CHAPTER __________ Using multiple data-sources to address taxonomic uncertainties Abstract In this chapter, I use numerous data-sources in conjunction to resolve two issues in sepsid taxonomy. In Part I, large differences between the genetic sequences of Sepsis flavimana (Sepsidae) specimens from USA and Germany suggested the possible presence of a 'cryptic' species. A follow-up investigation based on morphology, mating behaviour data and reproductive isolation experiments revealed that there were indeed two biological species. The American specimens were later revealed to be Sepsis pyrrhosoma, a 'cryptic' species that was previously synonymised under S. flavimana. For this study, I performed the morphological and taxonomic analysis, and contributed to the phylogenetic analysis. In Part II, specimens resembling Themira leachi (Sepsidae) were found in Neotropical Cuba, which is puzzling given that the species has only been recorded in the Holarctic. A morphological and genetic comparison with German specimens revealed that the Cuban specimens were indeed T. leachi, which confirms a disjunctive distribution in the species that was likely brought about by synantrophic processes. For this study, I similarly performed the morphological and taxonomic analysis. 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) DNA sequence data have recently gained much popularity in taxonomic research and is generally acknowledged today that they provide important evidence for delimiting species (Meier et al. 2008). DNA data can now be generated at a fast rate, with relatively low cost, and by personnel uninitiated with taxon-specific knowledge required for morphological research (Lee 2000, Scotland et al. 2003). However, increasingly, the widespread use of DNA sequences has also created problems in the form of so-called ‘cryptic species’ that are now routinely proposed when morphology and DNA sequence evidence – at least initially – yield different inferences about species boundaries (Bickford et al. 2007).The use of the term ‘cryptic species’ implies that the unit is already properly diagnosed as a species. However, this is rarely so and in most cases a resolution of the conflict between morphology and DNA sequence information is not even attempted. As a consequence, ‘cryptic species’ are accumulating in the literature and interfere with a proper classification and the A version of this chapter has been published as Tan DS, Ang YC, Lim GS, Ismail MRB, Meier R (2010) From ‘cryptic species’ to integrative taxonomy: an iterative process involving DNA sequences, morphology, and behaviour leads to the resurrection of Sepsis pyrrhosoma (Sepsidae: Diptera). Zoologica Scripta 39, 51-61, where I performed the morphological and taxonomic analysis, and contributed to the phylogenetic analysis. 129 assessment of biodiversity (e.g. resulting in an undercount of species numbers). Here I demonstrate how an iterative process based on multiple sources of data can move a ‘cryptic species’ from being only a putatively new species-level taxon to being formally recognized as a species based on sufficient evidence (Petersen et al. 2007). It is sometimes assumed that this process of iterative taxonomy only requires enough data, but this not necessarily be the case, as the same data may yield different species inferences under different species (Laamanen et al. 2003, Tan et al. 2008). Many authors avoid this issue due to the vitriol related to species concept discussions. However, it is precisely when data are in disagreement that it is important to be explicit about species concepts, because in these cases species concepts can matter (Tan et al. 2008). Here I suggest that the best solution is applying a two-step process. One can first evaluate the available data based on the species concept that is favoured by the authors. Afterwards, the same data can be discussed under alternative species concepts (Laamanen et al. 2003). This approach will ensure that species are clearly defined given that the authors’ opinion based on their species concept will be binding under nomenclatural rules. At the same time the treatment is transparent and allows proponents of alternative species concept to draw their own conclusions. Most species in entomology are recognized based on morphological characters. Sepsid flies are no exception but the use of morphology for some species can be problematic because of the bewildering amount of phenotypic variability present in this family (Pont and Meier 2002). In sepsids most of this variability is related to environmental factors, such as the amount of food available to the larvae (Meier 1995a). In these cases DNA sequences are particularly useful for clarifying species 130 boundaries, because the sequences are not affected by the environmental variables. In other cases the observed intraspecific variability is at least partially genetic (Reusch and Blanckenhorn 1998). Here, DNA sequences can still be used as additional evidence, but any observed sequence variability across allopatric populations can be difficult to interpret (Memon et al. 2006, Petersen et al. 2007, Ang et al. 2008a) because recently diverged species can share barcodes and may thus be incorrectly lumped into one species (Meier et al. 2006, Meier et al. 2008). Similarly, allopatric populations within old species may have distinctly different sequences and DNA evidence may erroneously suggest that they should be split into multiple species (Meier 2008, Meier et al. 2008). Here I demonstrate the value of an iterative approach using multiple sources of data by clarifying the species boundaries of Sepsis flavimana Meigen, 1826. As with many similar cases in recent literature (Bickford et al. 2007), my taxonomic problem started with finding unexpectedly high levels of COI divergence between what appeared to be allopatric populations that were collected from various locations in North America. Based on recently published identification keys (Ozerov 2000, Pont and Meier 2002), these specimens all keyed out to one species, S. flavimana. This particular species is one of the most morphologically variable sepsids, with much of its variability related to size (Munari 1993, Pont and Meier 2002). Not surprisingly, this species has spawned a large number of synonyms [eleven; Ozerov (2005)]. Among others Ozerov (2000) synonymised four Nearctic species with S. flavimana when revising the North American fauna (S. vicaria Walker, 1849, S. pyrrhosoma Melander and Spuler, 1917, S. melanopoda Duda, 1926 and S. kertezsi Duda, 1926). 131 However, the unexpectedly high level of genetic variability that I found within the North American populations of what appeared to be S. flavimana motivated me to re-investigate the morphology in order to test whether these genetically distinct populations may also be morphologically distinct. As additional sources of data, I was also able to study the mating behaviour (under laboratory conditions) and test for reproductive isolation based on cultures that had been established for two genetically distinct populations from North America and Europe. Materials and Methods Collection, rearing and morphology. Sepsis ‘flavimana’ specimens were collected from six American populations and stored in 100% ethanol for subsequent morphological and genetic study (Fig. 5.1). In addition, live specimens from New Orleans (LA, USA), Kevelaer (NRW, Germany), and Ahrensfelde (Schleswig-Holstein, Germany) were reared in laboratory cultures using sucrose syrup as a carbohydrate source and cow dung as a breeding substrate. Cow dung was initially frozen at -80˚C for several days to kill any insects infesting the dung prior to collection. Fly cultures were maintained at 25-28˚ C in 2l plastic containers. Compound microscopy and highfidelity microscopic photography (Visionary Digital ™ BK+ system using a Canon EOS MkIII fitted with Infinity Optics K2 Long Distance Microscope on CF4P3 objective settings) were used to study the morphology of specimens from all eight localities in detail. DNA sequences. A 778bp fragment of cytochrome oxidase c subunit I (COI) was amplified and sequenced, including the DNA barcoding region from 50 individuals 132 representing multiple populations of five nominal Sepsis species (S. biflexuosa, S. duplicata, S. flavimana, S. fissa, and S. ‘pyrrhosoma’) with S. fissa designated as outgroup based on Su et al. (2008). Genomic DNA was extracted from individuals via a modified phenol-chloroform method described by Petersen et al. (2007a), and amplification and sequencing protocols for COI followed Su and colleagues (2008). All sequences were aligned with CLUSTALX 2.01 (Thompson et al. 1997) and the alignment was free of indels. Phylogenetic analyses. Maximum likelihood and maximum parsimony were used to infer the gene-tree for the COI of S. ‘flavimana’ populations and related species. A new technology parsimony search was implemented in TNT 1.1 (Goloboff et al. 2008) with search level 55; the minimum tree length was found 10 times. Node support was assessed through jackknife resampling, with absolute frequency differences and 36% character deletion for 250 replicates. A maximum likelihood bootstrap tree was obtained with GARLI 0.951 (Zwickl 2006). Using the Akaike Information Criterion (AIC), MrModeltest (Nylander 2004) selected the GTR + Γ + I model for COI. The analysis was automatically terminated if there was no improvement of a log likelihood of 0.01 or more after 50,000 generations. Support was obtained as maximum likelihood bootstrap with 250 replicates under the same settings used to obtain the most likely tree. Observations of mating behaviour. Virgin flies were obtained from each culture by isolating a petri-dish of larvae-infested dung from the laboratory colony in an empty container and segregating males and females within hours of eclosion. Sepsid flies, at least in the flavimana group, acquire sexual maturity after two to five days (personal 133 observation). Flies were thus assumed to be sexually mature after five days as adults. To examine reproductive compatibility within and between populations, one virgin male was introduced to a 3.5cm plastic petri-dish containing a single virgin female, and the behaviour of both flies was recorded at 7X – 15X magnification with an analogue video recorder attached to a trinocular Leica MZ16A microscope. Recordings began upon introduction of both flies and ended either after successful copulation or after 60 minutes if copulation did not occur. The analogue recordings were then digitised and analysed frame-by-frame (25 frames per second) using the non-linear video editing software Final Cut Pro. Behavioural elements were then recorded to facilitate comparisons among populations. 10 and 12 mating trials were recorded for the two populations from North America and Europe respectively. Observations of mating behaviour. To examine the reproductive compatibility between populations of S. ‘flavimana’, we attempted to mate males and females from different continents. Five sexually mature virgin flies of each sex were placed in rearing containers under conditions identical to those in which cultures from individual collection localities live and breed successfully. Male and female flies originated from different continents, and the following reciprocal pairings were attempted: Ahrensfelde ♂ × New Orleans ♀; Ahrensfelde ♀ × New Orleans ♂. We also examined reproductive compatibility between the two European populations: Ahrensfelde ♂ × Kevelaer ♀ and Ahrensfelde ♀ × Kevelaer ♂. No flies died during the course of these trials. Each of these five male × female pairings was thrice replicated. The breeding substrate in each container (a 7cm petri-dish containing cow dung) was examined every other day for the presence of fertilized eggs or larvae. Substrate with fertilized eggs or larvae was removed and placed in separate containers for pupation of larvae. Where hybrid flies 134 were obtained, they were again segregated by sex within one day of eclosion to maintain their virginity. We then attempted to back-cross these hybrids with virgin flies from their parental cultures. To ascertain whether flies from failed back-crossing trials were fertile, we attempted to mate them with other flies from their own respective populations. Results Morphology We found two discrete morphotypes from the North American specimens that could be distinguished by a suite of morphological characters. One morphotype was indistinguishable from all European specimens of Sepsis flavimana, while the other morphotype has the following distinguishing features (Fig. 5.2): (1) the flies are consistently lighter in colour (especially on the thoracic pleura, face, gena and legs cf. Fig 2. A, G vs. H); (2) the male fore-tibia lacks a distinct ventro-basal bump on the tibia (C) as compared to the European morphotype (J); (3) the epandrium and base of the surstylus of the male is light in colour and only the tip is dark (lateral view A, dorsal view F); (4) the surstylus has a sub-medial tooth (D). Features and are consistent with the description of S. pyrrhosoma by Melander and Spuler (1917) which mentions that the species is “largely reddish along the sides” with “face and cheek yellowish,” and with a male foretibia “slightly decreasing in size towards the tip and bearing a very weak and setulose tubercle on the underside near the base.” Features 1–3 are also visible on the holotype of S. pyrrhosoma (W. Mathis, pers. comm.). For convenience, I refer to this as the ‘pyrrhosoma’ morphotype. North American 135 Figure 5.1: Consensus tree of Sepsis flavimana group. Parsimony jackknife percentiles are given above branches and maximum likelihood bootstrap percentiles below. 136 Figure 5.2: A–G. Sepsis pyrrhosoma (♂ unless otherwise noted). A. Habitus, lateral view, showing hypopygial capsule (hyp). B. Fore-femur, posterior view. C. Fore-tibia, posterior view. D. Surstylus, dorsal view. E. Thorax, lateral view, showing pruinosity pattern on postprotonotum (ppn), preepisternum (pest), anepisternum (aepst), ketepisternum (kepst), anepimeron (aepm), katatergite (kat), meron (m) and metepimeron (mepm). F. Postabdomen, ventral view, showing 4th sternite (4th st.) and hypopygial capsule. G. ♀ habitus, lateral view. H–K. Sepsis flavimana (♂). H. Habitus, lateral view, showing hypopygial capsule. I. Fore-femur, posterior view. J. Fore-tibia, posterior view. K. Surstylus, dorsal view. Scale bars (A, E, G, H): 1mm; (B, C, F, I, J): 0.5mm; (D, K): 0.1mm 137 THESIS CONCLUSIONS The future of morphology is exciting. Traditionally, morphology has been associated with long hours peering through a microscope and painstakingly detailing minute morphological features with the help of a camera lucida – especially if the morphologist happens to be an entomologist. However, there are now many new opportunities for a morphologist given all the recent technological advances in bioimaging and information technology. I hope that the studies in my thesis have illustrated this point. Here, I have used a variety of bioimaging tools to acquire detailed morphological data for studying the evolutionary biology, systematics, and taxonomy of Sepsidae. I also demonstrate how information technology can be used to digitize and disseminate taxonomic information via the internet. The pronounced sexual dimorphism in Sepsidae has been known for some time, and it has generated much interest. However, the morphology of Sepsidae were hitherto documented based on more traditional morphological methods. For example, studies of the sepsid sternite appendages were based on drawings and specimen dissections (Eberhard 2001d). This destroys the specimen and prevents future analysis. In order to illustrate how morphology can take advantage of recent developments in bioimaging techniques, I explored the use of a non-invasive technique, SRµCT to document the internal structures. More importantly, the volume reconstructions provide valuable 3D data which would otherwise be lost in the process of a physical dissection. Additionally, these data can be presented as 3D models which allow the user to manipulate and virtually 'dissect' the specimen, based on a embedded PDF object. I 181 used these data to describe how a ‘boring’ structure such as a sternite plate can evolve into a complex, moveable appendage, which is testament to the creative powers of sexual selection. In further exploring the powers of sexual selection, I also investigated another dimorphic structure that is under sexual selection: the sepsid male foreleg. It allowed me to quantify how sexual selection accelerates morphological change when compared to viability selection only, but I also found that only sexual selection of contactstructures led to fast change while structures that not physically interact with the female body evolve relatively slowly. This study provides further evidence for the creative powers of sexual selection. After all, the ventral region of the foreleg and the sternite region (where the sternite appendages are found) are in close contact with the female body. The male foreleg study also documented how the use of photomicrography has made it easier to quickly document a large number of specimens: 69 sepsid and 20 coelopid species were studied. To acquire the same data with a camera lucida would have been slower and details of chaetotaxy and microsetosity would have been lost. In the second half of the thesis, I attempt to make contributions to a “taxonomy in the 21st century”. I believe that it will be dominated by virtual specimens, abundant image data, and wikis as long as they are automatically generated by formal, descriptive papers. In this section of the thesis, I use similar techniques as in the evolutionary studies. Using photomicrography, I created ‘virtual specimens’ suitable for online dissemination in the form of a digital reference collection (Sepsidnet). When combined with new software that can facilitate the dissemination of large images 182 without requiring large bandwidth, Sepsidnet is a convenient identification tool for sepsid taxonomists. Photomicrography also becomes an important source of information in more traditional species description. Modern digital photography allows for the capture of abundant morphological information in the form of habitus images of multiple views. It can help safeguard a description from becoming inadequate should the diagnostic features listed in its treatment become insufficient for species delimitation once new species are found or new character systems are explored. Data abundance is not limited to morphology. I also demonstrate how morphology, when used in conjunction with other sources such as DNA data, mating behaviour records and reproductive isolation, can resolve some of the issues that are more commonly faced by taxonomists these days. This includes so-called ‘cryptic’ species based on DNA data. However, the increasing availability of modern bioimaging techniques does not mean that traditional morphological techniques are obsolete. For example, scientific illustrations remain an important mean for illustrating specimens, and they have the advantage that the illustrator can highlight pertinent details and summarize intraspecific variability. Drawings will thus remain an essential component of many species descriptions and modern bioimaging techniques are only a much welcome addition to the repertoire given that they can generate fairly accurate images of individual specimens rapidly and without the requirements of taxonomic expertise in the subject group. In addition to the digital reference collection, I also presented an example of species descriptions that become more accessible by the use of simultaneous 183 publication in journals and wikis. Wiki-based platforms such as Wikipedia (www.wikipedia.org) have many advantages, mainly because they are extremely efficient at disseminating information. This has led to dedicated species databases such as Encyclopedia of Life (EoL; http://eol.org/) to even harvest data from such resources, albeit with a disclaimer on the authority of the content, especially if the wiki is open to editing by the public (Page 2010). For example, Perochaeta cuirassa (Diptera), a new species described in my Zookeys publication, has a taxonomic entry in EoL (http://goo.gl/z9IcRf). The data in this entry was automatically trawled by EoL from the Species-ID entry. Another interesting feature of wiki-based platforms is that they allow edits on an entry, which can protect taxonomic work from becoming outdated should new data become available after publication. Of course, this editing feature is not without controversy - most wikis allow any author to make contributions, and the ease in which a contributor can edit an article can quickly yield vicious 'edit wars' when contributors from rival views constantly make changes to each other's edits on a page [e.g., Viegas et al. (2004)]. However, this is less likely to be a problem when wiki entries are linked to an authoritative source (such as a published article), and edits are only allowed by registered contributors (i.e., professional scientists, students, and amateurs but no anonymous access). Furthermore, wikis keep a freely-available and complete history of edits, which also becomes an impartial reflection of debate should an 'edit war' happen (Page 2010). With all these new opportunities opened by developments in bioimaging and data-sharing over the internet, it is no wonder that many morphologists are getting excited and exploring new avenues and aspects to morphological study. 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VA, and Dyar Pasture, GA, are morphologically indistinguishable from European S flavimana, and are henceforth referred to as the ‘flavimana’ morphotype Molecular Data We obtained COI barcode sequences of ca 778 bp from 50 specimens (GenBank accession numbers EU 435 804, EU 435 807, EU 435 808, EU 435 818, GQ354410, GQ388 730 – GQ388774) The parsimony analysis found 125 trees with a length of 211 steps and the. .. grassland along Leake Avenue near Mississippi River, New Orleans, LA), ca 5m ASL, 29˚ 55' 48 .34 " N 90˚ 8' 4.17" W 2008 (Coll R Meier); in Raffles Museum of Biodiversity Research, Singapore (RMBR) Other samples from Raleigh, NC, New York and Athens, GA Etymology The specific name first given by Melander and Spuler in their original description of the species (Melander and Spuler 1917), derived from the. .. reminiscent of their home climate (McGlynn 1999) Here I report the occurrence of a primarily Holarctic dipteran species, Themira leachi (Meigen), in Neotropical Cuba This discovery suggests that the species may have a large disjunct distribution, as the next closest record lies almost 3, 500 km to the north in Nearctic Newfoundland, Canada (Ozerov 1998) The genus Themira comprises 35 species and belongs to the. .. fore-legs and claspers (Pont and Meier 2002) Sepsis pyrrhosoma could therefore be easily mistaken as a S flavimana which probably explains why the former had been synonymised In order to further strengthen the morphological and genetic evidence for my hypothesis that S 145 pyrrhosoma is a valid species, we studied the mating behaviour and reproductive isolation and the morphological evidence corroborates... developed and have been modified into a raised, anteriorly open crater on the 2nd sternite and a pronounced protrusion on the 3rd sternite These sternite modifications are unique to T leachi and the only difference between the specimens is minor (more hook-like protrusion on the European specimen) Overall, the foreleg, sternite, and hypopygial capsule morphology are very similar between the European and. .. specimens using the DNA extraction, amplification, and sequencing protocols described in Su et al (2008) These sequences were submitted to Genbank (EU 831 274 – EU 831 277) and compared to a known sequence of T leachi from Europe (Genbank: EU 435 8 23) as well as COI sequences for ten other Themira species (Su et al 2008) Pairwise distances between the European and Cuban sequences were 0.5% to 0.8% Whether such... platform, SpeciesID I then conclude with a discussion of the distribution of Perochaeta and the three Sepsis species 155 Introduction 5 There are numerous advantages of wiki-based platforms for taxonomy This includes good accessibility and ability for regular, small edits In this chapter, I describe three new species and present two new records of Sepsidae for Vietnam in Zookeys These taxonomic data... listed at the start of each taxonomic description Such an arrangement greatly facilitates access to the species descriptions For example, there is no need to download and read the actual journal article in full if only the description is needed Furthermore, this allows other biodiversity data aggregators such as Encyclopedia of Life to harvest and display the information In fact, illustrations and other... lobo and can only be reliably distinguished from the latter based on the 4th sternite [cf P cuirassa (Fig 6.1) and P lobo (Fig 6.6)]: The sternite in P cuirassa lacks distinct lobes on the posterior end of the 4th sternite, while the sternite brush is thick and squat (as opposed to long and thin in P lobo), and the main scleral plate is much broader (long as wide) than in P lobo (twice long as wide) The. .. 6.4) and P lobo (Figs 6.7 – 6.9)] is also distinct, with P cuirassa bearing a large median, decussating protrusion on the dorsal side of the surstylus, while P lobo has a sub-median protrusion on the ventral side of the surstylus Perochaeta cuirassa is also readily distinguished from all other Perochaeta species based on the morphology of the 4th sternite and hypopygial capsule: The sternites brush of . G vs. H); (2) the male fore-tibia lacks a distinct ventro-basal bump on the tibia (C) as compared to the European morphotype (J); (3) the epandrium and base of the surstylus of the male is light. sequences of ca. 778 bp from 50 specimens (GenBank accession numbers EU 435 804, EU 435 807, EU 435 808, EU 435 818, GQ354410, GQ388 730 – GQ388774). The parsimony analysis found 125 trees with a length of. (RMBR). Other samples from Raleigh, NC, New York and Athens, GA. Etymology. The specific name first given by Melander and Spuler in their original description of the species (Melander and Spuler