Characterizing DNA preservation in degraded specimens of Amara alpina (Carabidae: Coleoptera)

DNA preserved in degraded beetle (Coleoptera) specimens, including those derived from dry‐stored museum and ancient permafrost‐preserved environments, could provide a valuable resource for researchers interested in species and population histories over timescales from decades to millenia. However, the potential of these samples as genetic resources is currently unassessed. Here, using Sanger and Illumina shotgun sequence data, we explored DNA preservation in specimens of the ground beetle Amara alpina, from both museum and ancient environments. Nearly all museum specimens had amplifiable DNA, with the maximum amplifiable fragment length decreasing with age. Amplification of DNA was only possible in 45% of ancient specimens. Preserved mitochondrial DNA fragments were significantly longer than those of nuclear DNA in both museum and ancient specimens. Metagenomic characterization of extracted DNA demonstrated that parasite‐derived sequences, including Wolbachia and Spiroplasma, are recoverable from museum beetle specimens. Ancient DNA extracts contained beetle DNA in amounts comparable to museum specimens. Overall, our data demonstrate that there is great potential for both museum and ancient specimens of beetles in future genetic studies, and we see no reason why this would not be the case for other orders of insect.     

Feather colours of live birds and museum specimens look similar when viewed by seabirds

Bird plumage and skin colour can be assessed from museum specimens. To determine whether these accurately represent the colours of live birds when viewed by birds themselves, we analysed the spectral reflectances of live and up to 100‐year‐old museum specimens of five seabird species (White‐faced Petrel Pelagodroma marina, Common Diving Petrel Pelecanoides urinatrix, Grey‐faced Petrel Pterodroma gouldi, Little Shearwater Puffinus assimilis and Fluttering Shearwater Puffinus gavia). Live birds had brighter colours than museum specimens, but there were no significant differences in the wavelengths reflected. Modelling indicated that seabirds would be able to detect colour changes in the skin, but not the feathers, of museum specimens, but only for species with blue or pink feet (Pelecanoides urinatrix and Puffinus assimilis). For seabirds, museum specimens are adequate proxies for feather colour but not for skin colour.     

Post-copulatory Mounting Behavior of the West Indian Sweetpotato Weevil, Euscepes postfasciatus (Fairmaire) (Coleoptera: Curculionidae)

Post‐copulatory associations between males and females have been found in a variety of insects and are often described as mate guarding. Males of the West Indian sweetpotato weevil Euscepes postfasciatus (Fairmaire) mount the female's back after copulation. Two hypotheses have been advanced to explain this behavior: mate guarding to prevent future copulations by rivals (hypothesis 1), and mate guarding to gain additional copulations (hypothesis 2). We conducted three experiments to test predictions from these hypotheses. Our results disproved hypothesis 1 because the duration of the post‐copulatory association was very brief in comparison with the length of the refractory phase all females showed after copulation. When we prevented females from resisting copulations during the post‐copulatory mounted phase males copulated again, while under normal conditions, a second copulation was never observed. This result may indicate the presence of a sexual conflict over mating. However, we propose an alternative interpretation of the result, namely that after mating, males test whether the copulation has successfully reduced female receptivity by attempting to remate. If females resist the mating, males leave.     

Effect of mating on survival at low temperature in females of the Japanese common grass yellow,Eurema mandarina

1. In some insects that overwinter as adults, mating occurs both before and after overwintering. Two hypotheses have been proposed to explain the adaptive significance of pre‐overwintering copulation of females. One is the bet‐hedging hypothesis, which explains pre‐overwintering copulation as a preparation for less chance of mating in the following spring. The other is the nuptial gift hypothesis, which states that secretions derived from males increase overwintering success of females. 2. In Eurema mandarina, both diapause autumn‐ and non‐diapause summer‐form male adults emerge with autumn‐form female adults in the last generation in a year. Most autumn‐form females mate with summer‐form males before winter, and re‐mate with autumn‐form males in the following spring. Because autumn‐form females have sufficient chances for mating after overwintering, the nuptial gift hypothesis has been regarded as the more probable hypothesis. 3. To test the nuptial gift hypothesis, the survival period was compared under short‐day conditions at 10 °C between mated and unmated females that had been reared on sucrose solution at 25 °C for 15–21 days. The mated females had significantly greater longevity than the unmated females, supporting the nuptial gift hypothesis. Body size also affected the survival period. 4. The results suggest that the nuptial gift is an important factor for the evolution of pre‐overwintering copulation in species in which females mate both before and after overwintering.     

Genital and body allometry in two species of noctuid moths (Lepidoptera: Noctuidae)

One‐size‐fits‐all and related hypotheses predict that static allometry slopes for male genitalia will be consistently lower than 1.0 and lower than the slopes for most other body parts (somatic traits). We examined the allometry of genitalic and somatic morphological traits in males and females of two species of noctuid moths, Spodoptera exigua (Hübner, [1808]) and Helicoverpa armigera (Hübner, [1808]). The relationship between genitalic traits and body size was generally strongly negative‐allometric in males but with no significant differences from 1.00 in females of the two species examined. However, in females, the slope of genital traits was also lower than the slopes for somatic traits. The relationship between somatic traits and the body size indicator was approximately isometric in most cases in males, except in four traits in S. exigua, in which the slopes showed slight negative allometry, and the hind tibia in H. armigera, in which the slope had positive allometry. However, in females, some somatic traits showed isometric and some other showed negative allometry in both species. The coefficients of variation (CV) for all structures in the males were low, not exceeding 10%. Genitalic traits showed significantly lower CV than somatic traits in males. In females, somatic traits showed lower CV than genitalic traits but with no significant difference in the H. armigera. Our observations of strongly negative allometry for genitalic traits in males are consistent with stabilizing selection on genital size and we suggest that male performance in interactions with females is the source of selection on male genital allometry. The difference in the degree of phenotypic variation between genitalic and somatic traits in the two studied species is attributed to the different developmental‐genetic architectures of these traits. Female genitalia showed a similar trend to the males, although the difference between genital and somatic traits was not significant in females. This finding suggests that selection is acting differently on male and female genitalia. Positive allometry of hind tibia in H. armigera may be a result of secondary sexual function.     

Sequence capture phylogenomics of historical ethanol‐preserved museum specimens: Unlocking the rest of the vault

Natural history collections play a crucial role in biodiversity research, and museum specimens are increasingly being incorporated into modern genetics‐based studies. Sequence capture methods have proven incredibly useful for phylogenomics, providing the additional ability to sequence historical museum specimens with highly degraded DNA, which until recently have been deemed less valuable for genetic work. The successful sequencing of ultraconserved elements (UCEs) from historical museum specimens has been demonstrated on multiple tissue types including dried bird skins, formalin‐fixed squamates and pinned insects. However, no study has thoroughly demonstrated this approach for historical ethanol‐preserved museum specimens. Alongside sequencing of “fresh” specimens preserved in >95% ethanol and stored at ?80°C, we used extraction techniques specifically designed for degraded DNA coupled with sequence capture protocols to sequence UCEs from historical museum specimens preserved in 70%–80% ethanol and stored at room temperature, the standard for such ethanol‐preserved museum collections. Across 35 fresh and 15 historical museum samples of the arachnid order Opiliones, an average of 345 UCE loci were included in phylogenomic matrices, with museum samples ranging from six to 495 loci. We successfully demonstrate the inclusion of historical ethanol‐preserved museum specimens in modern sequence capture phylogenomic studies, show a high frequency of variant bases at the species and population levels, and from off‐target reads successfully recover multiple loci traditionally sequenced in multilocus studies including mitochondrial loci and nuclear rRNA loci. The methods detailed in this study will allow researchers to potentially acquire genetic data from millions of ethanol‐preserved museum specimens held in collections worldwide.     

SPECIES ISOLATION,GENITAL MECHANICS,AND THE EVOLUTION OF SPECIES-SPECIFIC GENITALIA IN THREE SPECIES OF MACRODACTYLUS BEETLES (COLEOPTERA,SCARABEIDAE, MELOLONTHINAE)

The question asked was why male genitalic structures have diverged in three syntopic species of Macrodactylus beetles. Four hypotheses were evaluated: 1. The ways in which male genitalia mesh with internal female structures indicate that selection for species isolation via mechanical exclusion (“lock and key”) is unlikely to explain the genitalic differences. 2. The specific mate recognition hypothesis also clearly fails to explain genitalic differences due to the implausibility of postulated environmental effects on genitalia, and lack of postulated coevolution of male and female morphologies. 3. Selection for species isolation via differences in genitalic stimulation (sensory lock and key) is unlikely due to relatively infrequent cross-specific pair formation and intromission in the field, and “excessive” numbers of species-specific genitalic structures and male courtship behavior patterns which nevertheless occasionally fail. It also fails to explain the frequent failure of intraspecific copulations to result in sperm transfer. This hypothesis cannot, however, be rejected as confidently as the previous hypotheses. 4. Conditions under which sexual selection by cryptic female choice could take place are common. Females frequently exercise their ability to prevent sperm transfer by conspecific males even after intromission has occurred, and females generally mate repeatedly, probably with different males. Males behave as if cryptic female choice is occurring, courting assiduously while their genitalia are within the female. Sexual selection by female choice could thus contribute to the divergence in genitalic structures.     

Females gain survival benefits from immune‐boosting ejaculates

Females in many animal taxa incur significant costs from mating in the form of injury or infection, which can drastically reduce survival. Therefore, immune function during reproduction can be important in determining lifetime fitness. Trade‐offs between reproduction and immunity have been extensively studied, yet a growing number of studies demonstrate that mated females have a stronger immune response than virgins. Here, we use the Texas field cricket, Gryllus texensis, to test multiple hypotheses proposed to explain this postmating increase in immune function. Using host‐resistance tests, we found that courtship, copulation, and accessory fluids alone do not affect female immunity; rather, only females that acquire intact ejaculates containing testes‐derived components exhibit significant increases in survival after exposure to bacterial pathogens. Our data suggest that male‐derived components originating from an intact ejaculate and transferred to females during sex are required for the increased immune function characteristic of mated female crickets to occur.     

Three phyla—Two type specimens—One shell: History of a snail shell revealed by modern imaging technology

In a recent revision of the gastropod genus Bellardiella, it became evident that the neotype of B. crassilabris had numerous borings over its entire shell surface. Superficial and light microscopical observation of the shell showed that the borings were made by a bryozoan belonging to the genus Penetrantia. Conventional techniques such as endocasting with the dissolution of the shell are impossible for type material. The application of non‐destructive micro‐CT‐scanning of the shell revealed the three‐dimensional arrangement of the entire colony within the shell. Careful analysis of the structure of the autozooids of the colony revealed that the species could not be assigned to any known species; we accordingly describe it as Penetrantia bellardiellae Schwaha, n. sp. The application of micro‐CT not only allowed the depiction of the new bryozoan species but also revealed the remains of a hermit crab located within the shell. The shell of Bellardiella crassilabris thus represents the type specimen of two species and contains a member of a third phylum inside. To our knowledge, this is a unique case, which also emphasizes the usefulness of micro‐CT imaging for precious specimens as well as the importance of historical museum collections.     

Eye morphology and visual acuity in the pipevine swallowtail (Battus philenor) studied with a new method of measuring interommatidial angles

Because of the important role sensory systems play in the behaviour of animals, information on sensory capabilities is of great value to behavioural ecologists in the development of hypotheses to explain behaviour. In compound eyes, interommatidial angles are a key determinant of visual acuity but methods for measuring these angles are often demanding and limited to live animals with a pseudopupil. Here we present a new technique for measuring interommatidial angles that is less demanding in terms of technology than other techniques but still accurate. It allows measurements in eyes without a pseudopupil such as dark eyes or even museum specimens. We call this technique the radius of curvature estimation (RCE) method. We describe RCE and validate the method by comparing results from RCE with those from pseudopupil analysis for the butterfly Asterocampa leilia. As an application of RCE we measure the eyes of the butterfly Battus philenor, a species whose visually guided behaviour is well known but whose eye structure and visual acuity are unknown. We discuss the results of the eye morphology in B. philenor in relation to their behaviour and ecology. We contend that RCE fills a gap in the repertoire of techniques available to study peripheral determinants of spatial resolution in compound eyes, because it can be applied on species with dark eyes. RCE then opens up for sampling a larger number of specimens, which, in combination with being able to use museum specimens, makes it possible to quantitatively test ecologically and evolutionarily driven hypotheses about vision in animals in a new way.     

A non-sperm transferring genital trait under sexual selection: an experimental approach

Male genitalia are among the fastest evolving morphological characters, and at a general level sexual selection seems to be involved. But experimental determination of the functions of many remarkable genitalic elaborations is very rare. Here we present the first study to address experimentally the adaptive function of a male genital structure that is not involved in sperm transfer. Females of the orb-weaving spider Argiope bruennichi are sexually cannibalistic and polyandrous. The male increases his paternity by obstructing the female's insemination duct with a fragment of his complex genitalia (embolus tip). We manipulated males by detaching another species-specific structure, the median apophysis spur, and found that the spur promotes breakage of the embolus tip inside the female duct, but does not affect the probability and duration of copulation. These data are novel in that they suggest that a genitalic structure which does not transfer sperm nevertheless evolved in the context of sperm competition.     

Goings‐on inside a worm: functional hypotheses derived from sexual conflict thinking

Different interests between mating partners regarding the fate of their gametes can lead to sexual conflicts in many species. Although these conflicts can sometimes be dealt with pre‐copulatorily (e.g. by choosing with which partners to mate), they often extend beyond copulation. Post‐copulatory sexual conflicts are expected to be particularly strong in simultaneous hermaphrodites because an individual may have to accept sperm in order to obtain an opportunity to donate sperm, reducing the effectiveness of pre‐copulatory conflict resolution. The present study investigates the post‐copulatory interactions between male and female sexual traits of a highly promiscuous simultaneous hermaphrodite, the free‐living flatworm Macrostomum lignano. Using light and electron microscopy, we show the different levels of complexity of the sperm and the genitalia, and derive hypotheses about how the different traits may represent evolutionary responses to such sexual conflicts. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 370–383.     

Hybrid enrichment of poorly preserved museum specimens refines homology hypotheses in a group of minute litter bugs (Hemiptera: Dipsocoromorpha: Schizopteridae)

Although they are a valuable source of specimens, insect natural history collections continue to be under‐utilized in molecular systematics, mostly due to difficulties in obtaining DNA sequences. Old specimens or specimens stored under suboptimal conditions are intractable for traditional Sanger sequencing. In this study we use an inexpensive hybrid capture with in‐house generated baits to retrieve commonly utilized ribosomal and mitochondrial loci from old museum specimens and combine them with a Sanger‐generated dataset comprising recently collected material. We focus on the Corixidea genus group (Schizopteridae), which comprises rarely collected, small (1–2 mm) and primarily tropical insects of which only c. 10–20% of the species have been described. A molecular phylogeny is needed to resolve relationships and revise the genus‐level classification to correctly place the c. 150 yet to be described species. Applying this approach, we constructed a dataset, containing 101 taxa, 11 of which were preserved in low‐percentage ethanol, 48 are dry and point‐mounted, and 40 are > 20 years old at DNA extraction. The obtained data proved sufficient for reconstructing a well‐supported phylogeny with c. 50% of the predicted diversity, and for the oldest successfully sequenced specimen (95 years) to be unambiguously placed in that phylogeny. We confirmed monophyly of the Corixidea genus group, showed paraphyly of the genus Corixidea, and recovered nine well‐supported clades within the group. Ancestral character states of selected morphological features were inferred and used to re‐examine primary homology hypotheses and inform an upcoming taxonomic revision.     

A time-sequence functional analysis of mating behaviour and genital coupling in Drosophila: role of cryptic female choice and male sex-drive in the evolution of male genitalia

Male genitalia in Drosophila exemplify strikingly rapid and divergent evolution, whereas female genitalia are relatively invariable. Whereas precopulatory and post-copulatory sexual selection has been invoked to explain this trend, the functional significance of genital structures during copulation remains obscure. We used time-sequence analysis to study the functional significance of external genitalic structures during the course of copulation, between D. melanogaster and D. simulans. This functional analysis has provided new information that reveals the importance of male-driven copulatory mechanics and strategies in the rapid diversification of genitalia. The posterior process, which is a recently evolved sexual character and present only in males of the melanogaster clade, plays a crucial role in mounting as well as in genital coupling. Whereas there is ample evidence for precopulatory and/or post-copulatory female choice, we show here that during copulation there is little or no physical female choice, consequently, males determine copulation duration. We also found subtle differences in copulatory mechanics between very closely related species. We propose that variation in male usage of novel genitalic structures and shifts in copulatory behaviour have played an important role in the diversification of genitalia in species of the Drosophila subgroup.     

Micro‐CT X‐rays do not fragment DNA in preserved bird skins

Most zoological systematics studies are currently based on morphological features, molecular traits or a combination of both to reconstruct animals’ phylogenetic history. Increasingly, morphological studies of museum specimens are using X‐ray computed tomography to visualize internal morphology, because of its ‘non‐destructive’ nature. However, it is not known whether CT can fragment the size of DNA extracted from museum specimens, as has been demonstrated to occur in living cells. This question is of paramount importance for collections based research because X‐rays may reduce the amount of data obtainable from specimens. In our study, we tested whether exposure of museum bird skins to typical CT X‐ray energies (for visualization of the skeleton) increased DNA strand fragmentation, a key factor for the success of downstream molecular applications. For the present study, we extracted DNA from shavings of 24 prepared and dried bird skins (100⁺ years) footpads before and after CT scanning. The pre‐ and post‐CT fragmentation profiles were assessed using a capillary electrophoresis high‐precision instrument (Agilent Bioanalyzer). Comparison of the most common strand length in each DNA sample (relative mass) revealed no significant difference unexposed and exposed tissue (paired t‐test p = 0.463). In conclusion, we found no further quantifiable degradation of DNA strand length under standard X‐ray exposure obtained from our bird skins sample. Differences in museum preservation techniques probably had a greater effect on variation of pre‐CT DNA fragmentation.     

Copulation behaviour of Glossina pallidipes (Diptera: Muscidae) outside and inside the female, with a discussion of genitalic evolution

If species-specific male genitalia are courtship devices under sexual selection by cryptic female choice, then species-specific aspects of the morphology and behaviour of male genitalia should often function to stimulate the female during copulation. The morphology and behaviour of the complex, species-specific male genitalia of the tsetse fly, Glossina pallidipes Austen, were determined from both direct observations and dissections of flash-frozen copulating pairs; we found that some male genitalic traits probably function to stimulate the female, while others function to restrain her. The male clamps the ventral surface of the female's abdomen tightly with his powerful cerci. Clamping does not always result in intromission. Clamping bends the female's body wall and her internal reproductive tract sharply, posteriorly and dorsally, and pinches them tightly. The male performed sustained, complex, stereotyped, rhythmic squeezing movements with his cerci that were not necessary to mechanically restrain the female and appeared instead to have a stimulatory function. Six different groups of modified setae on and near the male's genitalia rub directly against particular sites on the female during squeezing. The designs of these setae correlate with the force with which they press on the female and the probable sensitivity of the female surfaces that they contact. As expected under the hypothesis that these structures are under sexual selection by female choice, several traits suspected to have stimulatory functions have diverged in G. pallidipes and its close relative, G. longipalpis. Additional male non-genitalic behaviour during copulation, redescribed more precisely than in previous publications, is also likely to have a courtship function. The elaborate copulatory courtship behaviour and male genitalia may provide the stimuli that previous studies showed to induce female ovulation and resistance to remating.     

Cryptic female choice via sperm dumping favours male copulatory courtship in a spider

Males of many animals perform ‘copulatory courtship’ during copulation, but the possible reproductive significance of this behaviour has seldom been investigated. In some animals, including the spider Physocyclus globosus (Pholcidae), the female discards sperm during or immediately following some copulations. In this study, we determined which of several variables associated with copulation correlated with paternity success in P. globosus when two males mate with a single female. Then, by determining which of these variables also correlated with sperm dumping, we inferred which variables may affect paternity via the mechanism of sperm dumping. Male abdomen vibration (a copulatory courtship behaviour) and male genitalic squeezing both correlated with both paternity and sperm dumping; so, these traits may be favoured by biased sperm dumping. Biased sperm dumping may also be the mechanism by which possible cryptic female choice favours another male trait that was the subject of a previous study, responsiveness to female stridulation.     

Old and new DNA unweave the phylogenetic position of the eastern Atlantic gall crab Detocarcinus balssi (Monod, 1956) (Decapoda: Cryptochiridae)

Phylogeny reconstructions based on mtDNA and nDNA have become the standard in studies on relationships between taxa. Difficulties in obtaining material, for example because of small endemic distributions, often lead to gaps in datasets. Collections in natural history museums can provide us with material to fill these gaps, but extracting DNA from historical specimens can be challenging. We used a PCR protocol for small amounts of sample material and high PCR yield on eggs of specimens of the coral‐dwelling gall crab family Cryptochiridae collected in 1984, including material from the eastern Atlantic species Detocarcinus balssi. We obtained DNA sequences from seven older museum specimens using newly developed primers, which we combined with COI sequences from recently collected material. Results show that D. balssi is closest to the Indo‐Pacific species Utinomiella dimorpha and not closely related to one of the other three Atlantic Cryptochiridae species. The remaining newly acquired DNA sequences from museum material cluster with the respective sequences from recently collected specimens.     

Museum specimens provide reliable SNP data for population genomic analysis of a widely distributed but threatened cockatoo species

Natural history museums harbour a plethora of biological specimens which are of potential use in population and conservation genetic studies. Although technical advancements in museum genomics have enabled genome‐wide markers to be generated from aged museum specimens, the suitability of these data for robust biological inference is not well characterized. The aim of this study was to test the utility of museum specimens in population and conservation genomics by assessing the biological and technical validity of single nucleotide polymorphism (SNP) data derived from such samples. To achieve this, we generated thousands of SNPs from 47 red‐tailed black cockatoo (Calyptorhychus banksii) traditional museum samples (i.e. samples that were not collected with the primary intent of DNA analysis) and 113 fresh tissue samples (cryopreserved liver/muscle) using a restriction site‐associated DNA marker approach (DArTseq^?). Thousands of SNPs were successfully generated from most of the traditional museum samples (with a mean age of 44 years, ranging from 5 to 123 years), although 38% did not provide useful data. These SNPs exhibited higher error rates and contained significantly more missing data compared with SNPs from fresh tissue samples, likely due to considerable DNA fragmentation. However, based on simulation results, the level of genotyping error had a negligible effect on inference of population structure in this species. We did identify a bias towards low diversity SNPs in older samples that appears to compromise temporal inferences of genetic diversity. This study demonstrates the utility of a RADseq‐based method to produce reliable genome‐wide SNP data from traditional museum specimens.