Long‐term stasis and short‐term divergence in the phenotypes of microsnails on oceanic islands

Phenotypic divergence is often unrelated to genotypic divergence. An extreme example is rapid phenotypic differentiation despite genetic similarity. Another extreme is morphological stasis despite substantial genetic divergence. These opposite patterns have been viewed as reflecting opposite properties of the lineages. In this study, phenotypic radiation accompanied by both rapid divergence and long‐term conservatism is documented in the inferred molecular phylogeny of the micro land snails Cavernacmella (Assimineidae) on the Ogasawara Islands. The populations of Cavernacmella on the Sekimon limestone outcrop of Hahajima Island showed marked divergence in shell morphology. Within this area, one lineage diversified into types with elongated turret shells, conical shells and flat disc‐like shells without substantial genetic differentiation. Additionally, a co‐occurring species with these types developed a much larger shell size. Moreover, a lineage adapted to live inside caves in this area. In contrast, populations in the other areas exhibited no morphological differences despite high genetic divergence among populations. Accordingly, the phenotypic evolution of Cavernacmella in Ogasawara is characterized by a pattern of long‐term stasis and periodic bursts of change. This pattern suggests that even lineages with phenotypic conservatism could shift to an alternative state allowing rapid phenotypic divergence.     

MOLECULAR AND REPRODUCTIVE CHARACTERIZATION OF SIBLING SPECIES IN THE EUROPEAN EARWIG (FORFICULA AURICULARIA)

The European earwig (Forficula auricularia) was formerly thought to present a mosaic of populations differing in their reproductive biology. We show that it is comprised of two as yet unrecognized sibling species. The molecular divergence between the two species, for a 627-bp amplified fragment overlapping the COI and COII mitochondrial loci, is six times larger than intraspecific variation. A species with two clutches a year lives predominantly in lowland and oceanic European habitats. A species with one clutch a year—except in the Mediterranean area where it has two clutches—lives predominantly in highland and continental European habitats. They both invaded North America during the 20th century, respectively, from the west and the east coasts, with no apparent mixing of their populations. The two species can occur in sympatry in Europe and are reproductively isolated by nearly complete failure to produce F₁ hybrids.     

A new genus of large hydrothermal vent‐endemic gastropod (Neomphalina: Peltospiridae)

Recently discovered hydrothermal vent fields on the East Scotia Ridge (ESR, 56–60°S, 30°W), Southern Ocean, and the South West Indian Ridge (SWIR, 37°S 49°E), Indian Ocean, host two closely related new species of peltospirid gastropods. Morphological and molecular (mitochondrial cytochrome c oxidase subunit I, COI) characterization justify the erection of G igantopelta gen. nov. within the Peltospiroidae with two new species, G igantopelta chessoia sp. nov. from ESR and G igantopelta aegis sp. nov. from SWIR. They attain an extremely large size for the clade Neomphalina, reaching 45.7 mm in shell diameter. The oesophageal gland of both species is markedly enlarged. Gigantopelta aegis has a thick sulphide coating on both the shell and the operculum of unknown function. The analysis of a 579‐bp fragment of the COI gene resulted in 19–28% pairwise distance between Gigantopelta and six other genera in Peltospiridae, whereas the range amongst those six genera was 12–28%. The COI divergence between the two newly described species of Gigantopelta was 4.43%. Population genetics analyses using COI (370 bp) of 30 individuals of each species confirmed their genetic isolation and indicate recent rapid demographic expansion in both species. © 2015 The Linnean Society of London     

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.     

Genetic variation in the soil phthiracarid mite, Steganacarus (S.) magnus (Nicolet, 1855) (Acarida, Oribatida) (Notulae Oribatologicae XL)

Genetic divergence and variability at 14 enzyme loci were examined in and between Italian populations of two edaphic Oribatid (Acarida, Oribatida) species, Steganacarus (Steganacarus) magnus (8 populations) and S. (S.) hirsutus (1 population). The seven populations belonging to S. (S.) magnus can be divided into two groups according to their phenotype, form anomala (A) (MON, ARG, AST, CDO) and form magna (M) (MAL, MAM, RIF) while another can be considered as hybrid between the two preceding groups (ZOC). Genetic identity (I) values between the S. (S.) magnus populations in spite of their morphological differences ranged from 0.977 to 1.000 showing the great genetic similarity of simple local populations while those of S. (S.) hirsutus indicated two distinct morphological species. The genetic distances between all the populations examined were very low despite the ecological differences and geographical distances between the collecting sites. Genetic variability estimates in all the populations of both species were very low when compared to those reported for most arthropods. Some explanatory considerations are suggested.     

Molecular and chemical characters to evaluate species status of two cuckoo bumblebees: Bombus barbutellus and Bombus maxillosus (Hymenoptera,Apidae, Bombini)

Many methods, based on morphological, molecular or chemical characters, have been used to address the question of species taxonomic status. Integrative taxonomy aims to define stronger supported taxonomic hypotheses by considering complementary datasets from different characters. By following an integrative approach, the present study includes molecular, chemical and morphological criteria to establish the taxonomic status of two rare and doubtful cuckoo bumblebee taxa: Bombus (Psithyrus) barbutellus and Bombus (Psithyrus) maxillosus. These two sympatric taxa are discriminated by few morphological criteria (mainly wing darkness and hair length). We used these morphological character diagnoses to establish an a priori status of our samples (23 specimens). We developed a combined molecular dataset from one nuclear gene, elongation factor 1α (EF‐1α), and one mitochondrial gene, cytochrome c oxidase subunit I (COI), spanning 1623 bp, and a chemical dataset of sexual marking pheromones (73 compounds). The molecular data were subjected to maximum‐likelihood and Bayesian phylogenetic inference under partitioned model and maximum parsimony. The chemical data were analysed by clustering and the two‐group k‐means method to test divergences between the two species. The resulting phylogenetic trees show no consistent divergence between the two taxa. Moreover, we found no divergence in the sexual marking pheromones in the clustering and two‐group k‐means analyses. These converging results support the conspecificity of both taxa. Nonetheless, our determinations using the traditional morphological criteria separated our samples into two taxa. We conclude that the morphological criteria seem to relate to intraspecific variations: B. maxillosus is regarded as a syn.n. of B. barbutellus.     

Phylogenetic analysis based on multiple gene sequences revealing cryptic biodiversity in Simulium multistriatum group (Diptera: Simuliidae) in Thailand

Phylogenetic relationships among four species of the Simulium multistriatum group (Diptera: Simuliidae) in Thailand were examined based on two mitochondrial genes (COI, COII) and one nuclear gene (18S/ITS1). Simulium takense was found to be genetically divergent (>20.3% for COI) from the other species, consistent with their distinctive morphology. Simulium chainarongi and S. chaliowae were monophyletic but were included in paraphyletic S. fenestratum. Simulium fenestratum was divided into three distinct lineages with high levels of genetic divergence. This suggests that S. fenestratum is a species complex. Neither morphological nor cytological examinations revealed evidence of sibling species. The clades derived from phylogenetic analyses were found to be correlated with the ecological conditions of larval habitat. Therefore, ecological adaptation may have played a role in black fly diversification and evolution. These results suggest the use of integrated, multidisciplinary approaches for fully understanding black fly biodiversity and systematics.     

ARE PARALLEL MORPHOLOGIES OF CAVE ORGANISMS THE RESULT OF SIMILAR SELECTION PRESSURES?

The amphipod Gammarus minus is present in both caves and springs, with cave populations showing elaborated (size and antennae) and reduced (eye) characters relative to spring populations. Earlier studies have shown that cave populations resulted from independent invasions of hydrologically isolated subterranean drainages and that there is genetic variation for both elaborated and reduced characters. In this study we tested the hypothesis that a similar pattern of selection on isolated cave populations is responsible for the parallel evolution of cave morphologies. We used variation in mating success and fecundity to test for the presence of directional selection on eye, antennal, and body size characters in a set of cave and spring populations during a series of seasonal cross-sectional samplings. We found significant directional selection for smaller eyes in caves and larger eyes in springs, which supports the hypothesis that selection is responsible for reduced eye size in cave populations. We also found selection for larger body and antennal size in cave populations, which is consistent with the hypothesis that parallel patterns of selection in caves are responsible for the elaboration of body and antennal size. However, we found selection for larger body and antennal size in spring populations that is not consistent with the observed divergence of spring and cave populations. We suggest that unmeasured components of viability selection could be more important in springs than in caves and may act against the selection for larger size found in spring populations.     

Incipient genetic divergence or cryptic speciation? Procamallanus (Nematoda) in freshwater fishes (Astyanax)

Hosts provide the main environmental traits parasites have to deal with, resulting in covariation between both associates at both micro- and macro-evolutionary scales; phylogenetic analyses of highly host-specific parasites have shown that parasite and host phylogeny might be highly congruent, and adaptation of a host species to new environments may lead to concordant changes of their parasites. Procamallanus (Spirocamallanus) neocaballeroi is a highly host-specific parasitic nematode of the Neotropical freshwater fish genus Astyanax in Mexico. One of the host species of the nematode is the emblematic Mexican tetra, A. mexicanus, which exhibits two contrasting phenotypes, a cave-dwelling morph (with troglomorphic features), and the surface-dwelling morph; other congeneric species inhabit rivers and lakes, and some of them occur in sympatry, displaying trophic specializations. Here, we explored the hypothesis that contrasting environments (surface rivers vs cave rivers), and host morphological divergence (sympatric ecomorphs in a lacustrine environment) might result in the divergence of their parasites, even though the hosts maintain a cohesive genetic structure as the same species. To test the hypothesis, several populations of Astyanax spp. were sampled to search for P. (S.) neocaballeroi. The nematode was found in 10 of the 52 sampled sites; two localities corresponded to cave populations. The phylogenetic analysis based on COI sequences yielded three major lineages for P. (S.) neocaballeroi. We found no concordance between the three lineages and the habitat where they occur in Astyanax mexicanus, even considering those living in drastic environmental conditions (caves), or between these lineages and lacustrine ecomorphs of Astyanax aeneus and A. caballeroi occurring in sympatry. Instead, genetic lineages of the nematode exhibit a clearer pattern of host species association and geographical distribution; our results showed that P. (S.) neocaballeroi is experiencing an incipient divergence although the morphological study of lineages shows no conspicuous differences.     

Molecular phylogeny and phylogeography of the Cuban cave-fishes of the genus Lucifuga: evidence for cryptic allopatric diversity

Underground environments are increasingly recognized as reservoirs of faunal diversity. Extreme environmental conditions and limited dispersal ability of underground organisms have been acknowledged as important factors promoting divergence between species and conspecific populations. However, in many instances, there is no correlation between genetic divergence and morphological differentiation. Lucifuga Poey is a stygobiotic fish genus that lives in Cuban and Bahamian caves. In Cuba, it offers a unique opportunity to study the influence of habitat fragmentation on the genetic divergence of stygobiotic species and populations. The genus includes four species and one morphological variant that have contrasting geographical distributions. In this study, we first performed a molecular phylogenetic analysis of the Lucifuga Cuban species using mitochondrial and nuclear markers. The mitochondrial phylogeny revealed three deeply divergent clades that were supported by nuclear and morphological characters. Within two of these main clades, we identified five lineages that are candidate cryptic species and a taxonomical synonymy between Lucifuga subterranea and Lucifuga teresinarum. Secondly, phylogeographic analysis using a fragment of the cytochrome b gene was performed for Lucifuga dentata, the most widely distributed species. We found strong geographical organization of the haplotype clades at different geographic scales that can be explained by episodes of dispersal and population expansion followed by population fragmentation and restricted gene flow. At a larger temporal scale, these processes could also explain the diversification and the distribution of the different species.     

Genetic structure among Charadrius plovers on the African mainland and islands of Madagascar and St Helena

Colonization of islands by long-distance dispersers has great impact on genetic diversification among populations and may spearhead speciation events. We investigated intra- and interspecific divergence in Charadrius plovers with populations on mainland Africa, Madagascar and St Helena. We analysed microsatellite loci and sequence data from four nuclear and two mitochondrial gene regions. Charadrius plovers are shorebirds with high dispersal and mobility. Our results confirmed genetic differentiation between Madagascar and mainland populations of three plover species (White-fronted Plover Charadrius marginatus, Kittlitz's Plover Charadrius pecuarius and, based on sequence data only, Three-banded Plover Charadrius tricollaris) but highlight substantial variation in levels of intraspecific divergence among the three species. Namely, the Kittlitz's Plover, a dispersive habitat generalist with a polygamous mating system, exhibited lower island–mainland differentiation (0.05% COI sequence divergence) compared with the two monogamous species, the White-fronted Plover (0.6% COI divergence) and Three-banded Plover (1.6% COI divergence). In addition, past colonization of the islands of St Helena and Madagascar by ancestors of today's Kittlitz's Plover has led to the evolution of two endemic island species, the Madagascar Plover Charadrius thoracicus and the more closely-related St Helena Plover Charadrius sanctaehelenae. We discuss the factors driving species differences in island–mainland divergence and highlight the importance of conserving genetically unique island populations and island habitats to safeguard future evolutionary potential.     

Molecular phylogeny and divergence time of the water penny genus Eubrianax (Coleoptera: Psephenidae) in Japan

The phylogenetic relationships among the Japanese members of the genus Eubrianax (Coleoptera: Psephenidae) were examined using the mitochondrial cytochrome oxidase subunit I (COI) gene and nuclear 28S rRNA gene sequences. Based on the molecular phylogeny as well as morphological features, the species status of Eubrianax brunneicornis Nakane, 1952 was proposed. The phylogenetic analyses recovered monophyly of the previously proposed pellucidus species group with four Japanese species, whereas a single Japanese species of the granicollis group was included in the lineage of the ramicornis group with five Japanese species. The divergence times of the species were estimated by dating the phylogenetic tree against the fossil record and a molecular clock based on the COI gene. The divergence of the Japanese species was inferred to have occurred during the Pliocene epoch.     

Integrative species delimitation reveals cryptic diversity in the southern Appalachian Antrodiaetus unicolor (Araneae: Antrodiaetidae) species complex

Although species delimitation can be highly contentious, the development of reliable methods to accurately ascertain species boundaries is an imperative step in cataloguing and describing Earth's quickly disappearing biodiversity. Spider species delimitation remains largely based on morphological characters; however, many mygalomorph spider populations are morphologically indistinguishable from each other yet have considerable molecular divergence. The focus of our study, the Antrodiaetus unicolor species complex containing two sympatric species, exhibits this pattern of relative morphological stasis with considerable genetic divergence across its distribution. A past study using two molecular markers, COI and 28S, revealed that A. unicolor is paraphyletic with respect to A. microunicolor. To better investigate species boundaries in the complex, we implement the cohesion species concept and use multiple lines of evidence for testing genetic exchangeability and ecological interchangeability. Our integrative approach includes extensively sampling homologous loci across the genome using a RADseq approach (3RAD), assessing population structure across their geographic range using multiple genetic clustering analyses that include structure , principal components analysis and a recently developed unsupervised machine learning approach (Variational Autoencoder). We evaluate ecological similarity by using large‐scale ecological data for niche‐based distribution modelling. Based on our analyses, we conclude that this complex has at least one additional species as well as confirm species delimitations based on previous less comprehensive approaches. Our study demonstrates the efficacy of genomic‐scale data for recognizing cryptic species, suggesting that species delimitation with one data type, whether one mitochondrial gene or morphology, may underestimate true species diversity in morphologically homogenous taxa with low vagility.     

Diversification process of stag beetles belonging to the genus Platycerus Geoffroy (Coleoptera: Lucanidae) in Japan based on nuclear and mitochondrial genes

Molecular phylogenies of the genus Platycerus in Japan were characterized based on the nuclear 28S ribosomal RNA and mitochondrial cytochrome oxidase subunit I (COI) genes. These analyses, combined with our previous morphological information, revealed a detailed diversification process of Platycerus in Japan, as well as estimates of their divergence times. Japanese species were monophyletic and were inferred to have diverged to the acuticollis species group and all other species ca 1.69 Mya. The acuticollis species group then appeared to split into P. viridicuprus and a group including P. takakuwai, P. albisomni and P. acuticollis ca 1.26 Mya. Other specific divergences have occurred primarily since ca 0.33 Mya. Comparing the molecular trees and the morphological tree, we also found introgression of the COI gene in some species. Genetic divergence of Platycerus has occurred intensely in southwestern Japan.     

Character displacement, frequency-dependent selection, and divergence of shell colour in land snails Mandarina (Pulmonata)

Endemic land snails of the genus Mandarina of the oceanic Bonin Islands offer an exceptional example of habitat and character divergence among closely related species. In this study, microhabitat differences between sympatric ground-dwelling species were studied by distinguishing habitats on the basis of vegetation and types of litter. In all sites where two ground species coexisted, segregation occurred with each species showing preference for the microhabitat in which they were found. When they were in sympatry, one species was predominant in relatively wet and sheltered sites and the other in relatively dry and exposed sites. Although most species can live in both types of habitat, occupation by one species is inhibited by occupation by another. This suggests that competitive interaction between sympatric species caused segregation. Except for populations that have undergone interspecific hybridization, no examples were found of sympatric populations of two ground species sharing a similar shell colour. Species that were predominant in relatively wet and sheltered sites possessed shells with dark coloration and their colour patterns were mostly of one type. Species that were predominant in relatively dry and exposed sites possessed shells with bright coloration and their color patterns were polymorphic. Most populations from areas in which single species were distributed had shells with medium coloration. Microhabitat differentiation between sympatric species possibly caused diversification of shell colour, because bright shells are advantageous in sites where snails are largely exposed, and dark shells are advantageous in sites in where they are mostly sheltered from sunlight. In addition, frequency-dependent selection by predators hunting by sight may have operated to maintain colour polymorphism in the populations which are restricted to exposed habitats by competition with other sympatric species. This reveals the importance of interaction among closely related species as a cause of diversification in ecological and morphological traits.     

Molecular phylogeography of Troglophilus cave crickets (Orthoptera,Rhaphidophoridae): A combination of vicariance and dispersal drove diversification in the East Mediterranean region

In this study, we investigated the molecular phylogenetic divergence and historical biogeography of cave crickets belonging to the genus Troglophilus (Orthoptera, Rhaphidophoridae) from caves in eastern Mediterranean and Anatolia regions. Three mitochondrial DNA genes (COI, 12S rDNA, and 16S rDNA) and two nuclear ones (18S rDNA and 28S rDNA) were amplified and partially sequenced to reconstruct phylogenetic relationships among most of the known Troglophilus species. Results showed a well‐resolved phylogeny with three main clades representing the Balkan, the Anatolian, and the Cycladian–Cretan lineages. Based on Bayesian analyses, we applied a relaxed molecular clock model to estimate the divergence times between these three lineages. Dating estimates indicate that radiation of the ingroup might have been triggered by the opening of the Mid‐Aegean trench, while the uplift of the Anatolian Plateau in Turkey and the changes of relief, emergence, and disappearance of orographic and hydrographical barriers in the Balkan Peninsula are potential paleogeographic events responsible for the initial diversification of the genus Troglophilus. A possible biogeographic scenario, reconstructed using S‐DIVA with RASP software, suggested that the current distribution of Troglophilus species can be explained by a combination of both dispersal and vicariance events that occurred in particular in the ancestral populations. The radiation of Troglophilus species likely started from the Aegean and proceeded eastward to Anatolia and westward to the Balkan region. Results are additionally compared to those available for Dolichopoda, the only other representative genus of Rhaphidophoridae present in the Mediterranean area.     

Unmasking alpha diversity,cladogenesis and biogeographical patterning in an ancient panarthropod lineage (Onychophora: Peripatopsidae: Opisthopatus cinctipes) with the description of five novel species

Speciation and biogeographical patterning in the velvet worm Opisthopatus cinctipes was examined under a null hypothesis that numerous discrete lineages are nested within the species. A total of 184 O. cinctipes specimens, together with a single specimen of each of the two congeneric point endemic sister species (O. roseus and O. herbertorum), were collected throughout the forest archipelago in the Eastern Cape, KwaZulu‐Natal and Mpumalanga provinces of South Africa. All specimens were sequenced for two partial mitochondrial DNA loci (COI and 12S rRNA), while a single specimen from each locality was sequenced for the nuclear 18S rRNA locus. Evolutionary relationships were assessed using maximum‐likelihood and Bayesian inferences, while divergence time estimations were conducted using BEAST. A Bayesian species delimitation approach was undertaken to explore the number of possible novel lineages nested within Opisthopatus, while population genetic structure was examined for the COI locus using ARLEQUIN. Phylogenetic results revealed that O. cinctipes is a species complex comprising seven geographically discrete and statistically well‐supported clades. An independent statistical approach to species delimitations circumscribed ca. 67 species. Results from divergence time estimation and rate constancy tests revealed near constant net diversification occurring throughout the Eocene and Oligocene with subdivision of ranges during the Miocene. Gross morphological characters such as leg pair number within O. cinctipes were invariant, while dorsal and ventral integument colour was highly polymorphic. However, scanning electron microscopy revealed considerable differences both between and within clades. The caveats associated with both morphological and algorithmic delineation of species boundaries are discussed. The five novel Opisthopatus species are described.     

Genetic divergence of Platycerus hongwonpyoi (Coleoptera: Lucanidae) in South Korea

We examined the genetic divergence of Platycerus hongwonpyoi Imura & Choe, 1989 in South Korea using the nuclear wingless (Wg) gene, internal transcribed spacer (ITS) region and mitochondrial cytochrome oxidase subunit I (COI) gene. We found no variation in Wg or ITS. Based on COI, P. hongwonpyoi was split into four well defined and one weakly supported clades, which were inferred to have diverged 2.11–1.33 Ma. The Platycerus hongwonpyoi population size seems to have decreased during the past several tens of thousands of years. The divergence times of major clades of P. hongwonpyoi were comparable with those involved in the speciation of certain Japanese species. Frequent overlapping of different clades at the same sites suggests the occurrence of secondary gene flow following differentiation in South Korea. In conclusion, the genus Platycerus underwent strikingly different divergence patterns in South Korea compared with Japan according to the disparate topographies of these two geographical areas.     

Morphological similarity and molecular divergence of Trochulus striolatus and T. montanus,and their relationship to sympatric congeners (Gastropoda: Pulmonata: Hygromiidae)

Shell morphology has played an important role in the differentiation of mollusc species. However, extensive morphological variation and the lack of readily available diagnostic characters often lead to confusion and controversy in taxonomy of closely related species, such as the genus Trochulus. Two nominal species Trochulus striolatus and T. montanus show only subtle differences in their reproductive systems and are indistinguishable on the basis of sole conchological traits. Therefore, we performed molecular and morphological analyses to establish robust species limits among the taxa. The canonical discriminant analysis (CDA) of shell characters confirmed strong similarity between the species. It also revealed high intraspecific variability of the shell morphology, which allowed the distinguishing of some populations as two extremely distinct forms, while simultaneously making it difficult to discriminate between the two species. Analysis of the genital morphology indicated that differences were continuous and practically negligible among the species. In contrast, phylogenetic analyses based on newly obtained 114 Trochulus sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and 55 homologues retrieved from the GenBank database showed clear genetic divergence between T. striolatus and T. montanus. Moreover, they revealed a close relationship between some T. montanus, some T. caelatus and T. clandestinus samples, which formed a monophyletic group. Some of their haplotypes were even identical. It suggested that their recent common origin or recurrent gene flow occurred between these populations. However, T. sericeus sequences were split into independent diverged lineages, which were separated by many unidentified Trochulus species. It suggested that this taxon may represent a paraphyletic species complex.