Male differentiation patterns in two polyphenic sister species of the genus Onthophagus Latreille, 1802 (Coleoptera: Scarabaeidae): a geometric morphometric approach

This paper focuses on morphological (both shape and size ) differences that quite similar polyphenic sister species evolve during divergence processes. Traits were analysed using a geometrical morphometric approach, which has the ability to evidence also very subtle differences in shape. As a case study, we considered males of the dung beetle sister species pair Onthophagus taurus and Onthophagus illyricus (Coleoptera, Scarabaeidae); these species represent a typical example of polyphenic trait expression concerning the facultative development of horns and considerable body size differences. External shape morphology failed to discriminate O. taurus from O. illyricus , whereas the reproductive system shape showed significant interspecific discrimination power. However, the head of O. taurus was significantly larger than that of O. illyricus and the reverse was true for the elytra. The two species also showed different allometric values of the head with respect to body size. This complex pattern of interspecific morphological divergence is discussed in the light of the differential trait divergence rate hypothesis. In both species, differences between major and minor forms concern the overall shape of head and pronotum: we suggest that such different forms, which likely reflect morphological readjustment to accommodate horns of considerable bulk and disproportionate length, may be nevertheless advantageously used by the two male morphs in their alternative reproductive tactics. Male genitalia sizes were virtually constant with respect to body size; however, the ratio between phallotheca and body size was significantly higher in minor males, in keeping with the hypothesis of a higher investment in genitalia borne by this morph.     

Epipharynx shape as a tool to reveal differentiation patterns between insect sister species: insights from Onthophagus taurus and O. illyricus (Insecta: Coleoptera: Scarabaeidae)

We evaluated the potential of the adult epipharynx to reveal interspecific differentiation patterns in closely related Onthophagus species. Although easy to analyse, this structure has received little attention in Scarabaeidae taxonomy, probably because a qualitative inspection of its shape often does not yield differences between congeneric species. The polyphenic sister species Onthophagus taurus Schreber, 1759 and O. illyricus Scopoli, 1763 were chosen as a case study. They are extremely similar, to the extent that in some cases they cannot be identified unambiguously without the help of biomolecular analysis. In this study, a combination of linear measurements and a landmark-based approach was employed to quantify inter- and intraspecific shape variation in the epipharynx of specimens sampled at the same study site. Our results showed that the epipharynx is a monomorphic structure: its shape does not vary as a function of sex or male phenotype. In males, epipharynx shape does not change with head shape or horn length. The close proximity of the epipharynx to the horns and the synchronous developmental patterns of these two structures suggest that a developmental trade-off may act between them. Despite these predisposing conditions, however, our results suggest that epipharynx size is not subject to costs associated with horn development, and that the trait is highly canalised. Surprisingly, when using geometric morphometrics the epipharynx appears to be a better tool than genitalia for discriminating between the two sister species.     

Assessing the taxonomic status of Osmoderma cristinae (Coleoptera: Scarabaeidae), endemic to Sicily,by genetic,morphological and pheromonal analyses

Resolving complexes of closely related and cryptic insect species can be challenging, especially when dealing with rare and protected taxa that are difficult to collect for genetic and morphological analyses. Until recently, populations of the genus Osmoderma (Scarabaeidae), widespread in Europe, were treated as a single species O. eremita (Scopoli, 1763) in spite of observed geographic variation in morphology. A previous survey using sequence data from the mtDNA cytochrome C oxidase I gene (COI) revealed the occurrence of at least two distinct lineages within this species complex: O. eremita in the west and O. barnabita Motschulsky, 1845, in the east. Interestingly, beetles confined to Sicily have been described as a distinct species, O. cristinae Sparacio, 1994, based on morphological traits. Only few Sicilian specimens were included in the former genetic analysis, and the results led to a still questionable taxonomic rank for these populations. To explore the robustness of the previous taxonomic arrangement for O. cristinae, a combination of genetic, morphological and pheromonal analyses was used. A 617‐bp fragment of the COI gene, aligned with O. cristinae and O. eremita sequences already available in GenBank, showed a clear genetic divergence between the two species (interspecific mean distance = 6.6%). Moreover, results from AFLP markers sustained the distinction of the two species. In addition, geometric morphometric analyses of the shape of male genitalia revealed a clear differentiation between the two species. Via scent analysis and field trapping, we demonstrated the production of the sex pheromone (R)‐(+)‐γ‐decalactone by males of O. cristinae, the attraction by conspecific individuals (mostly females) to this compound, and a lack of antagonistic effect of (S)‐(–)‐γ‐decalactone. The fact that O. eremita and O. eremita use the same compound for mate finding suggests that this sex pheromone has not undergone a differentiation and probably the allopatry of these two species compensates for the absence of a mechanism to avoid cross‐attraction. Our genetic and morphological data support the divergence of the two species and confirm the species status for O. cristinae, while sex pheromones are confirmed to be invariant among different species of the genus Osmoderma.     

Genetic and morphological differentiation patterns between sister species: the case of Onthophagus taurus and Onthophagus illyricus (Coleoptera, Scarabaeidae)

The present study investigated the morphological and genetic differentiation pattern between two sympatric dung beetle sister species, Onthophagus taurus and Onthophagus illyricus . The geometric morphometric approach coupled with the use of molecular markers allowed examination of the nature of interspecific relationships and an outline of the evolutionary and geographical processes that might have led to interspecific differentiation and the present-day partial sympatric and syntopic pattern of distribution. Geometric morphometrics failed to discrimininate the two species on the ground of external morphological traits, but revealed interspecific differences when the shape of the primary sexual traits was taken into account. The use of two different molecular markers (cytochrome oxidase subunit I gene and amplified fragment-length polymorphism) demonstrates that the two species are genetically well differentiated, forming two distinguishable lineages probably diverged during the Pliocene by allopatric speciation. No evidence of past or recent introgression and no support for hybridization were found, suggesting that sympatry was established subsequently, when speciation was accomplished. Both molecular markers and genital shape indicate that phenotypically intermediate individuals, despite their ambiguous appearance, are members of O. illyricus species rather than hybrids.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 197–211.     

Data on molecular taxonomy and genetic diversification of the European Hermit beetles, a species complex of endangered insects (Coleoptera: Scarabaeidae, Cetoniinae, Osmoderma)

A molecular analysis was carried out on the European hermit beetles (the Osmoderma eremita species complex) to explore their genetic diversification and the robustness of previous morphologically based taxonomic arrangements. Complete sequences of mtDNA cytochrome C oxidase I gene were obtained from 26 individuals. Mean levels of interspecific sequence divergence ranged from 0.044 to 0.186. The results indicate a clear-cut distinction between two clades. The first one includes the W-European O. eremita Scopoli 1763 ; and the two Italian endemic taxa Osmoderma italicum Sparacio, 2000 and Osmoderma cristinae Sparacio, 1994 ; from southern peninsular Italy and Sicily, respectively. The second one includes the widespread E-European Osmoderma barnabita ; and the southern Balcanic Osmoderma lassallei Baraud and Tauzin, 1991 from Greece and European Turkey. Within the two clades, molecular and morphological data well support a specific rank for O. lassallei and O. barnabita on one side, and for O. eremita and O. cristinae on the other side, while the taxonomic position of O. italicum , more closely related to O. eremita , is still uncertain. Current geographical distribution, interspecific genetic diversification, and very low levels of intraspecific genetic divergence in western European populations of O. eremita sensu stricto are hypothesized to be the result of multiple speciation events (mainly occurred in refugial forest areas of the Italian and Balkan peninsulas and Sicily before and during the Pleistocene glacial peaks), followed by fast post-glacial northward and westward expansion of some species.     

Genetic structure of natural populations of the California black abalone (Haliotis cracherodii Leach, 1814), a candidate for endangered species status

The black abalone (Haliotis cracherodii) has been severely depleted across much of its historic range by a combination of overexploitation and disease. Natural recovery of extirpated populations along the southern California coast will depend on the extent to which remnant populations can serve as larval sources to geographic locations formerly supporting abalone populations. Population genetic analyses of mitochondrial cytochrome oxidase subunit one (COI) DNA sequences, four nuclear microsatellites, and 142 amplified fragment length polymorphisms (AFLPs) were used to evaluate connectivity among populations of H. cracherodii sampled from the central California coast and four islands in the Southern California Bight. Global divergence among populations was significant at COI and the AFLP loci. The Hka28 microsatellite locus and AFLP data showed significant divergence in multiple pairwise population comparisons and exhibited a signal of isolation by distance. Although estimates of gene flow based on genetic analyses must be interpreted with caution, the observed level of interpopulation genetic divergence suggests that larval dispersal is restricted, and natural recovery of decimated H. cracherodii populations along the coast of California is unlikely to occur in the near future.     

Tylotus laqueatus,a new species of Dicranemataceae (Gigartinales,Rhodophyta) from the Hawaiian Islands

A rarely collected shallow‐subtidal Hawaiian macroalga has been determined anatomically and molecularly to belong to an undescribed species of Tylotus J. Agardh, the most widely distributed genus of the small, mostly Australian‐endemic family Dicranemataceae. Thalli are repent and imbricate on calcareous boulders at the type locality on O‘ahu, and are anchored both basally and by haptera arising marginally and ventrally on the (sub‐)dichotomous, linear axes. Simple or forked terete haptera can be a means of perennation by the occasional direct issuing of adventitious blades. Fronds are multiaxial and consist of a broad pseudoparenchymatous medulla of thick‐walled cells surrounded on both sides by a two‐ or three‐layered small‐celled pigmented cortex in which numbers of ‘glandular’ hairs are embedded. Tetrasporangia are zonate, and gametophytes are monoecious. Carpogonial branches are three‐celled, directed to the thallus surface, and borne laterally on inner‐cortical supporting cells; cystocarps are prominently protuberant and scattered sparingly on dorsal frond surfaces, the carposporophytes directed outwardly beneath an ostiolate pericarp and connected to the parent gametophyte across a broad placental base in which the remnant auxiliary cell persists centrally. The inner surface of the pericarp is unusual in producing extensive patches or isolated islands of short gonimoblast filaments with terminal carposporangia as an apparent result of the implantation of gonimoblasts into the tissue of the lining. Anatomy indicates that the new species is more closely related to the East‐Asian Tylotus lichenoides Okamura than to the only other described member of the genus, the type species T. obtusatus (Sonder) J. Agardh from southern Australia. An rbcL phylogeny supports placement of sequences for Hawaiian specimens within the genus Tylotus but distinct from all previously recorded sequences of Tylotus. As is widely reported in other molecular‐phylogenetic analyses of the Gigartinales, we find that support for generic and familial relationships within the order is strong whereas that for between‐family relationships is low.     

Molecular-genetic analyses reveal cryptic species of trematodes in the intertidal gastropod, Batillaria cumingi (Crosse)

Cryptic species of the digeneans, Cercaria batillariae (Heterophyidae) and an undescribed philophthalmid, were detected using polymerase chain reaction-based restriction fragment-length polymorphism methodology and sequence analyses. These digeneans were all collected from the same species of gastropod first intermediate host, Batillaria cumingi (=Batillaria attramentaria). The mitochondrial cytochrome oxidase subunit 1 gene (approximately 800bp) and nuclear internal transcribed spacer 1 gene (approximately 400bp) were used for species level discrimination. Restriction fragment-length polymorphism analyses of cytochrome oxidase subunit 1 gene showed that C. batillariae included 10 distinguishable fragment patterns, and the philophthalmid included five patterns. On the basis of subsequent sequence analyses, the restriction fragment length polymorphism patterns of C. batillariae were grouped into eight phylogenetically distinct lineages and those of the philophthalmid into three phylogenetically distinct lineages. There was no evidence of gene flow among the different lineages due to the lack of heterozygosity within the observed internal transcribed spacer 1 gene fragment patterns. This suggests that all of these lineages are different species. Most of these species were widespread, but some exhibited restricted geographic distributions. We discuss the implications of these findings for host specificity of these trematodes. These results demonstrate the utility of genetic analysis to distinguish species of morphologically similar trematodes. Hence, trematode species diversity may often be underestimated when species identifications are limited to morphological features.     

Larval morphology, genetic divergence, and contrasting levels of host manipulation between forms of Pomphorhynchus laevis (Acanthocephala)

Studies on parasite species with a wide geographic and ecological range may be confounded by still equivocal taxonomic identification. Here, we investigated genetic polymorphism and behavioural changes induced in a common intermediate host, in two different forms of Pomphorhynchus laevis based on the morphology of the larval infective stage (cystacanth). A 'smooth type' (S) and a 'wrinkled type' (W) of cystacanth were distinguished based on their surface and shape. We analysed sequence divergence at both nuclear (ribosomal gene 18S rDNA, and ribosomal internal transcribed spacers, ITS1/ITS2) and mitochondrial (cytochrome c oxidase subunit 1) genes of P. laevis cystacanths and adults at various geographical scales. A high level of sequence divergence at ITS1, ITS2 and cytochrome c oxidase subunit 1 (11, 8 and 20%, respectively) was found between these two forms. The divergence pattern consistently discriminated two groups independently of geographic origin or host, and was congruent with larval morphology. The two forms also strongly differed in the intensity of behavioural change induced in their common intermediate host, Gammarus pulex, with the S-type parasite inducing a positive phototactism, whereas W-type infected gammarids were as photophylic as uninfected ones. Overall, our data strongly support the specific status of these two forms. We suggest that smooth cystacanths correspond to P. laevis, whereas wrinkled cystacanths might correspond to the previously described and poorly documented, Pomphorhynchus tereticollis, considered a synonym of P. laevis. This study also confirms the value of a joint analysis of internal transcribed spacers and cytochrome c oxidase subunit 1 genes to biogeographic studies on these species. Finally, we emphasize the importance of linking morphological and biological characteristics of acanthocephalan cystacanths to molecular data, in the study of the evolutionary ecology and systematics of this group.     

Genetic and morphological studies of species status for poorly known endemic Trochulus phorochaetius (Bourguignat, 1864) (Gastropoda: Pulmonata: Hygromiidae), and its comparison with closely related taxa

The taxonomic status of Trochulus phorochaetius and its phylogenetic relationships to Trochulus plebeius and Trochulus hispidus were evaluated based on molecular, morphological, and genital anatomy data. The canonical discriminant analysis (CDA) of shell morphology allowed the clear differentiation between these three nominal species, whereas the genitalia revealed their high similarity. Analyses of cytochrome c oxidase subunit I (COI) sequences were not always congruent with the differentiation between these three species by shell characters. None of them formed a monophyletic group covering all its sequences. Instead, many sequences obtained from individuals classified to the same morphospecies, and/or usually collected from the same region or country, created highly supported separate clades and delimited clusters. Three distinct clades containing sequences of two morphospecies originating from the same country were identified in molecular phylogenetic and species delimitation studies: (1) T. plebeius + T. hispidus from Great Britain; (2) T. plebeius + T. hispidus from Poland; and (3) T. phorochaetius + T. hispidus from France. In the latter case some of the sequences were even identical. Their genetic similarity could indicate the ability to hybridize, which may be evidenced by the lack of major differences in their reproductive system. The assignment of distinctive morphospecies, and thus existing taxonomic names, to genetically defined evolutionary lineages is premature and arbitrary to some extent at this stage. © 2013 The Linnean Society of London     

Phylogenetic relationships of the enigmatic ornate shiner,Cyprinella ornata,a species endemic to Mexico (Teleostei: Cyprinidae)

The systematics and taxonomy of North Americancyprinid fishes has historically been said tobe in a chaotic state of affairs. Much of theconfusion as to relationships of species restsin the lack of explicit phylogenetic hypothesesof species and reliance upon degree ofdistinctiveness of taxa or their overallsimilarity for generic placement. Some specieshave had more turbulent or variable taxonomichistories than others. The ornate shiner, Cyprinella ornata, is one of those species,having been placed in the genera Notropis,Codoma, and Cyprinella within the last20 years and found in current texts in any ofthese three genera. Most of the confusionregarding placement of this species has beenrelated to lack of explicit phylogenetichypotheses to formulate its classification, butto a certain degree some researchers haveignored phylogenetic studies and preferred torely upon its morphological distinctiveness orreproductive behavioral traits of questionablehomology to place it in a monotypic genus andalign it with the genera Pimephales andOpsopoeodus.I present a phylogenetic analysis based oncomplete Cytochrome b sequences fromornate shiners, other species of Cyprinella, purported relatives, and severalother species of the Shiner Clade to determinethe phylogenetic affinities of this enigmaticspecies. Molecular analysis reveals the ornateshiner to be more closely related to species ofCyprinella than to a Pimephalesplus Opsopoeodus clade as previouslydiscussed in one morphological analysis, or asargued by Page and Johnston (1990), Johnstonand Page (1992) and Page and Ceas (1989) basedon observations of spawning behaviors andhypotheses of homology between crevice-spawningand egg-clumping behaviors. This molecularanalysis is more consistent with earliermorphological phylogenetic hypotheses of Mayden(1989) wherein these two clades are notconsidered to be closely related and the ornateshiner is a member of the genus Cyprinella.     

Fine-scale spatial genetic structure and gene flow in a small, fragmented population of Sinojackia rehderiana (Styracaceae), an endangered tree species endemic to China

Populations of Sinojackia rehderiana are highly threatened and have small and scattered distribution due to habitat fragmentation and human activities. Understanding changes in genetic diversity, the fine-scale spatial genetic structure (SGS) at different life stages and gene flow of S. rehderiana is critical for developing successful conservation strategies for fragmented populations of this endangered species. In this study, 208 adults, 114 juveniles and 136 seedlings in a 50 × 100-m transect within an old-growth forest were mapped and genotyped using eight microsatellite makers to investigate the genetic diversity and SGS of this species. No significant differences in genetic diversity among different life-history stages were found. However, a significant heterozygote deficiency in adults and seedlings may result from substantial biparental inbreeding. Significant fine-scale spatial structure was found in different life-history stages within 19 m, suggesting that seed dispersal mainly occurred near a mother tree. Both historical and contemporary estimates of gene flow (13.06 and 16.77 m) indicated short-distance gene dispersal in isolated populations of S. rehderiana. The consistent spatial structure revealed in different life stages is most likely the result of limited gene flow. Our results have important implications for conservation of extant populations of S. rehderiana. Measures for promoting pollen flow should be taken for in situ conservation. The presence of a SGS in fragmented populations implies that seeds for ex situ conservation should be collected from trees at least 19-m apart to reduce genetic similarity between neighbouring individuals.     

Clonal and spatial genetic structure in natural populations of Luohanguo (Siraitia grosvenorii), an economic species endemic to South China,as revealed by RAPD markers

Clonal growth is generally expected to have significant effects on the spatial genetic structure within populations. In this study, random amplified polymorphic DNA (RAPD) markers were used to reveal clonal and spatial genetic structure of four natural populations of Luohanguo (Siraitia grosvenorii), an economic vine species endemic to South China. A total of 351 ramets were assigned to 76 distinct multi-locus genotypes (i.e. genets), with the G/N varying from 0.121 to 0.350. No widespread genet was found across different populations. The clonal diversity (D) and evenness (E) ranged from 0.333 to 0.828 and from 0 to 0.741, respectively. While most genets consisted of fewer than five ramets, we observed some dominant genets that had much more (up to 69) ramets and spread over large areas. Spatial autocorrelation analyses revealed a spatial genetic structure (i.e. significant positive autocorrelation within 20 m and negative autocorrelation beyond 40 m) in one population, but not in other three populations with smaller population size. This study highlights the importance of clonal growth in shaping the spatial genetic structure in Luohanguo, which may have complex effects on the dynamics and evolution of its declining populations.     

Ancient lakes as hotspots of diversity: a morphological review of an endemic species flock of <Emphasis Type="Italic">Tylomelania</Emphasis> (Gastropoda: Cerithioidea: Pachychilidae) in the Malili lake system on Sulawesi,Indonesia

The viviparous freshwater gastropod Tylomelania (Caenogastropoda: Cerithioidea: Pachychilidae) endemic to the Indonesian island Sulawesi has radiated extensively in two ancient lake systems. We here present the first systematic species-level review of taxa in the five lakes of the Malili lake system, which contains the most diverse and best studied freshwater fauna on Sulawesi. Our results indicate a significantly higher diversity of Tylomelania in these lakes than previously perceived based on morphological evidence for delimiting the taxa. We describe nine new species, thus increasing the number of taxa known from the Malili lakes to 25. Tylomelania species are inhabiting all available substrates in the lakes, and the diversity of habitats is reflected in an unparalleled range of radula types in this closely related group. Several species show a high intraspecific variability in some characters, and their closer investigation will probably lead to the discovery of more cryptic species. As it is, this species flock on Sulawesi is among the largest freshwater mollusc radiations known. Since the Malili lake system also contains other large endemic species flocks of e.g. crustaceans and fishes, it is a major hotspot of freshwater biodiversity in Asia to become a conservation priority. Handling editor: K. Martens