Speciation Process Research Articles

Molecular and Morphometric Update on Italian Salicornia (Chenopodiaceae), with a Focus on the Species S. procumbens s. l.

Salicornia is a highly taxonomically problematic genus due to the reduced morphological observable characters. Ten Eurasian species are currently recognized: S. alpini, S. europaea, S. fruticosa, S. hispanica, S. lagascae, S. perennans, S. perennis, S. persica, S. procumbens, and S. pruinosa. In addition, eleven subspecies are accepted, mainly based on their distribution areas. Along the Venetian coasts and in Sardinia, in the past, an endemic species called S. veneta was recognized, but this name was later synonymized with S. procumbens subsp. procumbens. The aim of the present research is investigating different Italian Salicornia populations by a molecular point of view, using the nuclear ribosomal external transcribed spacer ETS and the plastid psbA-trnH intergenic spacer. A particular focus is on the comparison between Venetian (including those occurring in locus classicus of S. veneta) and Sardinian S. procumbens and other Italian populations of this species. The molecular analyses based on the plastid marker highlight that the Italian S. procumbens populations form two well distinct groups. In particular, some of the Venetian (Locus classicus of S. veneta) and all the Sardinian specimens are genetically distinct (=plastid haplotype 1) from the other investigated populations (=plastid haplotype 2). This indicates that the psbA-trnH haplotype 1 glassworts represent a distinct entity, which we suppose to coincide with the former S. veneta. Therefore, we suggest to recognize this taxonomic entity at the subspecies rank, as S. procumbens subsp. veneta comb. and stat. nov. However, contrary to the results found with the plastid psbA-trnH intergenic spacer, the ETS locus does not show a separation into two distinct clades for S. procumbens, probably due to a different evolution of the two loci. Nevertheless, in the ETS phylogenetic reconstruction, the Sardinian specimens (=ribotypes 2 and 3) are placed, together with a Moroccan sample, in a subclade separated from all the other S. procumbens. These results suggest that the Sardinian populations can represent a subspecies/incipient speciation process, probably due to geographic isolation. In the light of this, morphometric analyses (k-means, MANOVA, PCA, DA, and Box-Plot) have been carried out on the Sardinian and Venetian populations to verify if this distinction is detectable also by a morphological point of view. The morphometric analyses highlight the existence of two groups, concerning both the nuclear and plastid trees. Six characters were found to be diagnostic.

High resolution modeling of aluminium transport in a fjord estuary with focus on mean circulation and irregular flow events

Environmental impact assessments of trace metals and radionuclides in estuarine waters will benefit from numerical transport models that can provide detailed and accurate predictions of concentrations of harmful physico-chemical forms of contaminants at adequate spatial and temporal resolution. Aiming to study the potential of aluminium (Al) exposure to biota, a transport model (OpenDrift) including dynamic speciation and transformation processes was improved and applied, using three-dimensional hydrodynamic flow fields from a numerical ocean model (ROMS) at high horizontal resolution (32 m). Al transport and concentration was computed along the Sandnesfjorden Fjord, south-eastern Norway, from river outlet to open coastal waters. Validation of the circulation model with 29 hydrographic profiles from Sandnesfjorden showed substantial improvements compared to previous studies due to optimized model configuration (salinity overestimation decreased from >7 psu to <4 psu). Modeled Al data compared well with observed surface Al concentration from 12 locations and the along-fjord decreasing trend in Al-concentration was well reproduced (error ratios were <2 in Sandnesfjorden). Except in the channel area, both salinity and Al concentration estimates lie well within the expected variability. However, the transport modeling gave a more detailed site-specific picture of the Al concentration, suggesting more scattered and variable fields than indicated by observational data (variations of a factor 3–4 over short spatiotemporal scales). Reversed flow events (surface flow into the fjord) caused considerable mixing and redistribution of water masses, affecting both horizontal mixing of river discharges with coastal water as well as vertically as surface water mixed with deeper water masses. These blocking events strongly changed properties and distribution of the water masses giving rise to local and short-term high Al-exposure episodes (variations of a factor of 10 over a 12 h period) in the fjord that may pose risks to biota and therefore should be taken into account in impact and risk assessments.

Taxonomic Revision of Tribe Aleurocanthini Takahashi 1954 stat. rev. Using Consortium Gene Analysis (Mito-Nuclear-Primary Endosymbiont) with the First Evidence for Mitochondrial Recombination in Whitefly (Hemiptera: Aleyrodidae)

The discovery of the Aleurocanthus cf. Aleurocanthus spiniferus (Tea spiny whitefly spiniferus morphotype; previously found in Tokyo) for the first time in Shizuoka Prefecture raised the possibility that this species had invaded Honshu Island, Japan. Unlike the allied species, Aleurocanthus spiniferus and A. camelliae, which have been intercepted from China to Japan, the origin of the current species remains unclear. Despite the status of this species as a minor pest on the ornamental plant, Eurya japonica, the cryptic diversity among the black spiny whitefly is fascinating to be elucidated, specifically how the primary endosymbiont of whiteflies, Portiera aleyrodidarum, coevolved and contributed to the classification of whiteflies. The current study examines the taxonomic status of five species of whiteflies, i.e., A. spiniferus (Quaintance), Aleurocanthus aff. A. camelliae, Aleurocanthus cf. A. spiniferus, A. camelliae Kanmiya and Kasai, Aleurotrachelus camelliae Kuwana, and A. ishigakiensis Takahashi. Using consortium molecular typing targeting mitochondrial DNA (COI and 16S of mitoribosome), the nuclear gene (ITS1), and the ribosomal gene of Portiera, the phylogenetic clustering analysis has been conducted and revealed that the genus Aleurotrachelus sensu lato was clustered together with Aleurocanthini Takahashi, 1954 stat. rev. and reinstated Crenidorsum ishigakiensis comb. nov. due to crescent-shaped scallops being clearly defined. The current study also unveiled several putative species in the A. spiniferus species complex, molecularly. In addition, the recombination event was not detected in Portiera but has been detected in the mtCOI genes of the A. spiniferus cryptic species and the A. woglumi sequences deposited in the NCBI database. The mitochondrial recombination gives an insight into the speciation process among this species complex.

Back to the Sea: The Long and Winding Road of the Seagrass Species &lt;i&gt;Posidonia oceanica&lt;/i&gt;

&lt;em&gt;Posidonia oceanica&lt;/em&gt; (Linnaeus) Delile is one of the most important endemic species of the Mediterranean Sea. The long phylogenetic pathway of this species deserves special attention because its evolutionary history allows us to think back over the debated issue of life evolution on Earth. Hence, this marine plant, by terrestrial origin, was born from an ancient ancestor named &lt;em&gt;Posidonia cretacea&lt;/em&gt; Hosius &amp; Von der Marck. This seagrass species, currently extinct, lived in the coastal waters of the Tethys Ocean about 100 million years ago, during the geological times of the late Cretaceous. In the following Cenozoic Era, during the Miocene period, in the time lag from sixty to forty million years ago,a long process of allopatric speciation by separate areas led to eight species of Posidonia genus developed in the northern and southern hemispheres. &lt;em&gt;Posidonia oceanica&lt;/em&gt; is established in the boreal marine regions, so representing, within the long and winding road of life evolution on Earth, a typical example of paleo-Mediterranean relict. Nowadays, &lt;em&gt;Posidonia oceanica &lt;/em&gt;meadows perform important ecological functions in the Mediterranean Sea, not only for biodiversity conservation but also to mitigate climate change. In this way, meadows play a leading role in carbon sequestration, reducing the concentration of carbon dioxide in coastal waters. Lastly, the meadows of &lt;em&gt;Posidonia oceanica&lt;/em&gt; promote an effective removal process of plastic debris from Mediterranean sea waters.

Speciation without gene-flow in hybridizing deer.

Under the ecological speciation model, divergent selection acts on ecological differences between populations, gradually creating barriers to gene flow and ultimately leading to reproductive isolation. Hybridisation is part of this continuum and can both promote and inhibit the speciation process. Here, we used white-tailed (Odocoileus virginianus) and mule deer (O. hemionus) to investigate patterns of speciation in hybridizing sister species. We quantified genome-wide historical introgression and performed genome scans to look for signatures of four different selection scenarios. Despite ample modern evidence of hybridisation, we found negligible patterns of ancestral introgression and no signatures of divergence with gene flow, rather localized patterns of allopatric and balancing selection were detected across the genome. Genes under balancing selection were related to immunity, MHC and sensory perception of smell, the latter of which is consistent with deer biology. The deficiency of historical gene-flow suggests that white-tailed and mule deer were spatially separated during the glaciation cycles of the Pleistocene and genome wide differentiation accrued via genetic drift. Dobzhansky-Muller incompatibilities and selection against hybrids are hypothesised to be acting, and diversity correlations to recombination rates suggests these sister species are far along the speciation continuum.

The Spodoptera frugiperda Host Strains: What They Are and Why They Matter for Understanding and Controlling This Global Agricultural Pest.

Fall armyworm (Spodoptera frugiperda [J.E. Smith]) is a moth native to the Western Hemisphere where it is a major pest of corn (maize) and multiple other crops. It is now a global threat with its recent discovery and dissemination in the Eastern Hemisphere. Its broad host range is in part due to two subpopulations denoted as 'host strains' that differ in host plant specificity. Therefore, identifying the strains present in a location is critical to assessing what crops are at risk of infestation. However, much remains uncertain about how the strains differ and even on the fundamental issue of how they are identified. Complicating factors include the host strains are morphologically indistinguishable, the defining behavior of the strains (host plant specificity) is variable, and the existence of significant differences between geographical populations and laboratory colonies that are independent of strain identity. These factors contribute to substantial disagreements in the literature on presumptive strain differences. This paper presents a summary of strain characteristics and suggests the criteria that should be met before concluding a trait is 'strain-specific'. The intent is to facilitate a common understanding of what the strains represent and to develop a more consistent experimental framework for studies on strain phenotypes. Evidence is summarized that supports a primary role for Z-linked genes in strain identity, which has potential implications for genetic approaches to define the strains, and we discuss the possibility that the strains arose from allopatric (rather than sympatric) speciation processes.

Testing drivers of acoustic divergence in cicadas (Cicadidae: Tettigettalna).

Divergence in acoustic signals may have a crucial role in the speciation process of animals that rely on sound for intra-specific recognition and mate attraction. The acoustic adaptation hypothesis (AAH) postulates that signals should diverge according to the physical properties of the signalling environment. To be efficient, signals should maximize transmission and decrease degradation. To test which drivers of divergence exert the most influence in a speciose group of insects, we used a phylogenetic approach to the evolution of acoustic signals in the cicada genus Tettigettalna, investigating the relationship between acoustic traits (and their mode of evolution) and body size, climate and micro-/macro-habitat usage. Different traits showed different evolutionary paths. While acoustic divergence was generally independent of phylogenetic history, some temporal variables' divergence was associated with genetic drift. We found support for ecological adaptation at the temporal but not the spectral level. Temporal patterns are correlated with micro- and macro-habitat usage and temperature stochasticity in ways that run against the AAH predictions, degrading signals more easily. These traits are likely to have evolved as an anti-predator strategy in conspicuous environments and low-density populations. Our results support a role of ecological selection, not excluding a likely role of sexual selection in the evolution of Tettigettalna calling songs, which should be further investigated in an integrative approach.

Geographic variability of hybridization between red-breasted and red-naped sapsuckers.

Hybrid zones reveal the strength of reproductive isolation between populations undergoing speciation and are a key tool in evolutionary biology research. Multiple replicate transects across the same hybrid zone offer insight into the dynamics of hybridization in different environments, clarifying the role of extrinsic forces on the speciation process. Red-breasted and red-naped sapsuckers (Sphyrapicus ruber and Sphyrapicus nuchalis) have a long zone of contact over approximately 1,600 km from central British Columbia, Canada to central California, USA. We used Genotyping-by-Sequencing data from three independent sapsucker hybrid zone transects to compare hybridization dynamics between these species under variable geoclimatic conditions. We generated geographic clines of the genomic data to compare hybrid zone widths and used random forests models and linear regression to assess the relationship between climate and sapsucker ancestry along each transect. Our results show variation in the directionality of backcrossing, often indicative of moving hybrid zones. We note variable cline widths among transects, indicating differences in selection maintaining hybrid zone dynamics. Furthermore, random forests models identified different variables in close association with sapsucker ancestry across each transect. These results indicate a lack of repeatability across replicate transects and a strong influence of the local environment on hybrid zone dynamics.

A multivariate view of the speciation continuum.

The concept of a "speciation continuum" has gained popularity in recent decades. It emphasizes speciation as a continuous process that may be studied by comparing contemporary population pairs that show differing levels of divergence. In their recent perspective article in Evolution, Stankowski and Ravinet provided a valuable service by formally defining the speciation continuum as a continuum of reproductive isolation, based on opinions gathered from a survey of speciation researchers. While we agree that the speciation continuum has been a useful concept to advance the understanding of the speciation process, some intrinsic limitations exist. Here, we advocate for a multivariate extension, the speciation hypercube, first proposed by Dieckmann et al. in 2004, but rarely used since. We extend the idea of the speciation cube and suggest it has strong conceptual and practical advantages over a one-dimensional model. We illustrate how the speciation hypercube can be used to visualize and compare different speciation trajectories, providing new insights into the processes and mechanisms of speciation. A key strength of the speciation hypercube is that it provides a unifying framework for speciation research, as it allows questions from apparently disparate subfields to be addressed in a single conceptual model.

Origin and fate of dissolved inorganic carbon in a karst groundwater fed peatland using δ13CDIC

Continental hydrosystems and in particular peatlands play an important role in the carbon cycle of the Critical Zone (CZ). Peatlands are important sinks for organic carbon and have therefore been extensively studied. However, peatlands are not only important for the fate of organic carbon, but they also affect the cycle of Dissolved Inorganic Carbon (DIC) of the peatland and the surrounding watershed. The fate of DIC is particularly complex in peatlands in limestone-dominated regions, because bicarbonate concentrations in surface and groundwater are high and the interaction between peatlands and surrounding hydrosystems are facilitated by the presence of highly permeable karst aquifers. In the present paper we study the origin and the fractionation of DIC in a peatland located on top of a karst aquifer. The study is based on hydrochemical and isotopic (δ13CDIC) data from samples recovered during 2 campaigns (low flow, high flow) at various depths within the Forbonnet peatland (Jura Mountains, eastern France), at the peatland outlet and at adjacent karst springs representing the underlying aquifer. In order to evaluate secondary fractionation processes, the measured δ13CDIC compositions were compared to modeled values considering the origin of DIC and potentially associated fractionation and speciation processes. The main results are: (1) DIC is lost at the bog surface by CO2 outgassing. (2) The δ13CDIC compositions of deep catotelm pore waters from the bog were much heavier than the modeled values. We relate this discrepancy to methanogenesis and show that this process is favored by reduced conditions at pH ∼ 6 and a HCO3− content of ∼1 mmol/L, most probably due to punctual groundwater inflows at the base of the bog. Finally, contrasted δ13CDIC compositions between the bog and the fen of the peatland reveal an additional ecohydrological control on DIC speciation.

Phylogenetic Analyses of Some Key Genes Provide Information on Pollinator Attraction in Solanaceae.

Floral syndromes are known by the conserved morphological traits in flowers associated with pollinator attraction, such as corolla shape and color, aroma emission and composition, and rewards, especially the nectar volume and sugar concentration. Here, we employed a phylogenetic approach to investigate sequences of genes enrolled in the biosynthetic pathways responsible for some phenotypes that are attractive to pollinators in Solanaceae genomes. We included genes involved in visible color, UV-light response, scent emission, and nectar production to test the hypothesis that these essential genes have evolved by convergence under pollinator selection. Our results refuted this hypothesis as all four studied genes recovered the species' phylogenetic relationships, even though some sites were positively selected. We found differences in protein motifs among genera in Solanaceae that were not necessarily associated with the same floral syndrome. Although it has had a crucial role in plant diversification, the plant-pollinator interaction is complex and still needs further investigation, with genes evolving not only under the influence of pollinators, but by the sum of several evolutionary forces along the speciation process in Solanaceae.

Strong species structure but weak geographical structure in demersal Lake Victoria cichlids.

Studying phenotypic and genetic differentiation between very young species can be very informative with regard to learning about processes of speciation. Identifying and characterizing genetic species structure and distinguishing it from spatial genetic structure within a species is a prerequisite for this and is often not given sufficient attention. Young radiations of cichlid fish are classical speciation study systems. However, it is only during the past decade that population genomics based on next-generation sequencing has begun to provide the power to resolve species and distinguish speciation from spatial population structure for the youngest of these radiations. The Lake Victoria haplochromine cichlids constitute the youngest large cichlid fish radiation, probably <20,000 years old. Earlier work showed that communities of rocky reef cichlids are composed of many reciprocally monophyletic species despite their very recent origins. Here, we build on this work by studying assemblages of offshore demersal cichlids, adding analyses of within-species spatial structure to the sympatric species structure. We sampled seven multispecies communities along a 6-km-long transect from one side of the Mwanza Gulf to the other side. We investigated whether phenotypically diagnosed putative species are reciprocally monophyletic and whether such monophyly is stable across species geographic ranges. We show that all species are genetically strongly differentiated in sympatry, that they are reciprocally monophyletic, and that monophyly is stable across distribution ranges. We found significant differentiation between geographically distinct populations in two species, but no or weak isolation by distance. We further found subtle but significant morphological differences between all species and a linear relationship between genomic and morphological distance which suggests that differences in morphology begin to accumulate after speciation has already affected genome-wide restrictions of gene flow.

In memory of Judith Masters and Fabien Génin.

In memory of Judith Masters and Fabien Génin.

Distribution and phylogeny of mercury methylation, demethylation, and reduction genes in the Seto Inland Sea of Japan

Mercury (Hg) adversely affects human and environmental health. To evaluate the mercury (Hg) speciation (methylation, demethylation, and reduction) of microorganisms in coastal seawater, we analyzed the microbial functional gene sets involved in Hg methylation (hgcA and hgcB), demethylation (merB), and reduction (merA) using a metagenomic approach in the eastern and western parts (the Kii and Bungo channels, respectively) of the Seto Inland Sea (SIS) of Japan. We determined the concentration of dissolved total mercury (dTHg) and methylated mercury (dMeHg) in seawater. The metagenomic analysis detected hgcAB, merA, and merB in both channels, whereas the phylogenies of these genes differed between them. A correlation between Hg concentration (both dTHg and dMeHg) and the relative abundance of each gene was not observed. Our data suggests that microbial Hg methylation and demethylation could occur in the SIS and there could be a distinct microbial Hg speciation process between the Kii and Bungo channels.

Hydrogeochemistry of thallium and other potentially toxic elements in neutral mine drainage at the decommissioned Pb-Zn Raibl mine (Eastern Alps, Italy)

Decommissioned mines represent a worldwide concern due to the potential long-term effects related to the dispersion of potentially toxic elements (PTEs) in the environment. In this study, 176 water samples were collected in the period 2018–2021 at the carbonate-hosted Pb-Zn Raibl mine which is affected by neutral mine drainage (NMD). The post-flotation tailings are the main source of PTEs (Zn, Pb and especially Tl) in the river drainage system. Compared to other dissolved PTEs (Zn, Pb, Cd), Tl was found to be more mobile, reaching concentrations up to 120 μg L−1 in waters flowing in the tailings impoundments. Modelling results suggest that Tl is mainly present in the Tl(I) ionic and mobile form thus suggesting that relevant natural attenuation processes for this element are not expected in the investigated area. In contrast, Zn and Pb attenuation pathways appeared to be governed by pH-dependent speciation and sorption processes, whereas elevated Zn concentrations were likely also limited by hydrozincite precipitation. Metalloids such as As and Sb were almost entirely released into the slightly alkaline waters of the mine drainage system, which are generally characterised by longer residence time or standing waters whereas As and Sb concentrations were negligible in the tailings-seepage waters. However, intense rainy events may increase PTE concentrations of one order of magnitude, especially in the tailings impoundment groundwater as a result of a rise in the water table, and PTE total dissolved loads of three orders of magnitude, in the main stream during high flow events, thus representing the most critical factor in regulating the remobilisation and downward dispersion of PTEs in the river drainage system.

Species delimitation using genomic data to resolve taxonomic uncertainties in a speciation continuum of pelagic seabirds

Speciation is a continuous and complex process shaped by the interaction of numerous evolutionary forces. Despite the continuous nature of the speciation process, the implementation of conservation policies relies on the delimitation of species and evolutionary significant units (ESUs). Puffinus shearwaters are globally distributed and threatened pelagic seabirds. Due to remarkable morphological status the group has been under intense taxonomic debate for the past three decades. Here, we use double digest Restriction-Site Associated DNA sequencing (ddRAD-Seq) to genotype species and subspecies of North Atlantic and Mediterranean Puffinus shearwaters across their entire geographical range. We assess the phylogenetic relationships and population structure among and within the group, evaluate species boundaries, and characterise the genomic landscape of divergence. We find that current taxonomies are not supported by genomic data and propose a more accurate taxonomy by integrating genomic information with other sources of evidence. Our results show that several taxon pairs are at different stages of a speciation continuum. Our study emphasises the potential of genomic data to resolve taxonomic uncertainties, which can help to focus management actions on relevant taxa, even if they do not necessarily coincide with the taxonomic rank of species.

Allopatric origin, secondary contact and subsequent isolation of sympatric rockfishes (Sebastidae: Sebastes) in the north-western Pacific

Abstract Understanding how speciation occurs is central to biology. Gene flow between diverging taxa is correlated with geography and other aspects of speciation; therefore, the examination of gene flow during divergence is a potent approach to understanding the nature of speciation. Here, we inferred the speciation process of the sympatric rockfishes Sebastes steindachneri and Sebastes wakiyai in the north-western Pacific and its marginal seas based on genome-wide single nucleotide polymorphism and mitochondrial DNA data. Model-based demographic inference showed that gene flow between the two species was absent in the initial and late stages of divergence and present only in the middle stage. Population expansion occurred before or during the period of gene flow. The estimated timings of the initial divergence and population expansion fell within the Pleistocene, during which the seas currently inhabited by the two species were repeatedly isolated and reconnected. Contemporary isolation was supported by the absence of hybrids and the shared mitochondrial DNA haplotypes. Our results suggest that the two species initially diverged in allopatry, followed by secondary contact and introgression and by the completion of reproductive isolation. Given that complete isolation following secondary contact has rarely been tested or documented in marine organisms, we highlight the importance of careful consideration of alternative divergence scenarios to be tested, which should take into account the geological and environmental settings.

Contrasting continental patterns of adaptive population divergence in the holarctic ectomycorrhizal fungus Boletus edulis.

In the hyperdiverse fungi, the process of speciation is virtually unknown, including for the > 20 000 species of ectomycorrhizal mutualists. To understand this process, we investigated patterns of genome-wide differentiation in the ectomycorrhizal porcini mushroom, Boletus edulis, a globally distributed species complex with broad ecological amplitude. By whole-genome sequencing 160 individuals from across the Northern Hemisphere, we genotyped 792 923 single nucleotide polymorphismsto characterize patterns of genome-wide differentiation and to identify the adaptive processes shaping global population structure. We show that B. edulis exhibits contrasting patterns of genomic divergence between continents, with multiple lineages present across North America, while a single lineage dominates Europe. These geographical lineages are inferred to have diverged 1.62-2.66 million years ago, during a period of climatic upheaval and the onset of glaciation in the Pliocene-Pleistocene boundary. High levels of genomic differentiation were observed among lineages despite evidence of substantial and ongoing introgression. Genome scans, demographic inference, and ecological niche models suggest that genomic differentiation is maintained by environmental adaptation, not physical isolation. Our study uncovers striking patterns of genome-wide differentiation on a global scale and emphasizes the importance of local adaptation and ecologically mediated divergence, rather than prezygotic barriers such as allopatry or genomic incompatibility, in fungal population differentiation.

Environmental features drive lineage diversification in the Aricidea assimilis species complex (Annelida, Paraonidae) in the Mediterranean Sea

Individuals identified as Aricidea assimilis Tebble, 1959 were collected from ten localities across the Mediterranean Sea from 0.5 to 225 m depth in order to have a wide coverage of the species habitats and geographic range and to assess the effects of environmental factors and biogeographical barriers on molecular and morphological diversity. Two mitochondrial and one nuclear markers were used to reconstruct phylogenetic relationships and test the occurrence of cryptic species. We observed two highly divergent lineages, one including all individuals from shallow, sandy environments (<10 m depth) and the other with the individuals from deeper muddy bottoms (30–225 m depth). Less pronounced divergence was detected between morphologically distinct brackish-water individuals and the remaining shallow-water individuals. The divergence observed between deep-water and shallow-water lineages is consistent with the hypothesis of distinct species. The ambiguous results of species delimitation tests applied to the two shallow-water sub-lineages might instead suggest a process of incipient speciation, even if this hypothesis needs additional evidence. These results suggest that sediment represents the main factor driving genetic divergence and ultimately cryptic speciation in A. assimilis, while other depth-associated factors and geographical barriers do not seem to significantly contribute to the genetic architecture of this species, suggesting the occurrence of wide-range larval dispersal.

Reproductive isolation via divergent genital morphology due to cascade reinforcement in Ohomopterus ground beetles.

Secondary contact between incipient species and selection against maladaptive hybridization can drive reinforcement between populations in contact and result in reproductive character displacement (RCD). Resultant divergence in mating traits within a species may generate downstream reproductive isolation between populations with displaced and non-displaced traits, referred to as the cascade reinforcement hypothesis. We examined this hypothesis using three allopatric populations of the ground beetle Carabus maiyasanus with a genital lock-and-key system. This species shows RCD in male and female genital morphologies in populations in contact with the sister species C. iwawakianus. In a reciprocal mating experiment using three allopatric populations with differences in male and female genital sizes, insemination failure increased as the difference in genital size increased. Based on the reproductive isolation index, insemination failure was the major postmating-prezygotic isolation barrier, at least in one population pair with comparable total isolation to those of other species pairs. By contrast, there was only incomplete premating isolation among populations. These results suggest that RCD in genital morphologies drives incipient allopatric speciation, supporting the cascade reinforcement hypothesis. These findings provide insight into the roles of interspecific interactions and subsequent trait diversification in speciation processes.