MODERN MORPHOLOGICAL TECHNIQUES AND THE EVOLUTIONARY BIOLOGY AND TAXONOMY OF SEPSIDAE (DIPTERA) YUCHEN ANG (B.Sc. (Hons), NUS) DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2013 MODERN MORPHOLOGICAL TECHNIQUES AND THE EVOLUTIONARY BIOLOGY AND TAXONOMY OF SEPSIDAE (DIPTERA) YUCHEN ANG (B.Sc. (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ___________________ Yuchen Ang 16 August 2013 ACKNOWLEDGEMENTS The first person I must thank would have to be my supervisor and long-time mentor, Prof. Dr. Rudolf Meier. I have known The Teutonic Terror of S2 for close to nine years now, having been under his wing for both my B.Sc. and current Ph.D. In this time, I have benefitted so much, learning from him the ‘tools of the trade’, and his frank, critically constructive feedback has only helped to toughen my already-thick hide and more importantly develop my own set of critical thinking skills. Through numerous ‘coffee breaks’ and a lot of cajoling and nudging, I managed to get through and tackle the great academic Ph.D. beast. Sometimes I feel undeserving of all the trust, opportunities and help that he has given me, and ll this I am grateful for – especially his contagious passion in research and sepsids that I have contracted as well. Summarily I would liken him to the Gordon Ramsay of Science: doing research under him is tough and a little frightening because he demands so much, but ultimately, much more rewarding and inspiring as he imparts his knowledge and attitude that helps you achieve your own potential. Thank you Prof, this time I’m writing it for you. Next of course are my labmates whom I’ve come a long way with: Amrita, coding sleuth and occasional stats-tutor whom Sepsidnet and I are so in debt with for all the prompt and late-night help – I owe you so much tea now! Denise ‘Little-Red’ Tan, old-bird Mammafly with all the crazy long work-fevered nights we’ve shared, I’m glad you’ve found a way to permanently damage your head in Florida ☺ (sorry I couldn’t make it to your mega-sendoff all because of this). Kathy Su, I’m glad you’ve come back to evolab – it’s good to have a Science-first author doctor around in the consultation room pantry that I can rely on to critically examine my work, and also listen to my yammerings. Diego, for all the innuendo jokes and bouncing off interesting ideas – it really energizes me to explore more in my work. Ahlek Wong, I really appreciate all the help in the Chiro project rearing and stuff, sorry if I’m a little slow with the imaging. Jay, always with cookies and chips, please make more of that yummy dessert you being to lab. Lei, Mindy, Youguang, Nesibe, Ivy, Darren, plus all the others who have left the lab, all you guys and girls have made the lab the quirky and fun place to work in! Of course, I cannot forget my loved ones, who have made so many sacrifices; I really would not have made it here without your support. Ma and Pa, thank you for all the support in so many different ways you have given me; all your expressed worries I consider carefully, so try not to worry. Thank you for being so understanding about my regular late nights and not expecting me to pay the bills even when I’m already 30. I’m going to get an income soon. A job. With CPF. Promise! Mei, thanks for always offering to give me a lift home, both you and Di brighten my day with your silly antics and stupid 9GAG posts, specifically, “HEHEUHEHAHEAHUEH”. Grace, I’m sorry you’ve had to tolerate my crazy work-life schedule and thank you for all the care you’ve showered, all the little welfare packages of food and more snacks, coming all the way from the East to deliver hot food. And especially for that final push where you just pushed me on so much more. I’m sorry it had to come to this. Table of Contents THESIS SUMMARY List of Figures List of tables, videos and appendices . 10 List of publications used in this thesis . 11 List of species used in this thesis 12 CHAPTER 1: General Introduction 16 A plea for morphology in biology 16 Evolutionary biology and taxonomy of Sepsidae 19 CHAPTER 2: Phylogenetic origins and morphological evolution of the sepsid sternite appendage 23 Abstract 23 Introduction . 25 Part I: The evolutionary origins of the sternite appendages in the Sepsidae . 27 Materials and Methods . 27 Results and Discussion . 29 Part II: Co-option of muscles and sternites for the formation of the sepsid sternite appendage 37 Materials and Methods . 38 Results 39 Discussion 48 Appendix 1A . 50 Appendix 1B . 54 CHAPTER 3: Male sepsid legs evolve rapidly when in direct contact with female structures . 55 Abstract 55 Introduction . 56 Materials and Methods 62 Results 66 Discussion 73 Appendix 2A . 77 Appendix 2B . 81 CHAPTER 4: A plea for digital reference collections and other science‐based digitization initiatives in taxonomy: Sepsidnet as exemplar 82 Abstract 82 Introduction . 83 Digital reference collections . 86 Building the digital reference collection for Sepsidae 93 Conclusion . 99 CHAPTER 5: Using seemingly unnecessary illustrations to improve the diagnostic usefulness of descriptions in taxonomy . 101 Abstract 101 Introduction . 103 Materials and Methods 106 Results 108 Appendix . 127 CHAPTER 6: Using multiple data-sources to address taxonomic uncertainties . 128 Abstract 128 Part I: From ‘cryptic species’ to integrative taxonomy – an iterative process involving DNA sequences, morphology, and behaviour leads to the resurrection of Sepsis pyrrhosoma (Sepsidae: Diptera) 129 Materials and Methods . 132 Results 135 Discussion 145 Conclusion 148 Part II: Morphology and DNA sequences confirm the first Neotropical record for the Holarctic sepsid species Themira leachi (Meigen) (Diptera: Sepsidae)149 Materials and Methods . 150 Results and Discussion . 150 CHAPTER 7: Increasing taxonomic data accessibility by linking wiki-entries to species descriptions . 155 Abstract 155 Introduction . 156 Materials and Methods 159 Results 160 Key to species of the genus Perochaeta Duda, 1926 (males) 177 Discussion 178 THESIS CONCLUSIONS 181 References 185 THESIS SUMMARY In my thesis, I pursue my research interests in morphology by conducting a series of studies on the evolutionary biology and taxonomy of Sepsidae (Diptera), using various bioimaging techniques such as microcomputered tomography, photomicrography and scanning electron-microscopy. First, I document how de-novo moveable appendages evolved from male sternites and show that it evolved twice in sepsids. Second, I demonstrate that sexual selection highly increases morphological divergence by quantifying and comparing the rates of evolution between sexually dimorphic structures that are likely to be under sexual selection and monomorphic structures that are mostly under viability selection. Third, I explore the benefits of coupling taxonomy with information technology by creating a digital reference collection featuring 140 sepsid species and a web-tool for online species identification, as well as publishing species descriptions that are simultaneously featured as taxonomic entries in a wiki-based platform. Lastly, I show how morphological data-richness and iterative taxonomy can address inadequately diagnostic species descriptions as well as resolve 'cryptic' species proposed based on DNA sequences as well as disjunctive distributions in species. List of Figures Figure 1.1: Phylogenetic hypothesis of Sepsidae used in this study for Chapter 31 Figure 1.2: Phylogenetic hypothesis of Sepsidae parsimony and maximum likelihood support that appendages evolved twice and were lost three times .32 Figure 1.3A: Summary of female sternites across Sepsidae 33 Figure 1.3B: Summary of male sternites across Sepsidae 34 Figure 1.4. Photomicrographs of ventral abdomen with expanded view of sternites and 5, for Saltella sphondylli male, Themira lucida male, Themira superba male and Themira superba female, as well as the habitus of T. superba male 41 Figures 1.5 – 1.7 (Part 1): Three dimensional models showing various views for Saltella sphondylli male, Themira lucida male and Themira superba male (Views A-B) 44 Figures 1.5 – 1.7 (Part 2): Three dimensional models showing various views for Saltella sphondylli male, Themira lucida male and Themira superba male (Views C-E) 45 Figure 1.8: Three dimensional models showing various views for Themira superba female 46 Figure 1.9: 3D model schematic of relevant structures in Themira superba explaining how VM5-6 powers the arm 'raise' 46 Figure 2.1: ‘Wing clasp’ behavior in various sepsid species .60 Figure 2.2: Various ‘basitarsal thumbs’ in Coelopidae 61 Figure 2.3: Obtaining complexity scores from leg structures .65 Figure 2.4 (part 1): Forefemora and –tibiae for male and female sepsids, mapped onto the sepsid phylogenetic hypothesis .67 Figure 2.4 (part 2): Midfemora and –tibiae for male and female sepsids, mapped onto the sepsid phylogenetic hypothesis .68 Figure 2.5: Femora and tibiae for fore- and midlegs of male and female coelopids, mapped onto the coelopid phylogenetic hypothesis .69 Figure 2.6: graphical representation of tree length for various leg structures of and regions of sepsid males and females for quantitative and qualitative measures .71 Figure 2.7: graphical representation of tree lengthfor various leg structures of and regions of coelopid males and females for quantitative and qualitative measures .71 have been particularly small in this case, but misidentifications are not uncommon in museums and the proportion of unidentified specimens is generally large. Yet, many papers in the aforementioned Zookeys issue still promote wall-towall digitization of collections. This often comes in the form of drawer digitization with or without assigning physical or virtual unique specimen identifiers (Bertone et al. 2012, Blagoderov et al. 2012, Schmidt et al. 2012, van den Oever and Gofferje 2012). There is anecdotal evidence that this encourages loan activity (Bertone et al. 2012, Mantle et al. 2012), but given that the digitization of collections takes many peopleyears, a rigorous cost/benefit analysis would reveal that a few emails are probably more effective in encouraging loans. Of course, wholesale collecting of data without (much) quality control is not only found in museums. Instead it is also the technique du jour by data aggregators (e.g., GBIF, EOL http://eol.org/) and has also become common in DNA barcoding where it is now so rampant that the majority of DNA barcodes in GenBank had to be withdrawn because they were not identified to species [423,188 records: Kwong et al. (2012)]. In a museum environment, new material should be immediately digitized by capturing, for example, locality data and taxon membership, but the wholesale imaging of specimens should be considered more carefully. Fortunately, many museums have recognized that only well-curated parts of a collection should be digitized (Haston et al. 2012, Mantle et al. 2012, Tulig et al. 2012). In these institutions specimens destined for digitization are pre-selected and/or curated prior to digitization. The downside is that this requires considerable curatorial time that arguably should be spent on activities that generate new scientific insights. Indeed, it appears that in some museums the various 85 digitization activities now employ more personnel than research; i.e., wholesale digitization is in the process of changing the institutions’ character without helping to overcome the taxonomic impediment. It appears to me that healthier approaches are based on a mixture of on-demand digitization [see Mantle et al. (Mantle et al. 2012)] and integrating science and digitization (Meier and Dikow 2004, Schuh 2012, Wheeler et al. 2012). Such approaches often yield high quality data that are also more likely to justify the legacy costs associated with digital databases (Haston et al. 2012). These costs should be carefully considered in all digitization projects given that special funding may be available for the initial digitization of specimens/drawers but is unlikely to be available for the maintenance of databases. Cost-benefit analyses appear particularly lacking for drawer-digitization projects where maintenance is likely to be particularly labour-intensive. After all, when a specimen from a digitized drawer is (re)moved, the drawer has to be re-digitized and/or the specimen movement has to be tracked (Bertone et al. 2012, Dietrich et al. 2012). Digital reference collections Instead of selling a grand vision and discarding current taxonomic practice, I here promote a more modest cybertaxonomy tool that may have received little attention because of its lack of grandeur: the digital reference collection. Physical reference collections are important tools in taxonomy; they are used by many systematists for confirming identifications by comparing undetermined specimens with specimens that have been identified by taxonomic experts. Here I argue for digital reference collections which have the advantage that they are accessible to virtually all entomologists and can cover more species diversity than physical collections. I 86 demonstrate the value of such a digital collection by presenting one for Sepsidae (Diptera) that includes high-quality images for ca. 40% of the species-level diversity. Note that for ease of use, I believe that a digital reference collection should be a clean, simple, purpose-build tool for confirming species identification and not a collection of species pages with many fields that make it difficult to compare the images for multiple species (e.g., see AntWeb: http://www.antweb.org/). Online tools are particularly useful and needed for identification given that traditional tools can be subject to serious problems (Gotelli 2004). Among others these tools are (1) scattered across many journals thus creating an access problem, (2) of variable quality, (3) become outdated quickly (e.g., through the addition of new species and emended nomenclature including synonymization of species names), and (4) suffer from ontology problems when different authors not use the same terms for the same structure. Given these problems, it is not uncommon that biologists feel little confidence that a particular specimen has been identified correctly even if the most upto-date identification literature has been consulted. Hence, many specialists still maintain reference collections consisting of reliably identified specimens. These reference collections are used for confirming identifications through direct comparison of unidentified with identified material. However, few biologists have access to reference collections, of which most only contain a few species of merely regional significance. This is where digital reference collections can make a difference. Comparing an unidentified specimen under the microscope with a high-quality image on a nearby computer screen is in most cases sufficient and more convenient than seeking and retrieving a specimen from a traditional reference collection and juggling simultaneously with multiple pinned and/or ethanol-preserved vouchers. Digital 87 reference collections are also the best long-term solution for the problem of rarity. Many species have been collected only once and/or described based on a single specimen (Lim et al. 2012). These species are generally missing in physical reference collections while they can be included easily in digital collections. Having identification tools for these species is important because collectively they can constitute a large proportion of the species-level diversity in biodiversity samples (Longino et al. 2002) Based on cost-benefit arguments, digital reference collections are useful for taxa that need to be identified routinely by biologists from different backgrounds. A good example would be Sepsidae (Diptera) whose conspicuous sexual dimorphism, short generation times, and tolerance for laboratory conditions have attracted the attention of biologists from a variety of disciplines. For instance, researchers have used Sepsidae to address theoretical issues in systematics (Meier 1997, Su et al. 2008, Tan et al. 2010), to study the behaviour of these flies (Eberhard and Huber 1998, Eberhard 1999, 2000, Eberhard 2001a, Eberhard 2001e, 2003, Eberhard 2005, Puniamoorthy et al. 2008, Puniamoorthy et al. 2009, Tan et al. 2011), their development (Bowsher and Nijhout 2007, Bowsher and Nijhout 2009, Bowsher et al. 2013), genetics (Reusch and Blanckenhorn 1998, Muhlhauser and Blanckenhorn 2004, Hare et al. 2008) and evolution (Eberhard and Pereira 1996, Blanckenhorn et al. 1998, Blanckenhorn et al. 1999, Blanckenhorn et al. 2000, Eberhard 2001d, Blanckenhorn et al. 2002, Eberhard 2002a, b, c, Muehlhaeuser and Blanckenhorn 2002, Blanckenhorn et al. 2004, Muhlhauser and Blanckenhorn 2004, Blanckenhorn et al. 2007, Teuschl and Blanckenhorn 2007, Teuschl et al. 2007, Ingram et al. 2008, Puniamoorthy et al. 2010, Teuschl et al. 2010a, Teuschl et al. 2010b, Wild et al. 2011, Puniamoorthy et al. 2012). 88 Consequently, there is a large community of biologists with an interest in and need for up-to-date identification tools for Sepsidae. Digital reference collections can be created for any group of insects, but arguably the taxa most suited for easily generating one are those that require relatively few images for capturing the critical structures that are used for identification. In addition, the species diversity should be fairly well known overall so that the user can assess how much of the species diversity is covered by the collection. Sepsidae satisfy both conditions. Male foreleg and genital morphology is sufficient for identifying most species. In addition, given that the number of newly described species has dropped to 250 years ago [Sepsis cynipsea (Linnaeus, 1758)] and almost one sixth of the existing valid species before 1900 (Ozerov, 2005). As such, a substantial proportion of the descriptions are in old and/or discontinued 89 journals that are available only in a few libraries. Some of the most important, old publications and the recently published literature are available online via commercial journal subscriptions or publication digitization projects (e.g., Biodiversity Heritage Library: www.biodiversitylibrary.org/). However, much of the 20th century literature is less accessible due to of a toxic mix of lack of digitization and copyright restrictions. Access to primary type material is even more difficult. Despite having only about 350 described species, the types are distributed across ca. 50 institutions on all continents. Borrowing types is getting increasingly difficult due to the risk of damage or loss given that the proportion of old and fragile types is high and bound to increase because most species were described in the past centuries and the types are aging. Already many type specimens are so damaged that little morphological information can be obtained. This is particularly problematic in sepsids where many types lack legs or abdomina, and/or have claspers that are withdrawn into shrivelled bodies (e.g. see Sepsis kyandolirensis: http://goo.gl/LRIyVF). In addition to the access problems, there are numerous quality problems with the existing identification tools. More than 70 authors have contributed to species descriptions in the Sepsidae but the quality of the descriptions varies tremendously. Those from the 18th and 19th century are generally very brief and lack illustrations and information on genitalia. The descriptive literature of the 20th and 21st century is of higher quality, but some authors under- or overestimated the intraspecific variability, leading to some lumping and many synonymy problems [see Ozerov (1995) for the large number of synonyms created by Vanschuytbroeck]. Potentially all descriptions suffer from a lack of information on character systems that became relevant only after the species were described. For example, the authors of the 18th and 19th century did not 90 anticipate the need for information on genitalia, microtrichosity (“pruinosity”) patterns are rarely described in the literature of the early 20th century, and today’s descriptions are likely to suffer from yet unknown deficiencies. These quality problems can be partially overcome through digital photography (Cranston, 2005). High quality photographs provide similar amounts of information for all species. In addition, good photographs not only capture the morphological features that are regarded currently as important, but they may serendipitously also capture information that will only become relevant in the future. For example, if such images had been used by taxonomists of the early 20th century, microtrichosity patterns would have been captured. Of course, digital photography has also numerous shortcomings and drawings can be more effective in highlighting critical features and usually are better at summarizing intraspecific variability. Digital reference collections should thus consist of a mixture of photographs and other types of illustrations as available. An additional problem with the traditional literature used for specimen identification is that it becomes quickly outdated through the description of new species and new synonymy proposals. For example, in Sepsidae more than 650 species-level taxa have been described of which more than 300 have been subsequently synonymised. Many synonyms were proposed recently so that identification literature predating these proposals is outdated. The synonymy problem alone is widespread and can be quite severe for some species. For example, as of 2005, 87 of the 318 valid species had junior synonyms (Ozerov, 2005) and variable species such as Saltella sphondylii had attracted >20 separate descriptions (Pont & Meier, 2002). Digital reference collections can also become outdated if they are not updated regularly, but in contrast to the traditional identification tools, they can remain up-to-date and revision 91 dates can be specified on the webpage; that is, synonymy changes and descriptions of new species are more readily accommodated. Another much lamented problem with the identification literature is that it is incomprehensible to the non-specialist because of ontology problems (Gotelli, 2004). For example, sepsid taxonomists routinely differ in the terms used to describe the same morphological structure. The distribution of microtrichia on certain pleural sclerites is diagnostic for some sepsid species. Sometimes these sclerites are referred to as anepisternum, katepisternum and anepimeron (e.g., Ozerov, 1993a) whereas other authors use mesopleuron, sternopleuron and pteropleuron respectively [e.g., (Iwasa & Tewari (1990)]. The most direct way of overcoming such ontology issues is to agree upon a standard terminology, and many Dipterists use the terminology of the Diptera manuals (McAlpine et al., 1981; Courtney et al., 2000; Merz & Haenni, 2000; Sinclair, 2000). However, the published literature containing alternative ontologies still needs to be translated to the standard. With a digital reference collection, two solutions are apparent. First, the specimens can remain unlabeled. The user then directly compares the query specimen with the imaged specimen, thus potentially bypassing terminological problems. However, this could result in overlooking morphological features that are critical for species identification. Alternatively, the specimens can be labeled and an interactive glossary provided [see Hamilton et al. (2006)]. In Sepsidnet I opt for unlabeled images, but provide a glossary that allows the user to match terms in the identification literature with structures of a model sepsid species. 92 Building the digital reference collection for Sepsidae Specimen acquisition. I obtained material for 140 of the ca. 350 described species. This included type material for 32 species from several European institutions [Muséum National d’Histoire Naturelle, Paris, France (MNHN), Musée Royal de l’Afrique Centrale, Tervuren, Belgium (MRAC) and Royal Belgian Institute of Natural Sciences, Brussels, Belgium (RBINS)]. However, the specimens for most species were taken from rearings and cultures that were started from field-collected females as described in Ang et al. (2008) and Ang & Meier (2010). Most sepsid specimens in collections are pinned, but often it is preferable to preserve them in 80% ethanol so that the specimens are available for both morphological and molecular work (storage in a fridge/freezer is recommended). If cultures are needed, field-collected males usually can be killed immediately, while females are used to establish a culture. This ensures ample high-quality specimens with a definite male-female association and the option for additional work on behaviour, natural history, and reproductive isolation. Specimen identification. Sepsidnet is a community effort by sepsid workers from various parts of the world. This means that the species pages have been and continue to be checked by several experts in the group. Most specimens were identified initially based on the literature and then compared to the reference collection of the Evolutionary Biology Laboratory at the National University of Singapore (NUS). For most species a DNA barcode has been generated by collaborators that can be accessed via a Genbank link. Specimen imaging. Sepsid specimens were imaged first before extracting DNA. Males were preferred because they tend to show species-specific diagnostic characters. 93 Figure 3.1: Image of Nemopoda speiseri as seen in Zoomify™ viewer, showing the habitus, lateral view (a); abdomen, ventral view (b); and dissected hypopygial capsule, dorsal view (c). Scale bar denotes 1.0 mm. 94 Figure 3.2: Image of male Sepsis cynipsea fore femur and tibia (partial), anterior view. The resolution of the image is high enough to show the cuticular sculpturation detail on the femur (a) as well as the sexually dimorphic ornamentation on both femur and tibia (b). Scale bar denotes 0.5 mm. Females were imaged if the association with males was confirmed via a culture or rearing. Where possible, I documented the male lateral habitus (Fig. 3.1A; to view the leg ornamentation and thoracic pleura), the ventral view of the abdomen (Fig. 3.1B; to view any modified sternites), and where available, the dissected claspers (Fig. 3.1C). These structures were combined into a single image (as Fig. 3.1). Specimens from the reference collection at NUS as well as type material from RBINS were imaged serially at different focal distances using a Visionary Digital™ BK Plus Lab System. The images were then focus-stacked using Helicon Focus™ Pro (ver. 5.2.16). The image resolution generated with this system is sufficient enough to show cuticular sculpturation (Fig. 3.2). It is comparable in quality to observations by eye through a good quality stereomicroscope. Specimens from the European museums (MNHN and MRAC) were imaged and focus-stacked with the Auto-Montage® system using a Leica® DFC450 microscope. Image quality from this imaging system also is adequate 95 for diagnostic features. Composite images were edited with Adobe® Photoshop® CS4 to remove background noise and optimize brightness, contrast and colour balance. In addition to providing digital photographs, I included line drawings from the literature for all species. Sepsidnet design. Edited photographs were exported in Zoomify™ format (as a Zoomifyer) and embedded into the Sepsidnet website. The Zoomifyer is a specialized Flash object that allows users to stream high-magnification images of structures that are critical for identification: it divides an image into a series of smaller-sized picture tiles at different resolutions and sizes that are presented onto a fixed frame. Because the viewer frame requires only few picture tiles to be loaded at any time, viewing is fast and smooth. As Zoomifyers comprises a simple package of HTML code, small image files and a simple Flash movie code; it can be played readily on any browser with Flash support. Mobile Apple devices such as iPhones and iPads can similarly view Zoomifyers once additional plugins are installed, while Android™ devices will readily load the Flash object. A sepsidnet page is presented in two columns. The main specimen image for the target species (usually a male) is shown in a 700-by-700 pixel Zoomifyer on the top of the left column, with a link to specimen label information directly below it. Below the zoomifyer are diagnostic illustrations for the species. On the right column are several drop-down tabs that when selected give access to additional information. From top to bottom, the first gives access to taxonomic information that is drawn mostly from Pont & Meier (2002) and Ozerov (2005). It also includes links to external sources (literature: PubMed and Biodiversity Heritage Library; DNA sequences: GenBank). The second 96 Figure 3.3: A visual guide to using Sepsidnet. At the home page (top right panel), clicking on a species image (A) will open the species page. Here, the main habitus is displayed in a Zoomifyer (B), along with published illustrations (C) and any additional specimen images (D). On the right navigation pane (E) are options to view taxonomic information (F), known species distributions (G), an interactive morphoglossary (hovering your mouse over a structure will highlight the terminology on the left; H) as well as a drop-down species-list to navigate to other species pages (I). Clicking the species comparator button (J) will pull-up a four-windowed screen for comparison of four specimens simultaneously (K). Options for other comparator resolutions (e.g., 2X1, 2X3 windows) are also available by clicking the button on the top left. 97 tab provides information on species distribution, while the third opens up into an interactive morpho-glossary that has separate sections for "head", "thorax", "abdomen (male)" , and "abdomen (female)". The decision to include only data on taxonomic material and known distribution is a practical one: based on the fact that these are the most common information usually needed for identifying a species. Additional information such as natural history can then be further searched based on the external resources links. Below these three tabs are the species comparison and navigation tools. By clicking the "Compare with other species" button, a new comparison frame appears with four separate Zoomifyers for quadruple species comparison. Additional frame options for other screen resolutions are also offered (from two to six species comparisons available), and the comparison frame is easily closed to revert back to the species page. Underneath the comparison tab are three buttons for navigation. The left "previous species" and right "next species" buttons direct to (alphabetically) antecedent and subsequent species pages respectively, while the middle ‘species list’ drop-down tab opens a list of all species available on Sepsidnet. In some species there is one last additional tab that links to additional images. Fig. 3.3 provides a visual guide to using Sepsidnet. To ensure permanency, Sepsidnet is hosted by the server of the Faculty of Science at the National University of Singapore under an account that is associated with the Raffles Museum of Biodiversity Research: http://sepsidnet-rmbr.nus.edu.sg/, and can be sustained easily by any member of the museum familiar with basic HTML. Furthermore, the most essential data (locality and taxonomic information as well as images and illustrations) remain stored in universal formats (text and .tiff images) which can then easily be imported to another platform should the current formats fall out of favour. For example, should the FLASH engine (that runs the Zoomifyer 98 function) become universally obsolete online, the original .tiff images can still be migrated into a more viable format in the future. Conclusion We here present a digital reference collection for 139 species of Sepsidae (Diptera). The collection is a work-in-progress in that it currently comprises ca. 40% of the described species-level diversity but I will continue to add species as they become available. For example, pages for ca. 30 species are currently in preparation. Currently, many species are represented by only one specimen. This does not justice to the intraspecific variability and the morphological differences between males and females, nor are the immature stages shown. Future additions to the database will address these deficiencies. Nonetheless, I believe that Sepsidnet demonstrates that digital reference collections are powerful taxonomic tools because they can provide visual data on many species without the need for establishing physical reference collections. Such 'virtual specimens' are a good substitute to the actual specimens and can even be a better choice given that many physical specimens are in poor condition. Specialists and nonspecialists alike can utilize these images for confirming identification that were initially made based on traditional tools; thus, Sepsidnet is not intended to replace, but complement existing identification keys. Instead it is a tool that should be used after a preliminary identification has been obtained, because keys are often more thorough and a taxonomic worker may miss out characters crucial for diagnosis should they only rely on certain key views of the specimen alone. As such Sepsidnet can also relieve sepsid specialists of the burden of routine identification and in many cases avoid the need for mailing specimens and/or consulting type specimens. I have also used the online 99 images on Sepsidnet to inform the public and other biologists about what kind of structures have to be imaged before sepsid species can be identified based on photographs. This has helped with reducing the number of identification requests based on inappropriate images. As pointed out by many authors (Scoble, 2004; Cranston, 2005; Godfray, 2005; Knapp, 2008), taxonomy could benefit if data were available online. One way to organise these data is in the form of online databases. Credible, well-managed online databases undoubtedly can provide much information in a user-friendly interface and include many images. However, proponents of cybertaxonomy have created a bewildering array of different online databases that are often cluttered with lots of features and empty fields (a prime example being the digital biodiversity website Encyclopedia of Life). Many also lack clearly defined goals and add to the taxonomic impediment by diverting time of taxonomists away from scientific research. I would argue that only tools that help with overcoming the taxonomic impediment should be subject to a digital upgrade. For example, digital reference collections make it easier to confirm species identifications after an initial identification. All subsequent work on species-level revision should also be online but this neither requires re-inventing taxonomic procedures nor are all the conceivable bells and whistles necessary or desirable. For example, until there is evidence for overwhelming demand, I will resist the temptation of adding “Tweet” and “Like” buttons on Sepsidnet pages. 100 [...]... 88 89 90 91 92 93 94 95 96 97 98 99 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 11 0 11 1 11 2 11 3 11 4 11 5 11 6 11 7 11 8 11 9 12 0 12 1 12 2 12 3 12 4 12 5 12 6 12 7 12 8 12 9 13 0 13 1 13 2 Sepsis barbata Sepsis biflexuosa Sepsis coprophila Sepsis cynipsea Sepsis defensa Sepsis dissimilis Sepsis duplicata Sepsis fissa Sepsis flavimana Sepsis frontalis Sepsis fulgens Sepsis kalongensis Sepsis kiribatensis Sepsis kyandolirensis... Meijere 19 06 Duda 19 26 Vanshuytbroeck 19 63 Ozerov 19 86 Melander et Spuler 19 17 Weidemann 18 24 Becker 19 03 Frey 19 08 Vanshuytbroeck 19 63 (Fabricius 17 94) Melander et Spuler 19 17 Ozerov 2 011 (Melander et Spuler 19 17) Ozerov 2003 Ang 2 010 Vanshuytbroeck 19 63 (Robineau-Desvoidy 18 30) Vanshuytbroeck 19 63 Meigen 18 26 Pont 19 87 (Meigen 18 26) (Becker 19 15) Andersson 19 75 Melander et Spuler 19 17 Duda 19 26 (Zetterstedt... (Duda 19 26) Duda 19 26 Silva 19 90 (Brunetti 19 10) (Iwasa 19 84) S eguy 19 38 (Wiedemann 18 30) (Melander et Spuler 19 17) (Melander et Spuler 19 17) (Curran 19 27) (Fall en 18 20) (Duda 19 26) (Zetterstedt 18 47) Meigen 18 30 (Silva 19 91) Ozerov 2004 (Becker 19 19) Ozerov 2004 (de Meijere 19 06) (Walker 18 59) Zuska 19 70 (de Meijere 19 06) (de Meijere 19 06) Vanshuytbroeck 19 61 Vanshuytbroeck 19 61 Vanschuytbroek 19 61. .. Heterocheila buccata 1 Helcomyza mirabilis 1 Willistoniella pleuropunctata Author Used in Chapter(s) Coelopidae McAlpine, 19 91 (Malloch, 19 33) (Hutton, 18 81) (Schiner, 18 68) (Fabricius, 18 05) Haliday, 18 38 (Wiedemann, 18 24) Cresson, 19 14 (Hutton, 19 02) McAlpine, 19 91 Malloch, 19 33 McAlpine, 19 91 McAlpine, 19 91 McAlpine, 19 91 (Lamb, 19 09) McAlpine, 19 91 (Haliday, 18 33) McAlpine, 19 91 McAlpine, 19 91 Heterocheilidae... (Zetterstedt 18 47) (Meigen 18 26) (Staeger 18 44) Ozerov 19 86 Melander et Spuler 19 17 14 4 2,3,4 4 2,3,4 4 2,3,4 2,4 2,3,4 2,3,4 2,3,4 2,3,4 4 4 4 2,3,4 2,3,4,7 2,3,4 4 4 2,3,4 2,3,4 2,3,4,7 2,3,4 2,3,4 4 2,3,4 2,3,4,6 4 2,4 2,3,4,7 47 4 2,3,4 4 2,3,4 2 2,4 2,4 2,4 2,4 4 4 2,46 2,4 4 4 13 3 13 4 13 5 13 6 13 7 13 8 13 9 14 0 14 1 14 2 14 3 14 4 14 5 14 6 14 7 Themira minor Themira nigricornis Themira notmani Themira pusilla Themira... caucasica Themira annulipes Themira arctica Themira biloba Themira flavicoxa Themira germanica Themira gracilis Themira leachi Themira lucida Themira lutulenta Themira malformans Becker 19 07 Strobl 18 93 de Meijere 18 06 (Linnaeus 17 58) Ozerov 19 85 Brunetti 19 10 Haliday 18 38 Becker 19 03 Meigen 18 26 Walker 18 60 Meigen 18 26 Vanshuytbroeck 19 63 Vanshuytbroeck 19 63 Vanshuytbroeck 19 63 Weidemann 18 30 Duda 19 26... Vanshuytbroeck 19 61 Vanshuytbroeck 19 61 Vanschuytbroek 19 61 Vanshuytbroeck 19 61 Vanshuytbroeck 19 61 Ozerov 19 93 (Walker 18 49) (Vanshuytbroeck 19 63) (Vanshuytbroeck 19 61) Ozerov 19 93 Vanshuytbroeck 19 61 Ozerov 19 93 Ang 2 010 Ang et al 2008 Ozerov 19 92 Ang 2 010 (de Meijere 19 13) Ozerov 19 92 Robineau-Desvoidy 18 30 (Schrank 18 03) Duda 19 26 13 3,4 3,4 3,4 2,3,4 2,3,4 4 2,3,4 2,3,4 2,3,4 3,4 2,3,4 3,4 2,4 2,4 4... ruwenzoriensis (Hennig 19 60) Ozerov 2000 (Vanshuytbroeck 19 63) (Vanshuytbroeck 19 63) (Vanshuytbroeck 19 63) (Vanshuytbroeck 19 63) (Vanshuytbroeck 19 62) (Vanshuytbroeck 19 62) (Wiedemann 18 24) (Walker 18 60) (Schiner 18 68) (Bigot 18 57) (Ozerov 19 93) (Thompson 18 69) (Duda 19 26) (Coquillet 19 04) Silva 19 93 (Schiner 18 68) (Loew, 18 61) (Vanschuytbroeck 19 63) Ozerov 19 96 (Ozerov 19 90) (Vanschuytbroeck 19 63) Ozerov 2000... Meijere 19 13) (Wiedemann 18 30) (Duda 19 26) Iwasa et Tewari 19 90 Ozerov 19 92 (de Meijere 19 11) (de Meijere 19 04) Iwasa 19 84 Ozerov 19 97 Ozerov 19 94 (Vanschuytbroeck 19 63) (Duda 19 26) Ozerov 19 96 (Vanchuytbroeck 19 63) 12 3,4 4 4 4 4 4 4 4 2,3,4 4 2,4 2,4 2,4 2,4 4 2,4 2,4 2,4 2 4 4 3,4 3,4 4 3,4 4 2,4 2,4 2,4 2,4 2,4 2,4 2,4 4 4 2,4 2,3 2,3 4 4 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61. .. utilize morphology to address the questions and studies evolutionary biology Evolutionary biology and taxonomy of Sepsidae In my thesis, I use the different morphological techniques described above (i.e., high-resolution photomicrography, scanning electron microscopy and microcomputered tomography) to document the morphology of the dung-fly family Sepsidae (Diptera, Acalyptratae) Sepsidae are a moderately . 2,46 13 0 Themira lucida (Staeger 18 44) 2,4 13 1 Themira lutulenta Ozerov 19 86 4 13 2 Themira malformans Melander et Spuler 19 17 4 15 13 3 Themira minor (Haliday 18 33) 2,4 13 4 Themira. Andersson 19 75 2,4 12 6 Themira flavicoxa Melander et Spuler 19 17 2,4 12 7 Themira germanica Duda 19 26 4 12 8 Themira gracilis (Zetterstedt 18 47) 4 12 9 Themira leachi (Meigen 18 26). Vanshuytbroeck 19 63 4 11 2 Sepsis punctum (Fabricius 17 94) 2,3,4 11 3 Sepsis pyrrhosoma Melander et Spuler 19 17 2,3,4,6 11 4 Sepsis sapaensis Ozerov 2 011 4 11 5 Sepsis secunda (Melander et