Fig 1 - uploaded by Ian Kitching
Content may be subject to copyright.
![Two of the Hyles moths used to test the method described. A–D: Paratype female H. calida hawaiiensis, Hawaii [Hawaii] , Kau [Kaau], BMNHE #271618 (BMNH sphingid genitalia preparation #3087). E–H: Male H. livornica tatsienluica, China [Sichuan], Ta-Tsien-Lu [Kangding], BMNHE #812379 (BMNH sphingid genitalia preparation #3039). A, B, E, F show the moths before DNA extraction and genitalia preparation; C, D, G, H show the same moths afterwards. The specimens are deposited in the Natural History Museum, London, U.K.](profile/Ian-Kitching/publication/228495795/figure/fig1/AS:393860187475975@1470914995172/Two-of-the-Hyles-moths-used-to-test-the-method-described-A-D-Paratype-female-H-calida.png)
Two of the Hyles moths used to test the method described. A–D: Paratype female H. calida hawaiiensis, Hawaii [Hawaii] , Kau [Kaau], BMNHE #271618 (BMNH sphingid genitalia preparation #3087). E–H: Male H. livornica tatsienluica, China [Sichuan], Ta-Tsien-Lu [Kangding], BMNHE #812379 (BMNH sphingid genitalia preparation #3039). A, B, E, F show the moths before DNA extraction and genitalia preparation; C, D, G, H show the same moths afterwards. The specimens are deposited in the Natural History Museum, London, U.K.
Source publication
The external and internal genitalia of Lepidoptera have long provided a wealth of taxonomic and phylogenetic characters. However, traditional genitalia preparation techniques destroy both DNA, which is increasingly being used in Lepidoptera phylogenetics and species discrimination, and the scale pattern of the abdomen. In this paper, we describe a...
Contexts in source publication
Context 1
... study into the phylogeny and evolution of this ge- nus (cf. HUNDSDOERFER et al. 2009;A.K. Hundsdoerfer et al., unpublished data). The selected moths, from the collections of the Natural History Museum, London, U.K. (BMNH) and the Natural History Museum Vi- enna, Austria (NHMV), were at least 45 years old, and one was even more than 100 years old. Fig. 1 shows the uppersides and undersides of two moths before and after macerate extraction. The Hawaiian female H. calida hawaiiensis (Fig. 1A-D) is a bred moth that was accessioned into the BMNH in 1907. The year of capture of the male H. livornica tatsienluica from Chi- na is also not recorded but the specimen (Fig. 1E-H) was part of the ...
Context 2
... moths, from the collections of the Natural History Museum, London, U.K. (BMNH) and the Natural History Museum Vi- enna, Austria (NHMV), were at least 45 years old, and one was even more than 100 years old. Fig. 1 shows the uppersides and undersides of two moths before and after macerate extraction. The Hawaiian female H. calida hawaiiensis (Fig. 1A-D) is a bred moth that was accessioned into the BMNH in 1907. The year of capture of the male H. livornica tatsienluica from Chi- na is also not recorded but the specimen (Fig. 1E-H) was part of the L.W. Rothschild collection, which was bequeathed to the BMNH in 1939 (and the specimen may be older than 1903). The two studied paratypes of ...
Context 3
... even more than 100 years old. Fig. 1 shows the uppersides and undersides of two moths before and after macerate extraction. The Hawaiian female H. calida hawaiiensis (Fig. 1A-D) is a bred moth that was accessioned into the BMNH in 1907. The year of capture of the male H. livornica tatsienluica from Chi- na is also not recorded but the specimen (Fig. 1E-H) was part of the L.W. Rothschild collection, which was bequeathed to the BMNH in 1939 (and the specimen may be older than 1903). The two studied paratypes of H. salangensis from Afghanistan (NHMV; not il- lustrated) were collected in 1965. Although there is a small amount of degradation of the vestiture, the main abdominal pattern ...
Context 4
... it is as invasive as the traditional KOH mac- eration technique for genitalia dissection and prepara- tion, the method proposed here better preserves the ap- pearance of the specimen as the abdominal segments can be reattached after DNA extraction (Fig. 1). If suf- fi cient care has been taken, then minimal damage will have been infl icted upon the abdominal pattern char- acters, which are then available for future study. Such is not the case with traditional genitalia preparation where all the scales are removed from the abdominal skin prior to mounting it on the same microscope slide ...
Similar publications
This paper proposes a concise and effective approach termed discriminative feature representation (DFR) for low dose computerized tomography (LDCT) image processing, which is currently a challenging problem in medical imaging field. This DFR method assumes LDCT images as the superposition of desirable high dose CT (HDCT) 3D features and undesirable...
Citations
... To our knowledge, there are not many protocols specifically designed for the extraction of hDNA for wholegenome re-sequencing (WGS) of Lepidoptera 34 , and many studies use Targeted Enrichment (TE) approaches, reducing the cost of large phylogenomics studies 35,36 . Analysing mitochondrial DNA (mtDNA) can be another cost-efficient but powerful approach, e.g., to conduct phylogenetic analyses and trace maternal lineages. ...
The millions of specimens stored in entomological collections provide a unique opportunity to study historical insect diversity. Current technologies allow to sequence entire genomes of historical specimens and estimate past genetic diversity of present-day endangered species, advancing our understanding of anthropogenic impact on genetic diversity and enabling the implementation of conservation strategies. A limiting challenge is the extraction of historical DNA (hDNA) of adequate quality for sequencing platforms. We tested four hDNA extraction protocols on five body parts of pinned false heath fritillary butterflies, Melitaea diamina, aiming to minimise specimen damage, preserve their scientific value to the collections, and maximise DNA quality and yield for whole-genome re-sequencing. We developed a very effective approach that successfully recovers hDNA appropriate for short-read sequencing from a single leg of pinned specimens using silica-based DNA extraction columns and an extraction buffer that includes SDS, Tris, Proteinase K, EDTA, NaCl, PTB, and DTT. We observed substantial variation in the ratio of nuclear to mitochondrial DNA in extractions from different tissues, indicating that optimal tissue choice depends on project aims and anticipated downstream analyses. We found that sufficient DNA for whole genome re-sequencing can reliably be extracted from a single leg, opening the possibility to monitor changes in genetic diversity maintaining the scientific value of specimens while supporting current and future conservation strategies.
... We used a semidestructive approach, i.e., we removed the abdomen for DNA extraction without grinding the tissue, thus preserving the genitalia for future preparation (Hundsdoerfer and Kitching 2010). Genitalia dissections are routinely done for Lepidoptera by boiling abdomens in KOH to remove soft tissue, thus destroying the DNA in the process, so our approach is less destructive than what is normally done. ...
Billions of specimens can be found in natural history museum collections around the world, holding potential molecular secrets to be unveiled. Among them are intriguing specimens of rare families of moths that, while represented in morphology-based works, are only beginning to be included in genomic studies: Pseudobistonidae, Sematuridae, and Epicopeiidae. These three families are part of the superfamily Geometroidea, which has recently been defined based on molecular data. Here we chose to focus on these three moth families to explore the suitability of a genome reduction method, target enrichment (TE), on museum specimens. Through this method, we investigated the phylogenetic relationships of these families of Lepidoptera, in particular the family Epicopeiidae. We successfully sequenced 25 samples, collected between 1892 and 2001. We use 378 nuclear genes to reconstruct a phylogenetic hypothesis from the maximum likelihood analysis of a total of 36 different species, including 19 available transcriptomes. The hypothesis that Sematuridae is the sister group of Epicopeiidae + Pseudobistonidae had strong support. This study thus adds to the growing body of work, demonstrating that museum specimens can successfully contribute to molecular phylogenetic studies.
... These methods are not applicable to Lepidoptera, as this kind of handling would destroy most of the habitus of the specimen (including the detachment of wings and loss of most of the scales). Applicable methods for DNA extraction in Lepidoptera included the usage of the whole abdomen [49,73,74]. In those approaches, the habitus of the specimen was altered by the removal of the abdomen, the dissection of the genitalia and loss of scales at the abdomen while handling and during maceration for genitalia preparation. ...
Here we present and justify an approach for minimal-destructive DNA extraction from historic insect specimens for next generation sequencing applications. An increasing number of studies use insects from museum collections for biodiversity research. However, the availability of specimens for molecular analyses has been limited by the degraded nature of the DNA gained from century-old museum material and the consumptive nature of most DNA extraction procedures. The method described in this manuscript enabled us to successfully extract DNA from specimens as old as 241 years using a minimal-destructive approach. The direct comparison of the DNeasy extraction Kit and the Monarch® PCR & DNA Clean-up Kit showed a significant increase of 17.3-fold higher DNA yield extracted with the Monarch Oligo protocol on average. By using an extraction protocol originally designed for oligonucleotide clean-up, we were able to combine overcoming the restrictions by target fragment size and strand state, with minimising time consumption and labour-intensity. The type specimens used for the minimal-destructive DNA extraction exhibited no significant external change or post-extraction damage, while sufficient DNA was retrieved for analyses.
... Extraction from dry collections followed a modified protocol (see later). When material was not available as a molecular sample preserved in the −80 ∘ C freezer, we located desired taxa in private and institutional collections, removed the abdomen with sterile forceps, soaked the tissue overnight at ∼56 ∘ C in a solution of proteinase K and genomic lysis buffer (modified from Hundsdoerfer & Kitching, 2010). See Table S1 for information pertaining to the body part of each sample that was utilized in this study. ...
Mimallonidae, the sack-bearer moths, are a family of predominantly Neotropical moths containing nearly 300 described species. Mimallonidae feed on over 40 host plant families and are found in a variety of environments, but phylogenetic relationships of species within the family have never been investigated. We sequenced 515 loci using anchored hybrid enrichment target capture on ethanol-preserved and dried museum specimens, with dates of collection ranging from 1985 to 2017. We sampled 47 species, representing 32 of the 36 described mimallonid genera. By incorporating 19 dry museum specimens, and recovering an average of over 400 loci for each, we illustrate the utility of natural history collections in anchored hybrid enrichment-based phylogenomics. Maximum likelihood and multi-species coalescent analyses provide robust support for the recognition of six higher-level groups within Mimallonidae, which we designate as subfamilies: Zaphantinae St Laurent & Kawahara subfam.n., Aurorianinae St Laurent & Kawahara subfam.n., Mimalloninae Burmeister, Lacosominae Dyar, Druenticinae St Laurent & Kawahara subfam.n. and Cicinninae Schaus stat.n. Our phylogenetic results also robustly support eight new tribes: Lacosominae: Trogopterini St Laurent & Kawahara tribe n., Lacosomini Dyar stat.n., Alheitini St Laurent & Kawahara tribe n.; Druenticinae: Luramini St Laurent & Kawahara tribe n., Druenticini St Laurent & Kawahara tribe n.; Cicinninae: Bedosiini St Laurent & Kawahara tribe n., Psychocampini St Laurent & Kawahara tribe n., Cicinnini Schaus stat.n. Three new genera are also described based on our phylogenetic results: Herbinalla St Laurent & Kawahara, gen.n., Ulaluma St Laurent & Kawahara, gen.n., Bedosiallo St Laurent & Kawahara, gen.n. Naniteta Franclemont, syn.n. is a synonym of Lacosoma Grote. Six genera are paraphyletic, and in total 19 new combinations are proposed: Macessoga laxa comb.n., Lacosoma elassa comb.n., Thaelia anysia comb.n., Thaelia subrubiginosa comb.n., Herbinalla caudina comb.n., Druentica brosica comb.n., Ulaluma valva comb.n., Cicinnus eminens comb.n., Roelmana pluridiscata comb.n., Roelmana laguerrei comb.n., Psychocampa joanna comb.n., Psychocampa unalca comb.n., Psychocampa hamata comb.n., Psychocampa marona comb.n., Bedosiallo eugenia comb.n., Bedosiallo forbesi comb.n., Bedosiallo moengus comb.n., Bedosiallo styx comb.n. and Bedosiallo sylvia comb.n. This study is the first to implement the LEP1 probe set on a comprehensive taxonomic dataset that includes many museum specimens, and our results demonstrate that museum specimens can be used in anchored hybrid enrichment studies. Importantly, these data produce a robust phylogeny that will serve as a foundation for future studies on mimallonid evolution, such as host plant relationships and biogeography.
... Insect genitalia structures are important source of diagnostic characters for several groups of insects. Male genitalia are typically dissected and described after prepared in an aqueous solution of potassium hydroxide (KOH) followed by removal of muscles and fat body (Gurney et al. 1964, Hundsdoerfer andKitching 2010). The KOH procedure is effective to hasten decomposition of soft tissues and some poorly sclerotized structures. ...
... Knölke et al. (2005) first proposed extracting DNA as a by-product of genitalia preparation in Lepidoptera, avoiding morphological damage, which is usually achieved when a leg is dissected for DNA extraction. The Proteinase K (PK) treatment preserved morphological traits in Lepidoptera during DNA extraction from genitalia, but the authors did not evaluate efficacy of diaphanization and structure preservation of genitalia after the PK treatment (Knölke et al. 2005, Hundsdoerfer andKitching 2010). Moreover, Lepidoptera are relatively large specimens and applicability of procedure in minute wasps as parasitoids have not been discussed. ...
Genitalia are rich source of characters in insect taxonomy. Usually, they are examined after dissection and cleaning with potassium hydroxide (KOH), procedure that may damage both genital morphological structures and intracellular molecular contents. Enzymatic procedure with Proteinase K has been used to clean muscle off the genitalia while extract DNA, but its damage to the genital structures has not been evaluated. Herein, we qualitatively compare the use of KOH and Proteinase K to prepare genital structures in minute insects (Hymenoptera: Bethylidae). We show that Proteinase K is better to preserve the genital structure and provides quality DNA.
... To address the above questions, we sought to sequence 'historic' DNA from one of the century-old types of Hyles calida hawaiiensis. The abdomen of the paratype reared at Kau by Perkins (BMNH(E) #271618) was macerated following the protocols described by Hundsdoerfer & Kitching [23] (the specimen is illustrated both before and after treatment in their Fig 1A-1D). It is not known exactly when this moth was collected but it came to the then British Museum (Natural History) in 1907 (accession number 1907-347) as part of an exchange. ...
Analysing historic DNA from museum specimens offers the unique opportunity to study the molecular systematics and phylogenetics of rare and possibly extinct taxa. In the Hawaiian fauna, the hawkmoth, Hyles calida calida, occurs on several of the main islands and is quite frequent, whereas Hyles c. hawaiiensis is restricted to the Island of Hawaii where it appears to be very rare. Analysis of mitochondrial DNA sequences shows that Hyles c. hawaiiensis differs from the nominotypical subspecies by an average p-distance of 2.8%, which is of a similar order of magnitude to that found between other species of Hyles, suggesting that Hyles c. hawaiiensis should perhaps be awarded species status, although more data are required for a formal taxonomic revision. Given the rarity of this taxon, these analyses should be undertaken urgently so that conservation measures can be implemented before it becomes extinct.
... DNA from museum specimens was extracted using an aDNA methodology in a dedicated laboratory. Such tissue consisted of either the abdomen, the contents of which were digested enzymatically following the procedures described by Hundsdoerfer & Kitching (2010), or one or two legs, which were finely chopped with small scissors prior to addition of lysis buffer from a sbeadex forensic kit (LGC Genomics, Berlin, Germany). Manufacturers' instructions were followed except that the proteinase-K digestion was left overnight and the final elution was performed with 60 lL of elution buffer. ...
Three high-elevation Hyles species of Central Asia have proven difficult to sample, and thus, only a limited number of specimens are available for study. Ancient DNA techniques were applied to sequence two mitochondrial genes from ‘historic’ museum specimens of Hyles gallii, Hyles renneri and Hyles salangensis to elucidate the phylogenetic relationships of these species. This approach enabled us to include the holotypes and paratypes. The status of H. salangensis as a species endemic to a mountain range north of Kabul in Afghanistan is confirmed by this study. It is most closely related to Hyles nicaea and H. gallii, and quite distant from the clade comprising the species from Hyles vespertilio to Hyles tithymali, despite this group and H. salangensis both completely lacking an arolium on the pretarsus. Our results show that the samples assigned to H. renneri and Hyles livornica tatsienluica are conspecific and so we reinstate Hyles tatsienluica stat. nov. as the valid name for this species and synonymize H. renneri syn. nov. with it. This study shows that the distribution range postulated for H. tatsienluica extends from Nepal well into the mountains of south-western China. The distribution ranges of H. livornica and H. tatsienluica overlap. The study confirms the previously proposed synonymies of Hyles nepalensis, Hyles gallii intermedia and Hyles gallii tibetanica with H. gallii. Extensive species sampling (over 80% of Hyles species) in this study allowed additional analyses. The dated phylogeny reveals the global Hyles hawkmoth radiation to be much more recent than previously thought: it began in the Late Miocene and culminated in a Pleistocene burst of diversification in the Northern Hemisphere. Ancestral ranges of basal nodes were reconstructed as highly equivocal, but the Neotropics has the highest probability in the two oldest nodes. Although the origins of the Madagascan and Australian species also remain ambiguous, a large crown clade of fifteen species was reconstructed to have originated in the Palaearctic. The wide distribution ranges of the two migratory species, H. livornica and H. gallii, appear to blur any traces of the biogeographic origin of the clades containing these species. Specialization in larval host plant use onto particular plant families from the ancestral condition ‘polyphagous’ may have led to an increased rate of speciation and phylogenetic diversification in three subgroups of Hyles (the Hawaiian clade, the Hyles centralasiae group and the Hyles euphorbiae complex).
... DNA was extracted from three legs taken from the right side of the thorax of specimens that were frozen or stored in 96% ethanol. If a specimen had been allowed to dry, DNA was extracted from the abdomen of the specimen using a modified method from Knölke et al. (2005) and Hundsdoerfer & Kitching (2010). The DNA extraction was performed using the DNeasy blood and tissue kit (Qiagen, Valencia, CA) following the manufacturer's instructions with modifications noted below (detailed DNA extraction methods are available from the authors upon request). ...
The lichen moth tribe Lithosiini is best known for its hypothesized larval feeding behaviour, lichenivory. The larvae of some species have been found to be capable of sequestering polyphenolics, and the adults of some species are unpalatable to vertebrate predators. However, the chemical basis for the defence is unknown. Here we reconstruct a phylogenetic hypothesis using likelihood methods (maximum likelihood, Bayesian inference) for 65 species of Lithosiini representing 37 genera and seven outgroup species using 2806 bp of sequence data obtained from two mitochondrial gene fragments (COI barcoding region, CytB), and two nuclear gene fragments (RpS5 and ribosomal gene region 28S). Lithosiine species representing four of the seven subtribes and unplaced taxa are included in the analysis. The deeper relationships within the tribe are not strongly supported, and the monophyly of three of the four subtribes is not supported. The placement of the fourth subtribe, Acsalina, is weakly supported. We also conduct a survey of the secondary metabolites present within adults of five species. The species are examined for the presence of lichen polyphenolics and plant secondary metabolites sequestered by arctiines. Seven lichen polyphenolics are identified in the taxa examined, but no plant secondary metabolites are found using either analytical method. The results of this study and prior reports in the literature of chemical sequestration and unpalatability are plotted onto the resulting Bayesian inference phylogeny to examine the evolution of chemical defence within Lithosiini. Species that sequester lichen polyphenolics and unpalatable species occur in each of the major clades recovered in the analysis.
... We conducted the laboratory work in a dedicated ancient DNA clean-laboratory with Class II Safety Cabinets (HERAsafe KSP9; Thermo Scientific, Waltham, USA) and 4 hours of UV-light decontamination between the working steps. Tissue sampling of abdomens followed the protocol of Hundsdoerfer & Kitching [71] whereby abdomens are broken off carefully, macerated and glued on again after drying. DNA was isolated with the AGOWA sbeadex Forensic Kit (AGOWA Genomics, Berlin, Germany) using 120 μL of macerate or one leg cut into small pieces. ...
... The definition of the mitochondrial lineages of the HEC is based upon sequences of the genes for CO I/II and the interposed gene for t-RNA leucine (2284 bp in total; [34]). Specific primers to sequence these genes from historical specimens in 13 overlapping fragments (fragments A to M) had already been designed for the entire genus Hyles, see [71]. In the present study, we used a subset of three fragments covering 794 bp in total (fragment B: 277 bp, H: 280 bp, L: 237 bp). ...
Background
Mitochondrial genes are among the most commonly used markers in studies of species’ phylogeography and to draw conclusions about taxonomy. The Hyles euphorbiae complex (HEC) comprises six distinct mitochondrial lineages in the Mediterranean region, of which one exhibits a cryptic disjunct distribution. The predominant mitochondrial lineage in most of Europe, euphorbiae, is also present on Malta; however, it is nowadays strangely absent from Southern Italy and Sicily, where it is replaced by 'italica'. A separate biological entity in Italy is further corroborated by larval colour patterns with a congruent, confined suture zone along the Northern Apennines. By means of historic DNA extracted from museum specimens, we aimed to investigate the evolution of the mitochondrial demographic structure of the HEC in Italy and Malta throughout the Twentieth Century.
Results
At the beginning of the Twentieth Century, the European mainland lineages were also present at a moderate frequency in Southern Italy and Sicily. The proportion of 'italica' then steadily increased in this area from below 60 percent to near fixation in about 120 years. Thus, geographical sorting of mitochondrial lineages in the HEC was not as complete then as the current demography suggests. The pattern of an integral 'italica' core region and a disjunct euphorbiae distribution evolved very recently. To explain these strong demographic changes, we propose genetic drift due to anthropogenic habitat loss and fragmentation in combination with an impact from recent climate warming that favoured the spreading of the potentially better adapted 'italica' populations.
Conclusions
The pattern of geographically separated mitochondrial lineages is commonly interpreted as representing long term separated entities. However, our results indicate that such a pattern can emerge surprisingly quickly, even in a widespread and rather common taxon. We thus caution against drawing hasty taxonomic conclusions from biogeographical patterns of mitochondrial markers derived from modern sampling alone.
... But how to act in cases where closely related species are also sympatric or when the type locality is imprecise or unkown? Besides, the development of new techniques of DNA extraction and amplification to access molecular data from specimens collected long ago is limited (e.g., Hundsdoerfer & Kitching 2010). The opposite problem may also take place, since the comparison between a given specimen and a species described solely based on DNA data would necessarily rely on the sequencing of this specimen. ...
The use of DNA sequences, including DNA barcoding, as a taxonomical tool has been happening for some time (Tautz et al., 2003; Hajibabaei et al., 2007; Packer et al., 2009). However, the description of new species based solely on DNA sequences is a new idea (Cook et al., 2010) and a new practice (Brower, 2010). Our aim is not to further polarize the war between advocates of strictly molecular or strictly morphological systematics (following, e.g., Pires & Marinoni 2010). The objectives here are (i) to present some arguments regarding the perils of the proposition of a model (theory) for solely DNA-based descriptions (Cook et al. 2010) and the actual publication (practice) of such descriptions (Brower 2010), (ii) to discuss some reasons why we believe that adopting strictly DNA taxonomy for species description, setting aside everything we have learned from classic taxonomy, may not be the best alternative and (iii) to present the point of view about these matters of a PhD candidate and a recently graduated PhD working with taxonomy in a developing country.
