine and estuarine sediments. Based on allozyme and developmental studies, it is now clear that C. capitata is a species complex consisting of multiple distinct lineages worldwide, including the recently described C. teleta, a model species for spiralian development. The coast of the northern Gulf of Mexico, with its numerous bays and estuaries and frequently occurring natural and anthropogenic disturbances, provides an appropriate region for such studies. We sequenced a fragment of the mitochondrial cytochrome c oxidase subunit I gene for individuals of C. cf. capitata and C. cf. aciculata (distinguished by acicular spines on the first two chaetigers) collected from Texas and Florida coasts and analyzed them in conjunction with data available in GenBank. Our results indicate the presence of a Gulf of Mexico clade that is distinct from populations in Canada and the Indo‐Pacific. Populations in the northern Gulf of Mexico are structured geographically, with support for Texas and Florida clades, and there do not seem to be clear boundaries between C. cf. capitata and C. cf. aciculata. This is corroborated by the fact that multiple specimens were morphologically intermediate between the two species. In future studies, we aim to clarify whether the intermediate morphologies represent ontogenetic stages, neutral morphological variation, phenotypic plasticity, or sexual dimorphism in a single species or whether several lineages with incomplete reproductive barriers are present.     

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.     

Recent speciation and hybridization in Icelandic deep-sea isopods: An integrative approach using genomics and proteomics

The crustacean marine isopod species Haploniscus bicuspis (Sars, 1877) shows circum-Icelandic distribution in a wide range of environmental conditions and along well-known geographic barriers, such as the Greenland-Iceland-Faroe (GIF) Ridge. We wanted to explore population genetics, phylogeography and cryptic speciation as well as investigate whether previously described, but unaccepted subspecies have any merit. Using the same set of specimens, we combined mitochondrial COI sequences, thousands of nuclear loci (ddRAD), and proteomic profiles, plus selected morphological characters using confocal laser scanning microscopy (CLSM). Five divergent genetic lineages were identified by COI and ddRAD, two south and three north of the GIF Ridge. Assignment of populations to the three northern lineages varied and detailed analyses revealed hybridization and gene flow between them, suggesting a single northern species with a complex phylogeographic history. No apparent hybridization was observed among lineages south of the GIF Ridge, inferring the existence of two more species. Differences in proteomic profiles between the three putative species were minimal, implying an ongoing or recent speciation process. Population differentiation was high, even among closely associated populations, and higher in mitochondrial COI than nuclear ddRAD loci. Gene flow is apparently male-biased, leading to hybrid zones and instances of complete exchange of the local nuclear genome through immigrating males. This study did not confirm the existence of subspecies defined by male characters, which probably instead refer to different male developmental stages.     

Deceptive single‐locus taxonomy and phylogeography: Wolbachia‐associated divergence in mitochondrial DNA is not reflected in morphology and nuclear markers in a butterfly species

The satyrine butterfly Coenonympha tullia (Nymphalidae: Satyrinae) displays a deep split between two mitochondrial clades, one restricted to northern Alberta, Canada, and the other found throughout Alberta and across North America. We confirm this deep divide and test hypotheses explaining its phylogeographic structure. Neither genitalia morphology nor nuclear gene sequence supports cryptic species as an explanation, instead indicating differences between nuclear and mitochondrial genome histories. Sex‐biased dispersal is unlikely to cause such mito‐nuclear differences; however, selective sweeps by reproductive parasites could have led to this conflict. About half of the tested samples were infected by Wolbachia bacteria. Using multilocus strain typing for three Wolbachia genes, we show that the divergent mitochondrial clades are associated with two different Wolbachia strains, supporting the hypothesis that the mito‐nuclear differences resulted from selection on the mitochondrial genome due to selective sweeps by Wolbachia strains.     

Collembola,the biological species concept and the underestimation of global species richness

Despite its ancient origin, global distribution and abundance in nearly all habitats, the class Collembola is comprised of only 8000 described species and is estimated to number no more than 50 000. Many morphologically defined species have broad geographical ranges that span continents, and recent molecular work has revealed high genetic diversity within species. However, the evolutionary significance of this genetic diversity is unknown. In this study, we sample five morphological species of the globally distributed genus Lepidocyrtus from 14 Panamanian sampling sites to characterize genetic diversity and test morphospecies against the biological species concept. Mitochondrial and nuclear DNA sequence data were analysed and a total of 58 molecular lineages revealed. Deep lineage diversification was recovered, with 30 molecular lineages estimated to have established more than 10 million years ago, and the origin almost all contemporary lineages preceding the onset of the Pleistocene (~2 Mya). Thirty‐four lineages were sampled in sympatry revealing unambiguous cosegregation of mitochondrial and nuclear DNA sequence variation, consistent with biological species. Species richness within the class Collembola and the geographical structure of this diversity are substantially misrepresented components of terrestrial animal biodiversity. We speculate that global species richness of Collembola could be at least an order of magnitude greater than a previous estimate of 50 000 species.     

Cryptic speciation in the Anadenobolus excisus millipede species complex on the Island of Jamaica

Anadenobolus excisus is a large species of millipede endemic to the Caribbean Island of Jamaica. Initial detailed morphological studies showed little or no discrete variation across this species' distribution in somatic or, in particular. genitalic morphology. However, a molecular survey based on approximately 1000 base pairs of the mitochondrial (mtDNA) 16S rRNA gene that examines 242 individuals sampled from 54 localities reveals three highly divergent mtDNA lineages. A lack of discrete morphological differentiation suggests that genetic and morphological divergence may be decoupled, a pattern inconsistent with a number of evolutionary models. In contrast to minimal morphological divergence, size variation among mtDNA lineages suggests that character displacement has occurred and that these lineages are cohesive in sympatry. We conclude that A. excisus is actually a complex of three cryptic species and that morphological approaches to delineating millipede species may sometimes underestimate evolutionary diversity.     

Ulva (Chlorophyta,Ulvales) Biodiversity in the North Adriatic Sea (Mediterranean,Italy): Cryptic Species and New Introductions

Ulva Linnaeus (Ulvophyceae, Ulvales) is a genus of green algae widespread in different aquatic environments. Members of this genus show a very simple morphology and a certain degree of phenotypic plasticity, heavily influenced by environmental conditions, making difficult the delineation of species by morphological features alone. Most studies dealing with Ulva biodiversity in Mediterranean waters have been based only on morphological characters and a modern taxonomic revision of this genus in the Mediterranean is not available. We report here the results of an investigation on the diversity of Ulva in the North Adriatic Sea based on molecular analyses. Collections from three areas, two of which subject to intense shipping traffic, were examined, as well as historical collections of Ulva stored in the Herbarium Patavinum of the University of Padova, Italy. Molecular analyses based on partial sequences of the rbcL and tufA genes revealed the presence of six different species, often with overlapping morphologies: U. californica Wille, U. flexuosa Wulfen, U. rigida C. Agardh, U. compressa Linnaeus, U. pertusa Kjellman, and one probable new taxon. U. californica is a new record for the Mediterranean and U. pertusa is a new record for the Adriatic. Partial sequences obtained from historical collections show that most of the old specimens are referable to U. rigida. No specimens referable to the two alien species were found among the old herbarium specimens. The results indicate that the number of introduced seaweed species and their impact on Mediterranean communities have been underestimated, due to the difficulties in species identification of morphologically simple taxa as Ulva.     

Large Scale Geographical Mapping of Essential Oil Volatiles in Heracleum (Apiaceae): Identification of Novel Compounds and Unraveling Cryptic Variation

Conspecific populations of plants in their native range are expected to show considerable variation due to long‐term ecological and evolutionary factors. We investigated the levels of secondary metabolites in Heracleum including H. persicum a valuable medicinal plant to depict the magnitude of cryptic variation and the potential significance of novel chemical traits. The essential oil volatiles from fruits of 34 populations from different species of Heracleum in Iranian distribution range and a native of H. sphondylium and an invasive population of H. persicum from Norway were analyzed with GC/MS. Out of 48 compounds identified, a contrasting pattern in the level of two major compounds, octyl acetate and hexyl butyrate was found among all studied species. Interestingly, a significant geographic pattern was observed; the hexyl butyrate/octyl acetate ratio was high (range 1.8 – 3.2) in the northwestern Iranian populations of H. persicum compared to that in northern and central populations (range 0.3 – 0.9). Four populations from Zagros mountains also exhibited a unique composition. Anethole was found in two populations of H. persicum from central Zagros, which has not been previously reported for essential oil of fruits of Heracleum so far. The results suggest high efficiency of large scale sampling from distribution range of species in identifying novel compounds. The unique pattern of geographic structuring also provides novel information to unravel cryptic variation in Heracleum.     

How chromosomal rearrangements shape adaptation and speciation: Case studies in Drosophila pseudoobscura and its sibling species Drosophila persimilis

The gene arrangements of Drosophila have played a prominent role in the history of evolutionary biology from the original quantification of genetic diversity to current studies of the mechanisms for the origin and establishment of new inversion mutations within populations and their subsequent fixation between species supporting reproductive barriers. This review examines the genetic causes and consequences of inversions as recombination suppressors and the role that recombination suppression plays in establishing inversions in populations as they are involved in adaptation within heterogeneous environments. This often results in the formation of clines of gene arrangement frequencies among populations. Recombination suppression leads to the differentiation of the gene arrangements which may accelerate the accumulation of fixed genetic differences among populations. If these fixed mutations cause incompatibilities, then inversions pose important reproductive barriers between species. This review uses the evolution of inversions in Drosophila pseudoobscura and D. persimilis as a case study for how inversions originate, establish and contribute to the evolution of reproductive isolation.