LABOULBENIALES - Agentschap voor Natuur en Bos

LABOULBENIALESEXPLORING AND TESTING DNAEXTRACTION PROTOCOLS, CARRIONBEETLE HOSTS AND SPECIES IN „DEKAAISTOEP‟ (THE NETHERLANDS)byDanny Haelewaters© February 2011, Faculty of Sciences – Research group MycologyAll rights reserved. This master thesis contains confidential information andconfidential research results that are property to the UGent. The contents ofthis master thesis may under no circumstances be made public, nor completeor partial, without the explicit and preceding permission of the UGentrepresentative, i.e. the supervisor. The thesis may under no circumstances becopied or duplicated in any form, unless permission granted in written form.Any violation of the confidential nature of this thesis may impose irreparabledamage to the UGent. In case of a dispute that may arise within the contextof this declaration, the Judicial Court of Gent only is competent to be notified.Pictures front page: (from left to right) Hesperomyces virescens from Harmoniaaxyridis (specimen 1c, thallus 1) collected in „De Kaaistoep‟ (Tilburg, TheNetherlands), new for the mycoflora of The Netherlands; picture by DannyHaelewaters (2010). Carrion beetle Saprinus semistriatus; picture by UdoSchmidt (2008). Laboulbenia flagellata from Agonum assimile (specimen DH4,thalli G and H) collected at Schellandpolderdijk (Hingene, Belgium); picture byDanny Haelewaters (2009).

PART IILABOULBENIALES: EXPLORING AND TESTINGDNA EXTRACTION PROTOCOLS311. INTRODUCTION 321.1. DIFFICULTIES FOR DNA EXTRACTION OF LABOULBENIALES 321.2. GENBANK 321.2.1. THE GENBANK SEQUENCE DATABASE 321.2.2. THE SEARCH FOR SEQUENCES OF LABOULBENIALES/LABOULBENIOMYCETES INGENBANK 322. MATERIALS & METHODS 342.1. FUNGUS, HOST AND ORIGIN 342.2. MORPHOLOGICAL PROTOCOL AND DNA EXTRACTION 342.2.1. INTRODUCTION 342.2.2. PROTOCOL I (BASED ON WEIR & BLACKWELL, 2001b) 342.2.3. PROTOCOL II (BASED ON WEIR & BLACKWELL, 2001a) 352.2.4. PROTOCOL III: PUREGENE KIT A 352.2.5. PROTOCOL IV: DNEASY EXTRACTION KIT 352.2.6. PROTOCOL V: DIRECT PCR 362.3. PCR AMPLIFICATION 362.3.1. ITS 362.3.2. POST PCR 372.4. SEQUENCING AND SEQUENCE ANALYSIS 372.4.1 SEQUENCING 372.4.2 SEQUENCE ANALYSIS 373. RESULTS 393.1. DNA EXTRACTION 393.2. AMPLIFICATION 393.2.1. PRIMER PAIR ITS5/ITS4-A 393.2.2. PRIMER PAIR ITS5/ITS2 404. DISCUSSION 415. CONCLUSION AND SUGGESTIONS 44PART IIILABOULBENIALES OF CARRION BEETLES:A PRELIMINARY STUDY451. INTRODUCTION 461.1. FORENSIC ENTOMOLOGY 461.2. ANIMAL CADAVERS 461.2.1. SOURCES OF BIODIVERSITY 461.2.2. THE „ROTTING‟ OF A CADAVER 461.2.2.1. The fresh stage 471.2.2.2. The bloat stage 471.2.2.3. The early decomposition 47P a g e | 2

PART IVPRELIMINARY CHECKLIST OF LABOULBENIALES IN„DE KAAISTOEP‟771. INTRODUCTION 781.1. NATURAL LANDSCAPE DE KAAISTOEP 781.1.1. DESCRIPTION OF THE SITE 781.1.2. BIODIVERSITY IN DE KAAISTOEP 781.1.2.1. Plants and fungi in De Kaaistoep 791.1.2.2. Animals in De Kaaistoep 791.2. LABOULBENIALES IN THE NETHERLANDS 791.2.1. CONTRIBUTIONS OF MIDDELHOEK 791.2.2. CONTRIBUTIONS OF BOEDIJN 812. MATERIALS & METHODS 822.1. HOSTS 822.1.1. COLLECTION OF HOSTS 822.1.1.1. Pitfall traps 822.1.1.2. Window traps 822.1.1.3. Bands and rings 822.1.1.4. Malaise trap 832.1.1.5. Light traps 832.1.2. IDENTIFICATION OF HOSTS 832.2. LABOULBENIALES 832.2.1. SCREENING FOR AND IMBEDDING OF LABOULBENIALES 832.2.2. IDENTIFICATION OF LABOULBENIALES 842.3. PRESENTING RESULTS 843. RESULTS 863.1. PARASITE-HOST LIST 863.2. HOST- PARASITE LIST 864. DESCRIPTION OF THE SPECIES AND DISCUSSIONS 874.1. LABOULBENIA CALATHI T. MAJEWSKI 874.2. LABOULBENIA EUBRADYCELLI HULDÉN 884.3. LABOULBENIA PEDICELLATA THAXT. 894.4. LABOULBENIA VULGARIS PEYR. 914.5. HAPLOMYCES TEXANUS THAXT. 924.6. HESPEROMYCES VIRESCENS THAXT. 934.7. RHACHOMYCES LASIOPHORUS (THAXT.) THAXT. 944.8. STICHOMYCES CONOSOMATIS THAXT. 954.9. STIGMATOMYCES MAJEWSKII H.L. DAINAT, MANIER & BALAZUC 965. CONCLUSION AND SUGGESTIONS 98PART VREFERENCES99P a g e | 4

ACKNOWLEDGEMENTSThe work presented in this master thesis is the result of many years of biologicalinterest, not just the few that constituted my studies.I am very grateful to my supervisor, Annemieke Verbeken, for what she hastaught me throughout my studies. Her enthusiasm and interest for mycologyplayed a huge role in me choosing my study path. Her expertise andknowledge have taught me more than I could ever have expected.I would also like to thank my second supervisor, André De Kesel, for teachingme all about the fascinating Laboulbeniales. His passion has inspired me tofinish this work and even start new chapters.My thanks go out to Paul Van Wielink and Natuurmuseum Brabant (Tilburg) forgiving me the chance to work in a group of professional entomologists. Theidentification of carrion beetles would not have been so pleasant at any otherplace. Special thanks for involving me in the project of „De Kaaistoep‟.I wish to thank Dirk Raes (Agentschap voor Natuur en Bos) and Yves Braet andSofie Vanpoucke (National Institue for Criminalistics and Criminology) forinvolving me in the project Dood doet Leven and for the possibility to work atthe laboratories of the National Institute for Criminalistics and Criminology.I have to thank Jorinde Nuytinck, Kobeke Van de Putte, Luc Crêvecoeur, MarcDelbol, Jan Willem van Zuijlen, Hans Huijbregts, Pedro Crous, Walter Rossi andMeredith Blackwell for sharing their expertise with me. More than once, onlysmall issues were bothering me. However, without their help I was naught.I cannot forget my friends who have supported me throughout difficult times,who were always there when I needed a word. Special thanks to Steven“Sevenless” Haesendonckx (doing my very best to see you becoming doctor),Evelyn Theuninck (we will meet again in Chantemerle-lès-Grignan), LiesbethVerlinden (for some Mazurka distraction), Lindsay De Decker (loving you, in anasexual way, although… ^^), Frederik Byttebier (you gave me inspiration and –recently – a new dream to live for), Annemieke Krammer (dreaming aboutcandles, reincarnation, and lots – LOTS – of paper), Merel Van Den Broucke(my best witch friend), Evelien De Wilde (for giving me a home) and all friendsof the Gentse Biologische Kring (I will never forget you).I will always be indebted to my parents, who have supported me at all times.Thanks dad;-) And mum, I hope that – wherever you are – you can be proud.Your belief in me, even now, was the outstanding factor always helping me toget through.P a g e | 5

SAMENVATTINGINLEIDINGLaboulbeniales zijn obligaat ectoparasitaire ascomyceten die voorkomen opArthropoda, meestal insecten. Er zijn meer dan 2000 soorten beschreven in141 genera. Laboulbeniales hebben geen mycelium; ze vormen kleine thalli,opgebouwd uit een receptaculum met perithecia, aanhangsels en antheridia.Laboulbeniales zijn gastheerspecifiek. Het parasiteren van een bepaaldegastheersoort is afhankelijk van zowel de eigenschappen van het integument,het microklimaat ter hoogte van het oppervlak van de gastheer en debeschikbaarheid van nutriënten als de eigen voorkeuren voor een bepaaldmilieu. Er zijn geen vrijlevende levensstadia. Enkel seksuele reproductie isgekend. Aandacht wordt besteed aan de morfologie, de classificatie en deecologie, met bijzondere aandacht voor de verschillende vormen vanspecificiteit van Laboulbeniales.BESTUDEERDE ONDERWERPEN EN CONCLUSIESSpecies-gerelateerde taxonomische problemen bij Laboulbeniales kunnenenkel sluitend bestudeerd worden door het combineren van klassiekemorfologische methoden en moleculaire protocollen. Vier DNAextractieprotocollenwerden getest; WEIR & BLACKWELL (2001a) en WEIR &BLACKWELL (2001b) vormden de basis voor twee protocollen, de anderen warengebaseerd op de Qiagen Puregene Kit A en de Qiagen Dneasy Plant Mini Kit.Bij het vijfde extractieprotocol werden thalli zonder enige modificatiegeamplificeerd.Er werden geen sequenties van Laboulbeniales gevonden; we kregen enkel temaken met contaminanten uit verschillende klassen. Één contaminant waszelfs een basidiomyceet. Op basis van deze resultaten kan wordengeconcludeerd dat de beschrijvingen in de reeds vermelde referenties (WEIR &BLACKWELL 2001a, 2001b) volledig noch betrouwbaar zijn.Suggesties voor verder onderzoek worden gegeven.Gericht onderzoek naar Laboulbeniales op kadaverkevers werd tot op hedennog niet verricht. In dit werk werden in het Zoniënwoud (België) kevers(Coleoptera) bemonsterd in de directe omgeving van kadavers; dit gebeurdein het kader van het project Dood doet Leven (Agentschap voor Natuur enBos). Additioneel materiaal werd bezorgd door het Nationaal Instituut voorCriminalistiek en Criminologie. Alle kevers werden geïdentificeerd engescreend op de aanwezigheid van Laboulbeniales.In deze studie werden geen Laboulbeniales gevonden. Mogelijkerwijs hebbenLaboulbeniales door hun eigen milieuvoorkeuren moeilijkheden om zich opkadaverkevers te ontwikkelen. Kadaverkevers beschikken namelijk oververschillende soorten habitats.P a g e | 6

Voorlopig is Asaphomyces tubanticus (Middelh. & Boelens) Scheloske de enigebekende soort van Laboulbeniales die systematisch voorkomt opkadaverkevers. Deze soort werd al waargenomen in België (RAMMELOO, 1986;DE KESEL & RAMMELOO, 1992; DE KESEL, 1997). Rhadinomyces pallidus Thaxt. werdook reeds geobserveerd op kadaverkevers (MAJEWSKI, 2003), maar is nog nietbekend in België.Suggesties voor verder onderzoek worden gegeven.In Nederland is er sinds de jaren ‟40 (BOEDIJN, 1923; MIDDELHOEK, 1941, 1942,1943a, 1943b, 1943c, 1943d, 1945, 1947a, 1947b, 1949) geen onderzoek meerverricht naar Laboulbeniales. „De Kaaistoep‟ wordt beschouwd als eenhotspot van biodiversiteit in Nederland en leent zich dan ook uitstekend voorgericht onderzoek naar Laboulbeniales. In dit werk werden insecten uit decollectie van het Natuurmuseum Brabant (Tilburg, Nederland), verzameld inDe Kaaistoep, gescreend op de aanwezigheid van Laboulbeniales.Deze bijdrage resulteerde in negen soorten Laboulbeniales, waarvan zessoorten nieuw zijn voor Nederland. Het gaat om Laboulbenia calathi T.Majewski, Laboulbenia eubradicelli Huldén, Hesperomyces virescens Thaxt.,Rhachomyces lasiophorus (Thaxt.) Thaxt., Stichomyces conosomatis Thaxt. enStigmatomyces majewskii H.L. Dainat, Manier & Balazuc.We verwachten dat er nog meer nieuwe soorten voor Nederland zullenworden ontdekt in De Kaaistoep, naarmate het onderzoek zich meer opLaboulbeniales zal richten.P a g e | 7

PART IGENERAL INTRODUCTIONTHESIS OUTLINEAIMSP a g e | 8

1. LABOULBENIALES1.1. DEFINITION OF LABOULBENIALESLaboulbeniales include over 2000 species in 141 genera (SANTAMARIA, 1998; KIRKet al., 2001; WEIR & BLACKWELL, 2005) of obligate, ectoparasitic fungi, which liveassociated with arthropods, mostly true insects. Laboulbeniales have nomycelium; their thalli are small and of determinate growth, bearing antheridiaand perithecia on a receptacle with appendages (TAVARES, 1985). Only sexualstages are known. Laboulbeniales are host-specific (THAXTER, 1896; SCHELOSKE,1969; TAVARES, 1985; MAJEWSKI, 1994; DE KESEL, 1996, 1997).The knowledge on Laboulbeniales is rather recent and has increased slowly.Large parts of their biodiversity are still unknown and many questions stillunresolved.1.2. HISTORICAL BACKGROUNDIn the 1840s, two French entomologists, Joseph Alexandre Laboulbéne andAuguste Rouget, did the earliest observations on Laboulbeniales resulting in afirst publication on the Laboulbeniales (ROUGET, 1850, ref. in TAVARES, 1985). Atfirst, the structures on Brachinus (Coleoptera, Carabidae) were thought to besupernumerary antennal segments. ROUGET (1850, ref. in TAVARES, 1985)suggested that they were living organisms. The recognition as fungi came in1852 and the genus Laboulbenia was raised within the Pyrenomycetes – anextensive group of Ascomycetes (ROBIN, 1853). Later on, specimens onwingless dipteran parasites of bats were still mistakenly described asArthrorhynchus, an acanthocephalan worm, by KOLENATI (1857).MAYR (1853) thought that the „hairlike structures‟ on Nebria (Coleoptera,Carabidae) were chitinous. Suggesting that they were outgrowths of theinsect‟s integument, he noticed differences between those structures onyounger and older beetles. On older individuals, there was always a variableswelling on the hair, whereas no such swelling was present on the hair ofyounger beetles.PEYRITSCH (1871) made extensive observations on the structure anddevelopment of the housefly Laboulbenia (L. muscae Peyr.). PEYRITSCH (1871)redescribed Arthrorhynchus as Laboulbenia nycteribiae Peyr. He laterestablished the family Laboulbeniaceae in the Ascomycetes (PEYRITSCH, 1873)and laid a solid foundation for our understanding of the biology ofLaboulbeniales (PEYRITSCH, 1875), based on observations of laboratory coloniesof houseflies and studies of Laboulbeniales in the field.A systematic study of the Laboulbeniaceae was begun by Roland Thaxter in1890, when he published his first in a series of papers describing hundreds ofnew species. In 1896, THAXTER‟S first monographic volume on theLaboulbeniaceae appeared. It was particularly a summary upon all previouswork (TAVARES, 1985). The monograph of Thaxter – five volumes, published in1896, 1908, 1924, 1926 and 1931 – constituted the basis for later studies of thegroup. Unfortunately Thaxter died only one year after the publication of theP a g e | 9

fifth volume, therefore the sixth volume – which was meant to be a synthesis –was never prepared. More than 40 years study resulted in the description of103 genera, approximately 1260 species and 13 varieties (BENJAMIN, 1971). Upto now, about two thirds of all the known species have been described byThaxter (HULDÉN, 1983).THAXTER (1896) first separated the family of the Laboulbeniaceae into two„groups‟ (Table I below): the Endogenae and the Exogenae (based on thedevelopment of spermatia in the antheridia). The Endogenae included twoorders: the Laboulbenieae (with simple antheridia; 15 genera) and thePeyritschielleae (with compound antheridia; 11 genera). Those orders wouldcorrespond to tribes in modern classification systems. In the second volumeTHAXTER (1908) replaced the terms Endogenae and Exogenae by the subordinalnames Laboulbeniinae and Ceratomycetinae (Table I below); the majorsubdivisions from the former group Endogenae were substituted by families,Laboulbeniaceae and Peyritschiellaceae. Thaxter did not recognize aseparate family within the Ceratomycetinae. The name Ceratomycetaceae –now universally accepted – first has been introduced by MAIRE in 1916.Volume I Family LaboulbeniaceaeTable I: Classification in Thaxter‟s monographs I and II„Group‟ Endogenae ExogenaeOrder Laboulbenieae PeyritschielleaeVolume II Order LaboulbenialesSuborder Laboulbeniinae CeratomycetinaeFamily Laboulbeniaceae PeyritschiellaceaeP a g e | 10After Thaxter‟s death in 1932, various authors contributed to the knowledge bypublishing some short papers and/or describing new species and genera:Spegazzini, Picard, Maire, Chatton, Cépède, Giard and Istvánffi. The majorityof the numerous publications that have appeared were regional studies,collection reports and rather short (TAVARES, 1985; DE KESEL, 1997). However,when considered together, they have added extensive data on thedistribution of these fungi.Some researchers suggested a relationship between red algae andLaboulbeniales and thought that Dikaryomycota derived from red algae, withthe Laboulbeniales as an intermediate step KARSTEN, 1869; SACHS, 1874). Sexualreproductive structures, including a highly differentiated trichogyne and theabsence of mycelium, are unusual ascomycete characteristics andsuperficially resemble floridean algal features (BLACKWELL, 1994). CÉPÈDE (1914)believed the Laboulbeniales as a whole occupied a special place among thefungi; he placed them in the Phycascomycetes, a name he proposedbecause of the superficial similarities both to the Ascomycetes and to theRhodophyta (red algae). Until geneticists unquestionably proved thatAscomycetes derived from Chytridiomycetes-like ancestors, this “Red Algal (orFloridean) Hypothesis” persisted (JAMES et al., 2006). The analysis of JAMES et al.(2006) suggests that several clades of the Chytridiomycota (true fungi, stillproducing zoospores) form a paraphyletic assemblage at the base of the

Fungi, consistent with the view that the presence of flagella is an ancestralcharacter state in the Fungi.The first observation of a member of the Laboulbeniales in Belgium took place by Cépède andBondroit, in October 1910. It concerned a Laboulbenia rougetii Mont. & C.P. Robin, parasitizingBrachynus crepitans (COLLART, 1947).1.3. MORPHOLOGY OF THE THALLUS (TA VA R E S, 1985; MA J EWSKI, 1994)Figure I: Thallus of Laboulbenia calathi (from Calathus melanocephalus, specimen 3a, thallus 1).Abbreviations: f foot, I + II + III cells of primary axis of the receptacle, IV + V additional cells of thereceptacle, andr androstichum (complex of cells III-V), cell e insertion cell, ia inner appendage, oaouter appendage, VI stalk cell of the perithecium, VII secondary stalk cell, s stalk, p perithecium.Scale bar = 50 µm. Picture by Danny Haelewaters (2010).1.3.1. INTRODUCTIONThe thallus of the Laboulbeniales develops from a bicellular ascospore, by arestricted number of mitotic divisions, therefore resulting in a thallus with arestricted number of cells. The primary septum separates the larger andsmaller cell of the ascospore. It is often detected by its thickness and colour(slightly darkened).The main axis of the thallus is formed by the receptacle, which is attached atthe host‟s integument by means of a foot. Both the receptacle and the footoriginate from the larger – lower – cell of the ascospore. Additional divisions ofsome receptacle cells account for the perithecia, the only spore formingstructures within the Laboulbeniales (no asexual spore formation present). Thesmaller cell of the ascospore produces the primary appendage system – this isa prolongation of the receptacle axis. On the primary appendage, or on itsbranches, there is formation of antheridia, which produce spermatia.Thalli have a bilateral symmetry. This kind of symmetry occurs in many groupsof animals, but only exceptionally in plants and fungi.P a g e | 11

1.3.3. THE PERITHECI UMAscospores originate in the perithecium. TAVARES (1967, 1985) used peritheciumdevelopment and external structure of the perithecial wall to describe highertaxa. The perithecium is formed by divisions of one of the primary orsecondary receptacle cells. This gives at once the differentiation betweenprimary and secondary perithecium. In species without secondarily dividedreceptacle cells, the perithecium arises by divisions of cell II.We can distinguish the stalk and the perithecium:The stalk comprises two cells, the stalk cell (cell VI) and the secondary stalkcell (cell VII). The stalk cell is usually clearly visible; it subtends the basalcells of the perithecium (m, n and n’). Cell VII is directly derived from thestalk cell and is situated abaxially.The perithecium is more or less elongated and narrowed distally.Sometimes it is clearly differentiated into a rounded venter and a narrowneck (apex), terminating in an ostiole. The apex can rarely beasymmetric, so that the ostiole is situated subapical. The perithecial wallcells surrounding the ostiole often form distinct lips.The perithecium always consists of a well determined number of cells.Exceptions on this are genera in Herpomycetidae and Ceratomycetidae. Thelowest cells of the perithecium, just above the stalk cell and secondary stalkcell, are the basal cells. Usually, there are three such cells: the cell m is formedfrom cell VI, the cells n and n’ originate from cell VII. Divisions of cells n and n’result in three vertical rows of perithecial wall cells. The fourth row is formed bydivisions of cell m.The perithecial wall is double, i.e. composed of an interior and exterior part.The number and shape of the external wall cells plays an important role intaxonomy. The cells of the external wall are arranged in four vertical rows. Inprimitive genera, the number of cells in each of these rows is large; in othergroups, such as the Laboulbeniaceae, the vertical rows are composed of 4-5superposed cells. In contrast to the primitive genera, the cells in theLaboulbeniaceae are usually unequal in height, with the lower ones beingelongated and the apical ones being shortened.The basal part of the interior part of the perithecium contains the ascogonium.After fertilization through the trichogyne, it undergoes divisions resulting in oneto numerous ascogenous cells. The ascogenous cells give rise to new ascicontaining ascospores. The ascus wall is very thin; it disintegrates while still inthe perithecium during spore maturation.The trichogyne is an external thin appendage-like outgrowth of the youngperithecium. It is the place through which fertilization proceeds, a process inwhich different steps are recognized. At first, the trichophoric cell connectsthe trichogyne with the ascogonium within the perithecium. At an early stageof development, the perithecium is equipped with a trichogyne that receivesspermatia from the antheridia (TAVARES, 1985; DE KESEL, 1997). The next step inthe fertilization process is the fusion between the nuclei of the spermatium andthe ascogonium. The trichogyne deteriorates early after fertilization, except forthe basal part which often remains as a more or less prominent scar on thesurface of the mature perithecium.P a g e | 13

Figure II: Thallus of Laboulbenia calathi (from Calathus melanocephalus, specimen 3c, thallus 1),showing the trygogyne (arrow). Scale bar = 50 µm. Picture by Danny Haelewaters (2010).1.3.4. THE APPENDAGESThe upper – smaller – cell of the germinating ascospore produces the primaryappendage system only. The primary appendage is usually a directcontinuation of the receptacle axis. Its upper part sometimes carries a spine,the remains of the pointed spore apex. Extension of cytoplasma intoappendage branches takes place just below the spore apex, which is spinose.The failure of the spore apex to elongate directly upwards suggests that thespinose tip has a wall incapable of expansion. In Laboulbenia andStichomyces, on the contrary, no trace of the spore apex remains at maturity.The upper cell of the ascospore elongates directly upwards in these twogenera.Primary appendages present different ways of appearance. In some generathey consist of only one or two cells. Extensive primary appendage systemsoccur in Laboulbenia and some other genera. In Dipodomyces monstruosusand – probably – Chaetarthriomyces flexatus the primary appendagebecomes aborted.The basal cell of the primary appendage of Laboulbenia, also called theinsertion cell (cell e), is darkened and flattened. This insertion cell gives rise toand supports the outer and inner appendages. The outer (abaxial)appendage is simple or branched but usually sterile, whereas the inner(adaxial) appendage is shorter and bears flask shaped antheridia. The outerappendage may break off as the thallus develops.Little is known about the function of the sterile appendages. It is suggestedthat they play a role in water balance of the thallus (DE KESEL, 1996). However,no specific research has yet been done in this direction.The secondary appendages are less diverse. They include all appendages,derived from the lower spore cell. Secondary appendages can be simple orbranched, and sterile or bearing antheridia. In some genera, the secondaryappendage is two-celled. The distal cell is separated from the basal one by ablack septum and often breaks off.P a g e | 14

The identity of appendages, together with the way of regenerating in maturethalli, is very important when describing species.1.3.5. THE ANTHERIDIAIn the primitive representatives of the Laboulbeniales, spermatia aregenerated exogenously. However, for many primitive species, the manner offormation of spermatia has not yet been observed. Exogenous spermatialproduction is found mainly in species associated with aquatic hosts (WEIR &BLACKWELL, 2005).In the major part of the order, spermatia are endogenously formed within thespecialized antheridial cells. Antheridia can be found on both the primary andsecondary appendages. They often occur as an individual cell, usually with aslender neck functioning as a discharge tube. In Laboulbenia,Stigmatomyces, and many other genera, the antheridia are rather short andstout, with slightly narrowed necks. Herpomyces has very slender antheridia,which are taller than those in Laboulbenia and others. In some genera theappendage cells function as antheridia, with only the discharge tube beingfree (e.g. Chaetarthriomyces).In some species, old antheridia proliferate into sterile branchlets.frequently happens in many members of the genus Laboulbenia.ThisCompound antheridia only occur in representatives of the subfamiliesPeyritschielloideae and Monoicomycetoideae. Antheridial cells are arrangedso that the spermatia are released into a „chamber‟ (an intercellular space)with only one common exit. In the Monoicomycetoideae, the antheridia arerounded distally, with an indistinguishable pore. Compound antheridia with anelongated neck occur in the Peyritschielloideae. This shows that compoundantheridia originated more than once, as suggested by FAULL (1911).BENJAMIN (1971) wrote that of the 115 genera of Laboulbeniales thenrecognized, 25 genera had no known male sexual structures. Somedescriptions „with reservation‟ have been made.These three ways of spermatia formation – exogenously, endogenously insimple antheridia, endogenously in compound antheridia – were used byTHAXTER (1896, 1908) as main criterion in the classification of the orderLaboulbeniales.1.3.6. THE ASCOSPORESCharacteristics are: hyaline, elongate, spindle-shaped, surrounded by a thinmucilaginous envelope which makes them adhesive, immobile and relativelylarge (20-80 μm). The ascospores of Laboulbeniales are exclusively spread bythe activities of the host (DE KESEL, 1993; DE KESEL, 1996; DE KESEL, 1997) andtransmitted directly or indirectly (cfr. 1.6.1. TRANSMISSION OF SPORES).Ascospores are formed in the perithecium in such way that their larger cell isdirected upwards, and thus the first to be released from the ostiole.P a g e | 15

Figure III: Detail of thallus ofLaboulbenia pedicellata (fromBembidion guttula, specimen 12a,thallus 2), showing the peritheciumwith ascospores. Scale bar = 20µm. Picture by Danny Haelewaters(2010).Adherence to the host surface is beingfacilitated by the sticky envelope. Sporesalways consist of two unequal parts. Thelarger cell initiates the parasitic contactby forming the foot (also the receptacleoriginates from this cell). A septumseparates the basal cell from the smallerapical one. As mentioned before, thesmaller cell produces the appendagesystem.[For detailed information about the ontogeny of theLaboulbeniales, one is referred to TAVARES (1985), DEKESEL (1989) and WEIR & BEAKES (1996), whoextensively described the entire development (fromascospore to mature thallus).]1.4. CLASSIFICATION OF THE LABOULBENIALES1.4.1. POSI TION AMONG THE FUNGIThere has been much debate about the group the Laboulbeniales belong to.They have been considered to be acanthocephalans, basidiomycetes,zygomycetes, as well as ascomycetes. However, molecular analysis by WEIR &BLACKWELL (2001b) strongly supports the hypothesis that the Laboulbeniales-Pyxidiophora clade belongs within the Ascomycota. Depending on theauthor, this Laboulbeniales-Pyxidiophora clade is treated as a separate class(Laboulbeniomycetes) or as a subclass (Laboulbeniomycetidae) (cfr. Table IIbelow). Within the Ascomycota, the order of the Laboulbeniales can becharacterized by the absence of mycelium, absence of anamorphic stage,obligate parasitism on Arthropoda and individualized thick-walled thalliproducing only two-celled ascospores (BARR, 1983). Since their discovery, thereis agreement that these fungi form a separate group within the phylogenetictree. Especially the nearly invariable spore morphology suggests that thegroup is monophyletic (BENJAMIN, 1973); this monophyly is confirmed byphylogenetic analysis (WEIR & BLACKWELL, 2001b) (cfr. Figure IV below).Table II: Classification of the Laboulbeniales among the Fungi, according to KIRK et al. (2001) versusHIBBETT et al. (2007) and LUMBSCH & HUHNDORF (2007).Kingdom FungiKingdom FungiSubkingdom DikaryaPhylum AscomycotaPhylum AscomycotaSubphylum PezizomycotinaClass LaboulbeniomycetesClass AscomycetesSubclass LaboulbeniomycetidaeOrder LaboulbenialesOrder PyxidiophoralesOrder LaboulbenialesOrder PyxidiophoralesKIRK et al., 2001 HIBBETT et al., 2007; LUMBSCH & HUHNDORF, 2007P a g e | 16

Figure IV: Position of Laboulbeniomycetes among the Fungi, from The Tree of Life Web Project(SPATAFORA, 2007).1.4.2. CLASSIFICATI ON OF THE LABOULBENIALES“The structure of the perithecium and the relatively greater complication inthe general structure of both sexes (of Herpomyces) might be assumed toplace it higher in the scale than either Amorphomyces or Dioicomyces,although the occurrence of a series of forms on Blattidae, which are supposedto be representatives of one of the most ancient types of true insects, mightperhaps have been expected to be correlated with a more primitive type inthe parasite. But although the unisexual forms with simple antheridia might forsome reasons be assumed to be the more primitive, the present genus isdistinguished by a far more complicated structure than the other unisexualforms of the same type, and Amorphomyces still remains… by far the simplestin structure of all the Laboulbeniales.”– Roland Thaxter, 1908The order of the Laboulbeniales presently comprises over 2000 species in 143genera (SANTAMARIA, 1998; KIRK et al., 2001; WEIR & BLACKWELL, 2005; LUMBSCH &HUHNDORF, 2007), and it is expected that this number will increase by futurestudies. THAXTER (1896, 1908) used the different ways of spermatia formation –exogenously, endogenously in simple antheridia, endogenously in compoundantheridia – as main criterion in the classification.P a g e | 17

Table III: Classification of the Laboulbeniales, comparison between THAXTER (1908) and TAVARES (1985).THAXTER‟S classification (1908) TAVARES‟ classification (1985)Suborder Laboulbeniineae (Endogenae)Suborder HerpomycetinaeFamily LaboulbeniaceaeTribe HerpomyceteaeTribe AmorphomyceteaeTribe StigmatomyceteaeTribe IdiomyceteaeTribe TeratomyceteaeTribe CorethromyceteaeTribe LaboulbenieaeTribe RhachomyceteaeTribe ClematomyceteaeTribe CompsomyceteaeTribe ChaetomyceteaeTribe EcteinomyceteaeTribe MisgomyceteaeFamily PeyritschiellaceaeTribe DimorphomyceteaeTribe RickieaeTribe PeyritschielleaeTribe EnarthromyceteaeTribe HaplomyceteaeSuborder Ceratomycetineae (Exogenae)Tribe CeratomyceteaeTribe ZodiomyceteaeFamily HerpomycetaceaeTribe HerpomyceteaeSuborder LaboulbeniinaeFamily CeratomycetaceaeSubfamily TettigomycetoideaeSubfamily CeratomycetoideaeTribe ThaumasiomyceteaeTribe CeratomyceteaeSubtribe HelodiomycetinaeSubtribe CeratomycetinaeTribe DrepanomyceteaeFamily EuceratomycetaceaeFamily LaboulbeniaceaeSubfamily ZodiomycetoideaeSubfamily LaboulbenioideaeTribe CompsomyceteaeSubtribe CompsomycetinaeSubtribe KainomycetinaeTribe HydrophilomycetaeTribe CoreomyceteaeTribe TeratomyceteaeSubtribe TeratomycetinaeSubtribe RhachomycetinaeSubtribe ChaetomycetinaeSubtribe FilariomycetinaeSubtribe SmeringomycetinaeSubtribe ScelophoromycetinaeSubtribe HisteridomycetinaeSubtribe RhipidiomycetinaeSubtribe AmphimycetinaeSubtribe AsaphomycetinaeTribe LaboulbenieaeSubtribe LaboulbeniinaeSubtribe MisgomycetinaeSubtribe ChitonomycetinaeSubtribe ChaetarthriomycetinaeSubtribe StigmatomycetinaeSubtribe AmorphomycetinaeTribe EuphoriomyceteaeSubtribe EuphoriomycetinaeSubtribe AporomycetinaeSubfamily PeyritschielloideaeTribe PeyritschielleaeSubtribe PeyritschiellinaeSubtribe MimeomycetinaeSubtribe EnarthromycetinaeSubtribe DiandromycetinaeTribe DimorphomyceteaeTribe HaplomyceteaeSubtribe HaplomycetinaeSubtribe KleidiomycetinaeSubfamily MonoicomycetoideaeP a g e | 18This way of separating groups in the Laboulbeniales was universally accepted,until TAVARES (1967, 1985) applied a new way for classification using peritheciumdevelopment and perithecial wall structure as characteristics. Table III presentsthe comparison between THAXTER (1908) and TAVARES (1985).

LUMBSCH & HUHNDORF (2007) distinguish four families within the Laboulbeniales, asalready recognized by TAVARES (1985):Ceratomycetaceae 12 genera;Euceratomycetaceae 5 genera;Herpomycetaceae 1 genus;Laboulbeniaceae 125 genera.1.5. HOSTS1.5.1. GENERALLaboulbeniales are found on theintegument of living arthropods,mostly true insects. Table IV shows thedistribution of arthropod hosts beingparasitized by Laboulbeniales. Themajority of the Laboulbenialesparasitize representatives of thesubphylum Hexapoda, oftenColeoptera (beetles). About 80% ofthe described species ofLaboulbeniales parasitize beetles(WEIR & BLACKWELL, 2005).The vast majority of the beetle hostsare representatives of the twofamilies Carabidae (ground beetles)and Staphylinidae (rove beetles).Also other coleopteran families arebeing parasitized in minor degree,among them Alexiidae, Anthicidae,Apotomidae, Byrrhidae, Catopidae,Cerambycidae, Chrysomelidae,Ciidae, Clambidae, Cleridae,Coccinellidae, Corylophidae, Cryptophagidae,Cucujidae, Dryopidae,Table IV: Distribution of arthropod hostsparasitized by Laboulbeniales, persubphylum.Phylum ArthropodaSubphylum CheliceriformesClass ChelicerataSubclass ArachnidaOrder AcariSubphylum MyriapodaClass DiplopodaSubclass ChilognathaOrder CallipodidaOrder JulidaOrder SphaerotheriidaOrder SpirostriptidaSubphylum HexapodaClass PterygotaSubclass ExopterygotaOrder HemipteraOrder MallophagaOrder ThysanopteraOrder DictyopteraOrder OrthopteraOrder DermapteraOrder IsopteraSubclass EndopterygotaOrder HymenopteraOrder DipteraOrder ColeopteraDytiscidae, Elateridae, Endomychidae, Erotylidae, Gyrinidae, Haliplidae,Heteroceridae, Histeridae, Hydraenidae, Hydrophilidae, Lathridiidae,Leiodidae, Limnichidae, Microsporidae, Mycetophagidae, Nitidulidae,Passalidae, Phalacridae, Ptiliidae, Scarabaeidae, Scydmaenidae, Silphidae,Silvanidae and Tenebrionidae and Zopheridae (HINCKS, 1960; SCHELOSKE, 1969;MAJEWSKI, 1994, 2003; SANTAMARÍA et al., 1991; DE KESEL, 1997).[Name changes and updates on taxonomy were reviewed using VORST (2010). For example, manylaboulbenialean species have been recorded on members of the former family Pselaphidae,nowadays regarded as a subfamily Pselaphinae within the Staphylinidae.]P a g e | 19

Laboulbeniales only occur on adult hosts. BENJAMIN (1971) reports a fewcontrary studies, in which has been observed that representatives of theLaboulbeniales can grow also on immature stages. Herpomyces stilopygae,for example, grows on nymphs as well as on adults of their host (Blattaorientalis, suborder Blattodea, order Dictyoptera). However, infection is lessintense on nymphs and disappears completely with the molt (RICHARDS & SMITH,1955).1.5.2. ORDER COLEOPTERAThe Coleoptera form the most diverse group of extant Metazoa. Beetles arevirtually everywhere; they show an extreme diversity both ecologically andmorphologically. Their size ranges from tiny animals of nearby 0,3 mm(Ptiliidae) to „giants‟ of almost 18 cm (Titanus giganteus, the giant Amazonianlonghorn beetle) (PONS et al., 2010). The order consists of 370.000 speciesspread over 166 families (BURNIE, 2001), and is the largest of all insect orders. InWest-Europe, 5.000 species have been described.Coleopterans have two pairs of wings, of which the first pair is hard or leathery.The first wings – called elytra – touch each other dorsally. The second pair ofwings is membranous and lies folded up underneath the elytra. Sometimes thesecond pair of wings is not present or reduced (brachypterous); some specieseven have no wings at all (apterous).Mouth parts of coleopterans are always biting. This is a physical constraint;beetles, however, have invaded all available biotopes (including the sea) andopened up all possible food sources. The order comprises herbivores,detritivores, predators and parasites. Many species play a negative role forman: cockshafers and many other species harm our crops. Beetles, on theother hand, can be very useful allies in the battle against other threateninginsects. In particular lady birds, that eat greenflies, are important. All mouthparts, including the biting mandibles, are well developed. However, thisdoesn‟t mean that beetles are restricted to solid food; many species soak theirfood with digestive fluid before taking it up. Other species lick nectar, whilelarvae of even other species suck liquid food with their tube-like mandibles.The order of the Coleoptera consists of three suborders: Adephaga,Polyphaga and Archostemmata. The latter is not represented in Europe. Thesuborder of the Adephaga is the more primitive suborder and consists ofcarnivorous species. The name Adephaga refers to the habit to run after food(Latin ad = [go] to). The suborder of the Polyphaga is the larger one, and – ascan be expected (Polyphaga means „eating lots of things‟) comprises manyincoherent representatives. The phylogeny of this group is far from beingelucidated. Even after 20 years of research, there still are large and ratherheterogeneous superfamilies in the suborder Polyphaga.The family of the Staphylinidae (suborder Polyphaga) is parasitized by thegreatest number of genera of the Laboulbeniales (49 genera; TAVARES, 1979).The family of the Carabidae (suborder Adephaga) is parasitized by 15 genera(TAVARES, 1979). Also the family of the Hydrophilidae is parasitized by 15genera, but by a much smaller number of species (TAVARES, 1979).P a g e | 20

Substrate is the intermediate factor in the case of indirect (self-)transmission(cfr. Figure V below, right). The indirect transmission of L. slackensis is stronglyaffected by the age of the spores (DE KESEL, 1995b).It has been experimentally proven by DE KESEL (1995b) that the differencebetween direct and (lower) indirect transmission with L. slackensis is due to thelow output of ascospores towards the environment. Spores ooze out of theperithecium and form adherent thread-like structures, favouring the return tothe host (direct self-transmission) or a co-habitant with whom contact wasmade (direct transmission).Also the biology of the host is of great significance: Clivina fossor, host forLaboulbenia clivinalis Thaxt., leads a mainly subterranean way of life. Thereforethe importance of indirect transmission must not be underestimated (DE KESEL,1995a).1.6.2. MODEL OF ISLAND BIOGEOGRAPHY (MACA R T H U R & WI L S O N, 1967)The host populations of Laboulbeniales are probably similar to islands in themodel of Island Biogeography (DE KESEL, 1996, 1997): the divergence of hostpopulations will lead to divergence of the parasites by isolation of gene pools(reinforced ecological isolation).The great diversity in ecological adaptations of the Laboulbeniales within thefamily of the Carabidae is probably closely related to the different modes ofbehavior, habitat preferences and physiological requirements of the carabidhosts. Specialization of the Laboulbeniales is the result of 1/ obligateectoparasitism, 2/ host specificity and 3/ dependence on a specificenvironment.1.6.3. DISTRIBUTION PATTERNS (D E K E S E L, 1997)There are two distribution patters of Laboulbeniales:One specific host group parasitized by a large number of unrelatedparasite genera Staphylinidae;Many related parasite taxa parasitizing a single host family Carabidae.The adaptive radiation of the Laboulbenia genus within each tribe of thefamily of the Carabidae is indicated by the significant relationship betweenthe number of host species in each Carabidae-tribe and the number ofLaboulbenia species parasitizing hosts of that tribe.In general, it is likely that most epidemiological traits of host-parasiterelationships are controlled by complex interactions between the bothgenotypes (GHxGP interactions; VALE et al., 2008). Moreover, evenphylogenetic correspondence between parasite and host can be important.A history of parallel diversification between parasite and host has taken careof concordant (~ congruent) phylogenies between the both (as suggested byFUTUYAMA, 2005). Although no study has yet been conducted in whichspecifically phylogenies were compared, the above seems obvious: adaptiveradiation of Laboulbeniales depends upon the number of available hosts inthe host taxon.P a g e | 22

An all-inclusive discussion on the parallels between host phylogeny andLaboulbeniales phylogeny based alone on the host-parasite list is unrealistic.DE KESEL (1997) suggests that that the latter can be used in host cladistics, ifcombined with morphological, physiological, etc. data from the hosts.1.6.4. DETERMINING FACTORSAll factors with some influence on the occurrence and growth of thalli can besummarized in two groups: the host (1) and the (host‟s) environment. The lattercategory can be classified once more into abiotic (2) and biotic (3)environmental factors (DE KESEL, 1991; DE KESEL, 1993).Abiotic factors can be temperature, day length, humidity, substratecharacteristics, … Among biotic factors are beetle behavior (reproduction,activity), population density and population structure. Abiotic factors alwaysstrongly influence biotic factors.Observations of Clivina fossor during a complete year-cycle (DE KESEL, 1995a)suggest that the increasing temperature, host activity and the specificmicrohabitat selection of the host in spring enhances the thallus density ofparasitizing Laboulbenia clivinalis. DE KESEL (1995b, 1996) proved forLaboulbenia slackensis that the indirect transmission (1.6.1. TRANSMISSION OFSPORES) is not affected by the substrate.Artificial infections with L. slackensis showed that the parasite is potentiallyplurivorous on Carabidae (cfr. 1.7.3.2. Host specificity). The involvement of L.slackensis in an intimate association is determined by several factors, includingits own environmental preferences, characteristics of the host integument, themicroclimate in the host‟s surface and the availability of nutrients (DE KESEL,1996). Successful establishment of the parasite requires both the presence of apotential host and favorable environmental conditions for the fungus.[More detailed information on the ecology of Laboulbeniales can be found in SCHELOSKE, 1969 andDE KESEL, 1997.]1.7. PATHOGENICITYP a g e | 231.7.1. ARE LABOULBENIALES REAL P ARASITES?Undoubtedly, the Laboulbeniales are obligate ectoparasites of their arthropodhosts (SCHELOSKE, 1969; BENJAMIN, 1971; MAJEWSKI, 1994). In nature, they neveroccur apart from hosts, and attempts to grow members of the Laboulbenialesin axenic culture failed at early stages (BENJAMIN, 1971; WEIR & BLACKWELL,2001a). WHISLER (1967) did succeed in early development of the thalli to the 20-cell stage. To this end, ascospores from Fanniomyces ceratophorus (Whisler) T.Majewski were placed on a medium consisting mainly of brain-heart infusionagar. However, no development of the perithecial phase was initiated.Laboulbeniales cannot survive without nutrients of their host; there is noevidence of the host receiving something in exchange. Thus, most probably,they are parasites. However, since no precise experimental data on thenutrition of the Laboulbeniales is available (BENJAMIN, 1971; DE KESEL, 1997),things remain unclear. It could be a commensal or mutualistic relationship(depending on how much the host would attain in return).

CAVARA (1899, ref. in BENJAMIN, 1971) suggested that Laboulbeniales was ableto retrieve nutrients from the environment by means of the trigogyne or sterileappendages. SPEGAZZINI (1917), inspired by the absence of visible damage onthe host cuticle, suggested nearly the same idea: the Laboulbeniales retrievingtheir food from the environment, even without appendages, as there aremany species. However, these are hypotheses; more research is needed in thisarea. DE KESEL (1996) assumes that the appendages play a role in the waterbalance of the thallus.1.7.2. ATTA CHMENT TO THE HOS TFigure VI: Detail of thallus ofLaboulbenia calathi (from Calathusmelanocephalus, specimen 3a,thallus 1), showing the foot (arrow).Scale bar = 20 µm. Picture by DannyHaelewaters (2010).Thalli of nearly all species of theLaboulbeniales are attached to theirhost through the modified lowermostpart of the basal cell of the receptacle,the foot (cfr. Figure VI). In most species,the foot is black. The development ofthe foot occurs very early in thallusontogeny, starting with a thickeningmucilaginous sheath at one side of theascospore, probably as the firstdifferentiation of the basal cell;observed by TAVARES (1985) and DE KESEL(1989).Most Laboulbeniales do not penetrateinto living tissue of their host, but make contact through integument pores(TAVARES, 1985).In a few genera (e.g. Arthrorhynchus), the foot is absent and the basal cellpenetrates into the host hemocoel through well developed haustoria(MAJEWSKI, 1994). Herpomyces species make contact with the host by manyhaustoria of the secondary receptacle, penetrating only into the epidermalcell layer. Also the species without any visible haustoria probably take upnutrients from the host hemolymph (SCHELOSKE, 1969).P a g e | 241.7.3. SPECIFICITY OF THE LABOULBENIALES1.7.3.1. Position specificityLaboulbenia truncata Thaxt. and L. perpendicularis Thaxt. are located onrestricted areas of the body of their host, Bembidion picipes (= positionspecificity): L. truncata on the tarsi of the forelegs of males, L. perpendicularisbetween the coxae of the forelegs of female beetles. Both species alsodisplay a marked similarity of structure (BENJAMIN & SHANNOR, 1952). Whetherboth species are two different taxa or growth forms of one species will have tobe studied by using both morphological and molecular methods.Laboulbeniales can exhibit great phenotypic plasticity. Growth position oftenleads to the development of totally different morphs on one host (BENJAMIN &SHANOR, 1952; BENJAMIN, 1971; SCHELOSKE, 1976; HULDÉN, 1985, ref. in DE KESEL, 1997;MAJEWSKI, 1994; DE KESEL & VAN DEN NEUCKER, 2005; DE KESEL & WERBROUCK, 2008).Many of these morphs have been considered as different taxa. BENJAMIN &SHANOR (1952) suggest that position specificity may be the result of particular

PART IILABOULBENIALES:EXPLORINGANDTESTINGDNA EXTRACTION PROTOCOLSP a g e | 31

1. INTRODUCTION1.1. DIFFICULTIES FOR DNA EXTRACTION OF LABOULBENIALESMolecular studies of Laboulbeniales are challenging for the following reasons(WEIR & BLACKWELL, 2001a):The microscopic size of the thalli (length range [35 μm – 2 mm], on average200-300 μm) (SCHELOSKE, 1969; HULDÉN, 1983; SANTAMARÍA, 1998);Thalli require micromanipulation techniques in order to remove them fromtheir hosts;Thalli are heavily melanized and difficult to break open;The outer envelope comprises an adhesive mucilaginous componentinterfering with centrifugation;Recalcitrance to axenic culture.Many different approaches did not succeed in releasing DNA. Differentapproaches were used, most without any success: prolonged boiling of thalli(HANSON, 1992), microwave treatment (GOODWIN & LEE, 1993) and immersion inliquid nitrogen (HAUGLAND et al., 1999). Also direct addition of intact thalli toPCR mastermix was unsuccessful (e.g. pers. comm. DE KESEL, 2009).Beyond this, extra problems arose when working with hosts that are dried orpreserved in ethanol (95 %) (WEIR & BLACKWELL, 2001a).1.2. GENBANK1.2.1. THE GENBANK SEQUENCE DATABAS EDatabases form the basis for most applications in bioinformatics. The GenBanksequence database is an open access, annotated collection of all publiclyavailable DNA sequences. GenBank continues to grow at an exponential rate– in February 2008, there were approximately 82.853.685 sequences inGenBank; in October 2010 this number was already increased to 125.764.384(GENBANK RELEASE NOTES, 2010). This database is part of the InternationalNucleotide Sequence Database Collaboration, which comprises the DDBJ, theEMBL, and GenBank at the National Center for Biotechnology Information(NCBI). These three institutes exchange data on a daily basis.1.2.2. THE SEARCH FOR SEQUEN CES OF LABOULBENIALES/LABOUL-BENIOMYCETES IN GENBANKGenBank only includes 21 representatives of the order of the Laboulbeniales,shown in Table VII below.Of the 27 sequences in GenBank, categorized within the classLaboulbeniomycetes, 19 are the same as with the „Laboulbeniales‟ search.This could be the result of wrongly classified species. However, thisinconsistency can also be the consequence of the two different ways ofclassifying Laboulbeniales. The remaining 8 sequences are shown in Table VIIbelow.P a g e | 32

Table VII: Overview of all sequences in GenBank, found with both the „Laboulbeniales‟ search (first 21sequences) and the „Laboulbeniomycetes‟ search (last 8 sequences). Abbreviations: bp number ofbase pairs, rRNA ribosomal RNA, SSU small subunit, LSU large subunit, TEF1- translation elongationfactor-1 , RPB2 RNA polymerase II second largest subunit.Sequnce lengthSpeciesLocusAccession Publication[bp]Laboulbeniales sp. LM68 18S rRNA 1022 EF060444.1 (a)Rickia passalina 18S rRNA 490 AF432129.1 bCeratomyces mirabilis 18S rRNA 515 AF431764.1 bRhadinomyces pallidus 18S rRNA 515 AF431763.1 bCorethromyces bicolor 18S rRNA 502 AF431762.1 bCorethromyces sp. AW-2001 18S rRNA 515 AF431761.1 bBotryandromyces ornatus 18S rRNA 516 AF431760.1 bStigmatomyces rugosus 18S rRNA 514 AF431759.1 bStigmatomyces scaptomyzae 18S rRNA 518 AF431759.1 bStigmatomyces hydreliae 18S rRNA 516 AF431757.1 bRhachomyces philonthinus 18S rRNA 513 AF431756.1 bZodiomyces vorticellarius SSU rRNA 1083 AF407577.1 cStigmatomyces limnophorae SSU rRNA 1028 AF407576.1 cHesperomyces coccinelloides SSU rRNA 1038 AF407575.1 cHesperomyces virescens LSU rRNA 409 AF298235.1 dStigmatomyces protrudens LSU rRNA 401 AF298234.1 dHesperomyces virescens SSU rRNA 1085 AF298233.1 dStigmatomyces protrudens SSU rRNA 1081 AF298232.1 dPyxidiophora sp. IMI-1989 SSU rRNA 1103 AF313769.1 (e)Kathistes calyculata SSU rRNA 1089 AF313768.1 (e)Kathistes analemnoides SSU rRNA 1096 AF313767.1 (e)Pyxidiophora arvernensis is. AFTOL-ID 2197 TEF-1 678 FJ238412.1 (f)Pyxidiophora arvernensis is. AFTOL-ID 2197 RPB2 857 FJ238377.1 (f)Pyxidiophora arvernensis is. AFTOL-ID 2197 28S rRNA 1080 FJ176894.1 (f)Pyxidiophora arvernensis is. AFTOL-ID 2197 18S rRNA 1478 FJ176839.1 (f)Termitaria snyderi is. DAH 14 18S rRNA 1046 AY212812.1 gLaboulbeniopsis termitarius is. DAH 18 18S rRNA 998 AY212810.1 gPyxidiophora sp. 03 18S rRNA 1064 AY212811.1 gPyxidiophora arvernensis18S rRNA +type I intron424 U43339.1 (h)[(a) MAHDI & DONACHIE, 2006 – unpublished; b WEIR & HUGHES, 2002; c WEIR & BLACKWELL, 2001b; d WEIR & BLACKWELL,2001a; (e) WEIR & BLACKWELL, 2000 – unpublished; (f) SCHOCH, 2008 – unpublished; g HENK, WEIR & BLACKWELL, 2003; (h)Jones & Blackwell, 1995 – unpublished.]P a g e | 33

2. MATERIALS & METHODS2.1. FUNGUS, HOST AND ORIGINCarabid hosts of Laboulbenia species were collected in November 2008 and2009 (July – December) by means of hand capturing. All collecting sites aresituated in Hingene (Belgium, Prov. Antwerpen, N51°06‟ – E4°12‟), atSchellandpolderdijk along the river Schelde.The collecting sites can be categorized as alluvial areas (polders). These areasare not flooded daily, being protected by dykes; they have fine alluvial soilsthat can become relatively dry in summer. The sampled alluvial areas consistmostly of poplar plantations or mixed forests with mainly Alnus, Fraxinus,Quercus and Acer.2.2. MORPHOLOGICAL PROTOCOL AND DNA EXTRACTION2.2.1. INTRODUCTIONThe hosts were narcotized in a pot filled with chloroform gas (insects had nocontact with liquid chloroform). Screening of the hosts was done by using bothbinocular and stereomicroscope (50x). Separation of infected and uninfectedhosts had to be done with precision. Carabid hosts were identified to specieslevel, using the identification key of BOEKEN (1987).From this point, different strategies were tried out, in order to improve themethodology. The protocols will be discussed below, in chronological order.2.2.2. PROTOCOL I (BASED ON WEIR & BLACKWELL, 2001b)Thalli were aseptically removed from each host using a sterile micropin andtransferred to a 2 µL drop of Milli-Q H2O on a sterile microscope slide. An 18 x18 mm coverslip was placed over the thallus. Hosts were observed using aNikon SMZ800 stereoscopic zoom microscope (binocular); a Nikon Eclipse E600research microscope was used for visual inspection of the thalli. Thalli wereidentified to species level using MAJEWSKI (1994), and photographed with NikonDigital Camera DXM1200. After photographing, the thalli were crushedbetween the two slides. A razor blade was used to remove the coverslip.Visual inspection was needed in order to find the thalli on the coverslip or themicroscopic slide. The crushed material was hydrated in 20 μL of extractsolution (cfr. Figure VIII for composition).99 µL Milli-Q H2O + 1 µL Triton (100%) 100 µL 1% Triton detergent1 µL 1% Triton + 19 µL Milli-Q H2O 20 µL extract solutionFigure VIII: Composition of extract solution.P a g e | 34The thalli were transferred into 2mL tubes using a sterile micropin. Glass beads(0,25-0,50 mm in diameter) were added to the tube. The content of the tubewas additionally crushed in a bead beater (3 x 90 sec; 30x/s) (RETSCH Mixer MillMM200, Haan, Germany). Centrifugation took place for 2 minutes at 14.000rcf. 30 µL Milli-Q H2O was added to the tube, whereupon the tube was placed

in a heating block for 5 minutes at 100° C. Storage for short periods took placeat 2 – 7° C; for longer periods at -20° C.2.2.3. PROTOCOL II (BASED ON WEIR & BLACKWELL, 2001a)One thallus or a group of thalli inserted at the same place (two to nine) wereaseptically removed from each host using a sterile micropin and transferred toa 2 µL drop of 0.1xTE buffer (10 mM Tris.Cl, 0.1 mM EDTA, pH 8.0) on a sterilemicroscope slide. An 18 x 18 mm coverslip was placed over the thallus/thalli.Hosts were observed using a Nikon SMZ800 stereoscopic zoom microscope(binocular); a Nikon Eclipse E600 research microscope was used for visualinspection of the thalli. Thalli were identified to species level using MAJEWSKI(1994), and photographed with Nikon Digital Camera DXM1200. Afterphotographing, the thalli were crushed between the two slides. The slide wasimmediately placed on a bed of dry ice (Ice Man BVBA, Bottelare, Belgium)and allowed to freeze. A razor blade was used to remove the coverslip. Visualinspection was needed in order to find the thalli on the coverslip or themicroscopic slide. 2 μL of the extract solution (cfr. Figure IX for composition)was pipetted on top of the crushed material.99 µL Milli-Q H2O + 1 µL Triton (100%) 100 µL 1% Triton detergent2 µL 1% Triton + 18 µL 0.1xTE buffer 20 µL extract solutionFigure IX: Composition of extract solution.This material was again allowed to freeze on the bed of dry ice. When theextraction solution started to defrost, after removal from the dry ice, thethallus/thalli were transferred into a 2 mL tube, together with the remaining 18μL of extract solution. An additional 30 μL of 0.1xTE buffer was added,whereupon the tube was placed in a heating block for 15 minutes at 60° C.Storage took place at -20° C.2.2.4. PROTOCOL III: PUREGENE KIT ADNA was extracted using Qiagen‟s Puregene Kit A (Qiagen, The Netherlands)and the provided documents at Ghent University, research group Mycology.Qiagen, Gentra Puregene Handbook (2007).2.2.5. PROTOCOL IV: DNEASY EXTRACTION KITDNA was extracted using Qiagen‟s DNeasy Plant Mini Kit (Qiagen, TheNetherlands) and the provided documents at Ghent University, research groupMycology. The elution of DNA for all specimens was performed in duplicatewith 100 µL AE buffer. For every specimen, two elutions were carried out.In order to overcome eventual contaminations, single hosts were precedentlyplaced in a ultrasonic bath for 5 minutes.Qiagen, DNeasy Plant Mini and DNeasy Plant Maxi Handbook (2004).P a g e | 35

2.2.6. PROTOCOL V: DIRECT PCROne thallus or a group of thalli inserted at the same place (two to nine) wereaseptically removed from each host using a sterile micropin, crushed andwithout any modification transferred into a 2 mL tube and used directly in PCRamplification.This protocol, however, provides more risk for contamination. Therefore, singlehosts were placed in a ultrasonic bath for 5 minutes.2.3. PCR AMPLIFICATION2.3.1. ITSThe internal transcribed spacer (ITS) region of the nuclear rRNA was amplifiedby PCR using the primer sets ITS5/ITS4A and ITS5/ITS2.Table VIII: Characteristics of primers used in this study: F forward, R reverse, Tm melting temperature (in°C), Ta optimal annealing temperature (in °C), #bp number of base pairs, GC percentage of GC bases, Rreference).Primer name Sequence F/R Tm Ta #bp GC RITS5 5‟-GGAAGTAAAAGTCGTAACAAGG-3‟ F 63 58 22 40,9 aITS4-A 5‟-CGCCGTTACTGGGGCAATCCCTG-3‟ R 68 63 23 65,2 bITS2 5‟-GCTGCGTTCTTCATCGATGC-3‟ R 62 57 20 55,0 a[a WHITE et al., 1990; b LARENA et al., 1999.]PCR micture (cfr. Table IX) consisted of CoralLoad PCR Buffer II (10x), MgCl2 (25mM), dNTP (10 mM), forward + reverse primer (10 µM), Taq polymerase (5 u/µL),H2O MilliQ and DNA extract. The total volume was 50 µL. All PCR reactionscontained 5 µl DNA extract. The next protocol was applied:Table IX: Overview of PCR components, amplification protocol.Chemical Reference Concentration Volume/reaction [µl]PCR buffer II Qiagen Hilden, Germany 10x 5MgCl2 Qiagen Hilden, Germany 25 mM 0,5dNTP Eppendorf Hamburg, Germany 10 mM 1Forward primer Invitrogen TM Merelbeke, Belgium 10 µM 1Reverse primer Invitrogen TM Merelbeke, Belgium 10 µM 1Taq polymerase Qiagen Hilden, Germany 5 u/µL 0,3H2O MilliQ / 36,2DNA extract / 5TOTAL 50The amplification was conducted un der the following conditions: an initialdenaturation step at 94° C for 10 minutes, 35 cycli of denaturation (at 94° C for30 seconds), primer annealing (at 55° C for 30 seconds) and extension (at 70°C for 45 seconds), and a final extension step at 70° C for 10 minutes. Hereafter,the temperature was held constant at 20° C until PCR products were taken outof the thermocycler.Only with the direct PCR protocol (2.2.7. PROTOCOL V: DIRECT PCR), the thallus ora group of thalli inserted at the same place were directly added to the PCRreaction.P a g e | 36

2.3.2. POST PCRPCR products were analyzed by electrophoresis on 1% agarose gels in 1xTAEbuffer (Qiagen, The Netherlands). PCR products were run by 120 mV forapproximately 30 minutes using a 50 to 2000 bp molecular weight marker(Merck NV, Overijse, Belgium). After electrophoresis, gels were stained withethidium bromide (EtBr) and viewed under UV light (254-365 nm).2.4. SEQUENCING AND SEQUENCE ANALYSIS2.4.1 SEQUENCI NGThe obtained PCR products were purified using ExoSAP (USB ® , USA). The DNAsequencing reactions were performed with the ABI PRISM ® BigDyeTMTerminators v3.1 Cycle Sequencing Kit using the same primers on an ABI PRISM ®3130xl DNA Sequencer.2.4.2 SEQUENCE ANALYSISThe generated chromatograms were visualized using the programSequencher ® 4.9 (Gene Codes Corporation, Michigan, USA). These peak plotswere controlled and modifications were submitted manually.The modified sequences were compared with DNA databank GenBank. Theaim was to consider similarities with species already present in GenBank (1.2.2.THE SEARCH FOR SEQUENCES OF LABOULBENIALES/LABOUL-BENIOMYCETES IN GENBANK).Therefore, the program BLAST (Basic Local Alignment Search Tool) wasdeveloped. BLAST is supported by the National Center for BiotechnologyInformation (NCBI) and can be found at the following link:http://www.ncbi.nlm.nih.gov/BLAST.The BLASTn search enging was used, which is suitable for queries of 3000 or lessnucleotides in particular. All default algorithm parameters have been used,except the “low complexity regions”. With this filter working, sequences withlong series of A‟s or T‟s or repeating sequences – such as GTGTGTGT – will berecognized and deleted. This function is used to exclude false similarities, butalso “real” similarities are deleted, leading to a false top 50 of similarsequences in GenBank.Each matching sequence resulting from the BLASTn search is proved with:Accession number;Description;Max score;Total score;Query coverage;E value;Max identity.The accession number contains the link to view the entire sequence. The (max+ total) score is a number that counts for both the identities and gaps of thecompared sequences. The query coverage is the percentage of the querylength that is included in the aligned sequences. The E(xpect) value describesthe number of matches a particular sequence would be having by randomP a g e | 37

P a g e | 38chance. Thus, the lower the E value, the more significant the similarity of thequery sequence to another. The max identity is the highest percentageidentity for an aligned sequence to the query sequence (often referred to as„% similarity‟).

3. RESULTS3.1. DNA EXTRACTIONDNA was extracted from 48 specimens. Hereafter, DNA was used for PCRreaction; except for the direct PCR protocol in which the thallus or groups ofthalli directly were transferred into the 2 mL tubes. With every DNA extractionprotocol, a negative control was included to check for contaminations duringthe extraction. The negative controls were included in the PCR reactions.3.2. AMPLIFICATION3.2.1. PRIMER PAIR ITS5/ITS4-AThe adapted ITS sequences of specimens labo1111 [immature thallus, DH 3B],flag1126 [thallus DH5A], flag1129 [thallus DH 5E], flag1131 [immature thalli, DH11A + 11B], labo1132 [thallus DH 11E], coll1133 [thallus DH 11F], labo1136[immature thallus, DH 12A], flag1137 [two thalli, DH 13A + 13B] and flag1141[thallus DH 13J] match for 99% with Davidiella macrospora and for 98% withDavidiella tassiana. Davidiella is classified within the class of theDothideomycetes (Ascomycota).The adapted ITS sequence of specimen flag1115 [two thalli, DH 3H + 3I)matches for 99% with Cladosporium, a member of the Dothideomycetes(Ascomycota).The adapted ITS sequence of specimen flag1146 [two thalli, DH 15A + 15B]matches with several organisms for 97 to 99%: Cladosporium (Ascomycota,Dothideomycetes), Davidiella (Ascomycota, Dothideomycetes), Choiromyces(Ascomycota, Pezizomycetes) and Dioscorea (Spermatophyta).The adapted ITS sequence of specimen flag1152 [thallus DH 16C] matches for99% with Pichia sp., for 93% with Pichia castillae and media and for 90% withPichia stipitis (but with query coverage of 100%). Furthermore, the sequencematches for 91% with Candida fermenticarens and for 90-92% withDebaryomyces. All hits – Pichia, Candida, Debaryomyces – belong to theSaccharomycetes (Ascomycota).Table X: Top matches with GenBank for specimens amplified using primers ITS5 andITS4-A. Specimens identified only to genus level were immature.Specimen Species Protocol Length Comparison GenBank Referencelabo1111 Laboulbenia sp. I 595flag1115 Laboulbenia flagellata I 598flag1126 Laboulbenia flagellata III 608flag1129 Laboulbenia flagellata III 608labo1131 Laboulbenia sp. IV 608labo1132 Laboulbenia sp. IV 607Davidiella macrosporaDavidiella tassianaCladosporium sp.Cladosporium cladosporioidesDavidiella macrosporaDavidiella tassianaDavidiella macrosporaDavidiella tassianaDavidiella macrosporaDavidiella tassianaDavidiella macrosporaDavidiella tassiana-a-b-a-a-a-aP a g e | 39

coll1133 Laboulbenia collae IV 608labo1136 Laboulbenia sp. V 608flag1137 Laboulbenia flagellata V 608flag1141 Laboulbenia flagellata V 609flag1146 Laboulbenia flagellata II 505flag1152 Laboulbenia flagellata II 691Davidiella macrosporaDavidiella tassianaDavidiella macrosporaDavidiella tassianaDavidiella macrosporaDavidiella tassianaDavidiella macrosporaDavidiella tassianaCladosporium cladoporioidesCladosporium sp.Choiromyces venosusDavidiella tassianaDioscorea polystachyaCladosporium ossifragaPichia sp.Pichia castillaePichia mediaPichia stipitisCandida fermenticarensDebaryomyces hanseniiDebaryomyces pseudopolymorphus-a-a-a-a-cde-f-ggh-i-[a SIMON & WEISS, 2008; b BRAUN et al, 2003; c WIRSEL et al., 2002; d FERDMAN et al., 2005; e BUKOVSKA et al., 2010; fSCHUBERT et al., 2007; g VILLA-CARVAJAL et al., 2006; h JEFFRIES et al., 2007; i ARTEAU et al., 2010.]3.2.2. PRIMER PAIR ITS5/ITS2The adapted ITS sequence of specimen flag1151 [thallus, DH 16B) matches for92% with Mycena amabilissima (Basidiomycota, Agaricomycetes).Table XI: Top matches with GenBank for specimens amplified using primers ITS5 and ITS2Specimen Species Protocol Length Comparison GenBank Referenceflag1151 Laboulbenia flagellata II 359 Mycena amabilissima a[a MATHENY et al., 2006.]P a g e | 40

4. DISCUSSIONMolecular techniques are used to study symbionts and parasites, includingplant pathogens and lichenized and mycorrhizal fungi. However,Laboulbeniales show particular difficulties when it comes to DNA extraction:their microscopic size, the finite number of thick-walled cells in the thallus andfrequently heavily melanized regions, thus difficult to break open. Therefore,attempts to amplify ITS sequences of Laboulbeniales failed. Manycontaminations were found instead.For all 48 specimens, ITS amplification following the different protocols (WEIR &BLACKWELL, 2001a; WEIR & BLACKWELL, 2001b; 2.2. MORPHOLOGICAL PROTOCOL ANDDNA EXTRACTION) was not successful in releasing laboulbenialean DNA. For 12specimens, amplification with ITS5/ITS4-A led to sequences that could be usedfor comparison with GenBank. Matches were obtained from differentascomycotan classes: Dothideomycetes, Pezizomycetes and Saccharomycetes.For one specimen [flag1146], there even was a 99% similarity matchwith Dioscorea polystachya, a member of the Archaeplastida (while fungi areOpisthokonta). One specimen amplified using ITS5/ITS2 [flag1151] matched inGenBank with Mycena amabilissima (Basidiomycota, Agaricomycetes). Thephylogeny of the most common resulting matches in GenBank is presented inFigure X below.SpermatophytaAscomycota, DothideomycetesAscomycota, PezizomycetesBasidiomycota, AgaricomycetesAscomycota, SaccharomycetesFigure X: Phylogeny of the most common resulting matches in GenBank, based on 18S rDNA. Thephylogenetic tree is the result of a neighbor joining analysis of a dataset with 15 sequences.Sequences (with accession number between brackets) are (from top to bottom) Dioscoreapolystachya (FJ860063), Cladosporium sp. (GU214631), Davidiella tassiana (EU343349),Cladosporium cladoporioides (AY251074), Cladosporium ossifragi (EF679382), Davidiella macrospora(EU167591), Choiromyces venosus (AF435827), Mycena amabilissima (DQ490644), Pichia sp.(HM627157), Candida fermenticarens (FM178353), Pichia castillae (DQ409168), Pichia media(DQ409170), Pichia stipitis (CP000497), Debaromyces hansenii (GQ458025) and Debaromycespseudopolymorphus (EF198011).P a g e | 41

Cladosporium (Ascomycota, Dothideomycetes) is one of the largest, mostheterogeneous genera of Hyphomycetes, including endophytic, fungicolous,human pathogenic, phytopathogenic and saprobic species (CROUS et al.,2007). Davidiella is the teleomorph. It seems that incorrectly identifiedsequences are present in the GenBank database, as suggested by BROCK et al.(2009). Blasting the resulting match Dioscorea polystachya (FJ860063) givesDavidiella tassiana (99% identity), Cladosporium cladosporioides (99% identity)and Cladosporium sp. (99% identity) as most common matches, alongDioscorea alata and other Dioscorea polystachya specimens (from the sameresearch group). It is suggested that those sequences of Dioscoreapolystachya with accession numbers FJ860063, FJ860073, FJ860075, FJ860081and FJ860096 and Dioscorea alata with accession numbers FJ860064 andFJ860066 are incorrectly identified.The amplification with ITS5/ITS2 led to useful sequences for only on specimen[flag1151]. The resulting ITS sequence shows, compared with the GenBankdatabase, high similarity with Mycena amabilissima, a member ofbasidiomycotan Agaricomycetes.Amplification using ITS5/ITS2 led to shorter sequences than amplication withITS5/ITS4-A, since with ITS2 as reverse primer only the ITS1 region will beamplified (cfr. Figure XI) (WHITE et al., 1990).Figure XI: Schematic overview of the entire internal transcribed spacer region (ITS), flanking DNA andprimer positions (not drawn to scale).Protocol II was suggested to give consistent results (WEIR & BLACKWELL, 2001a).Yet, it may be stated that the protocol outline was not very clearly described,in both WEIR & BLACKWELL (2001a) and WEIR & BLACKWELL (2001b) on whichprotocol I of this research was based. We tried to communicate with Alex Weirand his students for additional information on the protocols but got no reply.WEIR & HUGHES (2002) suggest that the DNA extraction protocol described inWEIR & BLACKWELL (2001a) is reliable. However, since 2002, no other studies wereperformed using this protocol. The current research suggests that there is a lotof work in finding a real reliable and feasible DNA extraction protocol and PCRamplification procedure for Laboulbeniales.The DNA extraction protocol using Qiagen‟s Puregene Kit A (protocol III) wasunsuccessful, as the protocol using Qiagen‟s DNeasy Plant Mini Kit.Direct addition of thalli to the PCR reaction (protocol V) was unsuccessful,confirming previous reports (WEIR & BLACKWELL, 2001a).P a g e | 42

Suggestions are given regarding the DNA extraction protocol forLaboulbeniales.At first, after narcotizing in a pot filled with chloroform gas, infected hostsshould be put in a glass tube filled with EDTA (Ethylenediaminetetra-aceticacid), a divalent cation chelating agent, and placed in an ultrasonic bath for5 minutes. EDTA has been shown to inhibit Candida albicans (Ascomycota,Saccharomycetes) through its inhibitory effect on filamentation and hyphaldevelopment (RAMAGE et al., 2007). The ultrasonic bath provides a superficialphysical purification of the hosts: high frequency sound waves are used toagitate; cavitation bubbles induced by the agitation act on contaminants.Hereafter, the hosts should by washed with Milli-Q H2O whereupon they shouldbe put in a new glass tube filled with mannose and placed in an ultrasonicbath for 5 minutes. LEMAIRE et al. (2004) provide strong evidence that mannoseis an antagonist of the G protein-coupled receptor Gpr1 in Saccharomycescerevisiae (Ascomycota, Saccharomycetes). Gpr1 is important for thedetection of glucose and the detection of low sucrose concentrations (duringperiods of famine). This sensor system has a great importance for the survivalof C. cerevisiae.It is important not to forget that the success of molecular research not onlydepends on DNA extractions. It might be that our PCR amplificationprocedure was unsuccessful. Therefore, the design of laboulbenialean-specificprimer should be investigated.All together, the suggestions regarding both the DNA extraction protocol andthe PCR amplification procedure are subject for a doctoral study.The success of incorporating Laboulbeniales into a molecular phylogeny isindicated to be dependent on clean and pure cultures of taxa. The followingsuggestions are interesting topics for further research:The nutrients Laboulbeniales derive from their hosts are unknown (BENJAMIN,1971; DE KESEL, 1997). Labelling techniques of hosts using chemicals thatremain detectable in the parasite should be used.In order to be able to work with clean and pure cultures of taxa, a mediumin which Laboulbeniales can form stable populations should be created.Stable populations of Laboulbeniales have successful spore transmission,thallus development and spore production.The study of Laboulbeniales should make a shift towards the biochemicalresearch: What are the nutrients that the parasites derive from their hosts?How important are the sterile appendages to the water balance of thethalli and what are the consequences on the development of the abovementioned medium?P a g e | 43

5. CONCLUSION AND SUGGESTIONSMany attempts to amplify ITS sequences of Laboulbeniales failed, also theprotocols used in the current research. Contaminations have been found, likeothers did, belonging to Ascomycota, Dothideomycetes (Davidiella),Saccharomycetes (Pichia, Candida, Debaryomyces) and Basidiomycota,Agaricomycetes (Mycena).It is suggested that the protocol outline in both WEIR & BLACKWELL (2001a) andWEIR & BLACKWELL (2001b) is incomplete and unreliable.Different suggestions are given:Infected hosts should be treated with EDTA and mannose, as additionalpurification steps;Laboulbenialean-specific primers should be investigated;A medium should be created in which Laboulbeniales can form stablepopulations;Biochemical research should be done of Laboulbeniales in order to giveanswers about the appendages and water balance of the thalli.P a g e | 44

PART IIILABOULBENIALESOFCARRION BEETLES:A PRELIMINARY STUDYP a g e | 45

1. INTRODUCTION1.1. FORENSIC ENTOMOLOGYForensic science is a multidisciplinary field, comprising different kinds ofapproaches (MILDENHALL et al., 2006). One field of forensic science is that offorensic entomology, combining the study of insects and other arthropods withthe criminal investigation. When forensic entomology is involved, the insectsbecome part of the evidence.The major use of forensic entomology is to estimate the time of death or thepost mortem interval (PMI) of a corpse, based on the insects invading thedead body. Both the time it has taken for the insects to reach the body forlaying eggs and the rate of development need to be considered whenestimating a (minimum) PMI.Species identification is the most crucial element in forensic entomology.Species that appear almost the same to the naked eye may have differentgrowth rates, behaviours and habitat preferences. Members of the ordersDiptera and Coleoptera are vital in forensic entomology (cfr. Table XII).Major fly familiesCalliphoridaeSarcophagidaeMuscidaePiophilidaeScathophagidaeSepsidaeSphaeroceridaeStratiomyidaePhoridaePsychodidaeTable XII: Important fly and beetle families for forensic entomology.Major beetle families(order Diptera)(order Coleoptera)SilphidaeDermestidaeStaphylinidaeHisteridaeCleridaeTrogidaeScarabaeidaeNitidulidae1.2. ANIMAL CADAVERS1.2.1. SOURCES OF BIODI VERSI TYAn animal consists for the greater part of carbohydrates, lipids and proteins.When it dies, these high quality nutrients become available for otherorganisms. The cadaver offers a temporary living for many invertebrates.Animal cadavers represent the most important source of biodiversity (pers.comm. DIRK RAES, 2010). MELIS et al. (2004) suggest to leave cadavers in foreststo better stimulate a natural environment, since they have a significantecological impact.1.2.2. THE „ROTTING‟ OF A CADA VER (SC H I L T H UI Z E N & V A L L E N D U U K, 1998)The cadaver of an animal undergoes quick and considerable changes. This„rotting‟ takes place in a few stages:P a g e | 46

Figure XII: Stages of the „rotting‟ of different cadavers. Top left: fresh stage. Top middle, right: earlydecomposition. Bottom left: late decomposition. Bottom middle, right: dry stage. Pictures byAgentschap voor Natuur en Bos (2008) and Danny Haelewaters (2010).1.2.2.1. The fresh stageThe fresh stage begins immediately after the cessation of the heart. Thecadaver looks completely normal (cfr. Figure XII, top left); there‟s not yet any(bacterial) decomposition. During this stage, which lasts one to two days, thefirst flies and staphylinids start to encroach upon the cadaver. These beetlesare saprophagous, feeding on decaying and dead organic matter.Oxygen present in the body is quickly depleted by the aerobic organismsfound within. This creates the ideal environment for the anaerobic organisms,transforming carbohydrates, lipids and proteins to yield organic acids andgases. This process of microbial proliferation is referred to as putrefication andleads to the second stage.1.2.2.2. The bloat stageThis second stage is characterisized by bacterial decomposition, thus gaseswithin the cadaver are formed giving the cadaver a swollen appearance anda strong scent. As the pressure of the gases within the cadaver increases,fluids are forced to escape from natural orifices such as the nose, mouth andanus. This pressure may also cause rupturing of the skin. Fly eggs and younglarvae (maggots) are present. Maggot feeding, together with theaccumulation of gases, will lead to post-mortem ruptures of the skin, furtherallowing fluids to escape into the surrounding environment. Also the hair willdetach from the skin, due to maggot activity. The first carrion beetles arecoming to the cadaver: silphids, histerids and large staphylinids.1.2.2.3. The early decompositionThis stage is characterized by great mass losses, resulting from the feeding ofthe maggots and the purging of fluids into the surrounding environment, theso-called cadaver decomposition island (CDI) (CARTER et al., 2007). Thenumber of maggots is enormous: the whole cadaver can be filled withmaggots (cfr. Figure XII, top middle, right).P a g e | 47

Different groups of carrion beetles attain their maximum densities. The actualdecomposition of a corpse is a bacterial process which requires oxygen. Onlywhen the cadaver is riddled by the activity of insects (maggots), oxygen isable to reach the interior; aerobic microorganisms can do their work anddecomposition begins. The internal temperature increases significantly abovethe ambient temperature (MELIS et al., 2004), therefore creating amicroclimatic patch. The cadaver is starting to look very dirty now. Body fluidsare abundantly released, the skin detaches.1.2.2.4. The late decompositionIn this stage, all the meat has gone and the skin has completely released. Themaggots are migrating away from the cadaver to pupate (cfr. Figure XII,bottom left). Large staphylinids predate on the remaining maggots and largescavengers – fox, wild boar, badger, raven – appear around the cadaver.After three to nine weeks, the cadaver is largely desintegrated.The CDI will display an increase of soil carbon and nutrients.1.2.2.5. The dry stageThe last stage begins when the last maggot leaves the cadaver. All thatremains is hard, dry skin, cartilage and bones (cfr. Figure XII, bottom right).Some specialized fungi, such as hoof fungus (Onygena equina), can end upupon the remains. Until now, little is known about this. The remaining bonesare a rich source of calcium and phosphorus for animals.If all soft tissue has been removed from the cadaver, it will be referred to ascompletely skeletonized.Depending on the temperature, humidity, body volume and activity ofscavengers, the „rotting‟ process takes several weeks to many months.Until now, few is known about possible preferences of beetles associated withdecomposing cadavers of specific species. More research and moreover acomparative study between carrion beetles upon cadavers of deer, foxes,pigs and other species is needed.It is worth studying cadavers, at least if no artificial interventions occur: thebody should remain as intact as possible, free from any human disturbance.This way large numbers of insects and scavengers can be observed and animpression of the natural succession of species can be obtained (VAN WIELINK,2004).1.2.3. PROJECT DOOD DOET LEVENIn june 2008, Agentschap voor Nauur en Bos started the project Dood doetLeven in the Sonian Forest, in addition to a similar project in the naturallandscape Ooijpolder (Nijmegen, The Netherlands). Recently, the NationalInstitute for Criminalistics and Criminology (Brussels, Belgium) became a partnerof the project.P a g e | 48

When an animal dies in an accident, Dirk Raes, conservator in the SonianForest, is being informed by the police or fire department. The animal isplaced in the forest on a spot that is not accessible to the public. Dirk is theonly one who knows exactly where the cadavers are situated. Nearby eachcadaver a (hidden) camera is placed, recording every movement. Maggotsand beetles are collected by both hand capturing and simple pitfall traps andsubsequently identified up to species level.There is still some taboo around dead animals in forests. However, a deadanimal gives life: flies are the first ones to approach the cadaver, they lay eggsand thus give rise to maggots, which in addition attract beetles (pers. comm.DIRK RAES, 2010). Next to these smaller fauna, there are the foxes using thebones to sharpen their teeth and the buzzards eating some last pieces ofmeat. This way cadavers accounts for a welcome meal and/or residence fornatural scavengers.The project Dood doet Leven in the Sonian Forest has three intentions:To study the insect, beetle and mammal species present upon and/or in theneighborhood of cadavers;To describe and analyse the currents status of biodiversity, following theEuropean “Regional Nature Conservation Objectives” project (directive92/43/EEG);To make the public familiar with the presence of dead animals in nature.1.3. DESCRIPTION OF COLLECTING SITES1.3.1. SONIAN FOREST (DUTCH: ZONIËNWOUD)The Sonian Forest (N50°46‟ – E4°25‟) is a 4.421 ha forest, situated southeast ofBrussels. The forest lies in the Flemish towns Sint-Genesius-Rode, Hoeilaart,Overijse and Tervuren, the Brussels-capital region towns Ukkel, Watermaal-Bosvoorde, Oudergem and Sint-Pieters-Woluwe, and the Walloon townsTerhulpen and Waterloo.The Sonian Forest is part of the scattered remains of the ancient SilvaCarbonaria. The first mention of the Sonian Forest dates from the early MiddleAges (Soniaca Silva) (DUVIVIER, 1862). At the beginning of the 19th century, theforest had a surface of 10.000 ha (present: 4.421 ha). The loss of forest areahas been caused by large-scale deforestation during the first years of theBelgian independency (1830), recent landscape developments and theconstruction of racecourses, infrastructure, roads and railways.The forest consists mainly of European beeches (Fagus sylvatica) and oaks(Quercus sp.). Most of the trees are over 200 years old. The forest wasoriginally inhabited by 46 different mammal species. Of those, sevendisappeared due to human influence and impoverishment of the ecosystem:brown bear, wolf, hazel dormouse, red deer, badger and hare.1.3.2. MEERDAAL FOREST (DUTCH: MEERDAALWOUD)Together with the nearby Heverlee Wood, Meerdaal Forest (N50°48‟ – E4°42‟)represents a clear environmental beacon lying south of Leuven city. Both, aswell as the Sonian Forest, are remnants of the ancient Silva Carbonaria.P a g e | 49

The 1.319 ha Meerdaal Forest has been cut off from the smaller Heverlee Wood(650 ha) by the high way E40 (Brussels – Germany). This high way has resultedin the establishment of the association „Vrienden van Heverleebos enMeerdaalwoud‟.The main tree species are:Beech (Fagus sylvatica) (31%);Scots pine (Pinus sylvestris);English or pedunculate oak (Quercus robur);Northern red oak (Quercus rubra).In open areas, there is a lot of bracken (Pteridium aquilinum) and birch (Betulasp.).The shrub layer is absent. The soil vegetation is scattered and the herb layer ismainly constituted by bracken (Pteridium aquilinum), blackberry (Rubusfructicosus), lily-of-the-Valley (Convallaria maialis) and May Lily (Maianthemumbifolium).P a g e | 50

2. MATERIALS & METHODS2.1. SONIAN FOREST(PR O J E C T DOOD DOET LEVEN)Five cadavers of deer (Capreolus capreolus (Linnaeus)) were placed atdifferent sites by Dirk Raes, conservator in the Sonian Forest:Site 1 (ZO1): Site „oefenrenbaan‟. This site is an open beech forest.Site 2 (ZO2): Reserve Joseph Zwaenepoel, Haras area. This part is an halfopenoak forest.Site 3 (ZO3): Arboretum Groenendaal, location „educatief pad‟.Site 4 (ZO4) consists of beeches.Site 5 (ZO5): Reserve Joseph Zwaenepoel, Haras area. This part is an halfopenoak forest, with solitary dead – and banded – beeches.The cadavers were attached with one or more metal pins in the ground. Thecadaver at site 5 was even placed in a metal cage, so the fox could not dragthe cadaver away.The sites were inspected at 19/05 (site 1 and 2), 16/06 (site 1, 2 and 3), 5/08 (site4) and 13/08/10 (site 5). Inspection consisted at first of the observation withouttouching the cadaver. After about ten minutes, the legs and head were liftedand even the whole cadaver was replaced (in order to collect thecoleopterans underneath the cadaver).Five or six simple pitfall traps were positioned in a circle around each cadaver.All pitfall traps were made of flower pots, of about 10 cm wide and 8 cmdeep. Those were emptied while inspecting the cadavers. All insects weredirectly put into 90% ethanol for storage.Identification of beetlesIdentification of all beetles was done up to species level or at least genus level,using appropriate identification keys:BOEKEN (1987): Identification of Carabidae;FREUDE et al. (1964, 1974): Identification of Staphylinidae;FREUDE et al. (1979): Identification of Cleridae;JANSSENS (1960): Identification of Geotrupidae;SCHILTHUIZEN & VALLENDUUK (1998): Identification of Histeridae and Silphidae.Any name changes and updates on taxonomy concerning coleopterans werereviewed using VORST (2010).The material is deposited at the private collection of the author (Chantemerlelès-Grignan,France).Screening for LaboulbenialesAll beetles were screened for the presence of Laboulbeniales by using astereomicroscope (50x).P a g e | 51

2.2. MEERDAAL FOREST(DA TA NA TI O NA L INSTI TUTE FOR CRIMI NA LI S TI C S A ND CRI MI NO LOGY)In a period of almost three years (from August 2003 to March 2006), 13cadavers of the domestic pig (Sus scrofa Linnaeus) were exposed in a specificsite in the Meerdaal Forest, having the following characteristics:Sandy soil covered with beeches (Fagus sylvatica);Open areas with bracken (Pteridium aquilinum) and birch (Betula sp.).The sites were inspected at different moments and in different intervals: initiallyinspection occurred every day. After two weeks, the cadavers wereinspected only twice a week. After four to five weeks in total, the cadaver waschecked once per month for the presence of coleopterans.Inspection happened at first without touching the cadaver. After about tenminutes, the legs and head were lifted and even the whole cadaver wasreplaced (in order to collect the coleopterans underneath the cadaver).Generally, the cadavers were examined on all of their sides in order to collectlarvae and adults of insects (coleopterans and dipterans; the latter notincluded in this research). Most of the collecting happened by capturing byhand, although also some pitfall traps were used. Important for theinterpretation of the results is that only the insects with forensic interest werecollected. All insects were directly put into 90% ethanol for storage.Identification of beetlesIdentification of all beetles was done up to species level or at least genus level,using appropriate identification keys:BOEKEN (1987): Identification of Carabidae;FREUDE et al. (1964, 1974): Identification of Staphylinidae;FREUDE et al. (1966): Identification of Cerambycidae and Chrysomelidae;FREUDE et al. (1967): Identification of Nitidulidae;FREUDE et al. (1969): Identification of Scraptiidae, Scarabaeidae andTenebrionidae;FREUDE et al. (1971): Identification of Ptiliidae;FREUDE et al. (1979): Identification of Cantharidae, Melyridae, Cleridae,Elateridae, Buprestidae, Dryopidae and Dermestidae;FREUDE et al. (1981, 1983): Identification of Curculionidae;JANSSENS (1960): Identification of Geotrupidae;KLAUSNITZER (1997): Identification of Silphidae larvae;SCHILTHUIZEN & VALLENDUUK (1998): Identification of Histeridae and Silphidae;ZEEGERS & HEIJERMAN (2008): Identification of Cerambycidae;Any name changes and updates on taxonomy concerning coleopterans werereviewed using VORST (2010).The material is deposited at the collection of the National Institute ofCriminalistics and Criminology (Brussels, Belgium).Screening for LaboulbenialesAll beetles were screened for the presence of Laboulbeniales by using astereomicroscope (50x).P a g e | 52

3. RESULTS3.1. SONIAN FOREST3.1.1. GENERALDuring the five collecting moments (May 19 to August 13 2010), a total of 224coleopterans were captured by means of pitfall traps and hand capturing. Allof them were identified to species level. An overview of the number ofspecimens and the number of species per family is given in Table XIII below.The column BE / NL in Table XIII represents the number of known species perfamily in Belgium and The Netherlands. A total of 4163 species of coleopteransare recorded in The Netherlands, spread over 96 families (VORST, 2010).Table XIII: Overview of Coleoptera in the Sonian Forest, found upon cadavers of deer.The right column shows the number of species known in Belgium (BE) and TheNetherlands (NL), based on (1) LUC CRÊVECOEUR (pers. comm., 2011), (2) BELGIAN SPECIES LIST(2011) and (3) VORST (2010).Family # specimens # species BE / NLCarabidae 22 4 390 (2) / 372 (3)Cleridae 1 1 12 (2) / 12 (3)Geotrupidae 122 1 8 (2) / 7 (3)Histeridae 33 3 62 (2) / 64 (3)Silphidae 37 7 21 (2) / 21 (3)Staphylinidae 9 6 1060 (1) / 1057 (3)TOTAL 224 22 1553 / 1533Laboulbeniales on carrion beetlesAll of these specimens have been screened for the presence ofLaboulbeniales; no infections were found.Explanation of results:The families are placed in alphabetical order, as well as the subfamilies withineach family, the genera within each (sub)family and the species within eachgenus.An overview of the collected families is given. Per family the number ofspecimens, of species, and of species known from Belgium / The Netherlands ismentioned. For each family an overview is given on the collected species. Foreach species the number of specimens and the collecting period ismentioned, as well as the specific collecting area.For all families an introduction is given.The different sites at the Sonian Forest are abbreviated as follows: AG, site 3(Arboretum Groenendaal); JZH, site 2 and 5 (Reserve Joseph Zwaenepoel,Haras area); OR, site 1 („oefenrenbaan‟); 4, site 4.P a g e | 533.1.2. FAMILY CARABIDAECarabids, also knows as ground beetles, have long legs and typically largeeyes. This is a large family, with more than 40.000 species worldwide, of which

approximately 2.700 are found in Europe. Most species are carnivorous andactively hunt for any invertebrate prey. Only a few species are specialized, likeCalosoma upon caterpillars, Cychrus upon snails, some Dyschirius species uponstaphilinids and Lebia upon larvae of leaf beetles (family Chrysomelidae).Most carabids are nocturnal (Luff, 1978; SABO & POWER, 2002).Coleoptera undergo a complete metamorphosis: egg – larva – pupa - adult.Most species finish the development from egg to adult within one season andhibernate as adults. Members of this family can grow quite old, often a fewyears.Table XIV: Family Carabidae from cadavers in the Sonian Forest.Order ColeopteraSuborder AdephagaSuperfamily CaraboideaFamily Carabidae LatreilleSubfamily CarabinaeSpecies # specimens Location DateCollectionreferenceCarabus Linnaeusauronitens Fabricius 1 JZH 19/05/10 ZO2.5nemoralis Müller 1 JZH 19/05/10 ZO2.6violaceus Linnaeus 194JZHJZH05/08/1013/08/1013/08/10ZO4.5ZO5.4ZO5.9Subfamily HarpalinaeSpecies # specimens Location DateCollectionreferencePterostichus Bonelliniger (Schaller) 1 JZH 13/08/10 ZO5.10Order ColeopteraSuborder PolyphagaInfraorder CucujiformiaSuperfamily CleroideaFamily Cleridae LatreilleSubfamily KorynetinaeNecrobia Olivier3.1.3. FAMILY CLERIDAEMembers of this family, commonly referred to as checkered beetles, have avarierty of habitats and feeding preferences. Most genera are predaceousand feed on other beetles and larvae. However, others are scavengers oreven pollen feeders. Larvae of clerids are predaceous and feed vigorouslybefore pupation.Some species are occasionally found on cadavers in the late decomposition /dry stage.Species # specimens Location DateTable XV: Family Cleridae from cadavers in the Sonian Forest.Collectionreferenceviolacea (Linnaeus) 1 OR 16/06/10 ZO1.11P a g e | 54

Order ColeopteraSuborder PolyphagaInfraorder ScarabaeiformiaSuperfamily ScarabaeoideaFamily Geotrupidae LatreilleGeotrupes Latreille3.1.4. FAMILY GEOTRUPIDAEGeotrupidae include about 620 species distributed in temperate, subtropicaland Asian-tropical regions. Most excavate burrows in which they lay theireggs. Eggs are laid in or upon the provision mass and buried; the developinglarvae feed upon the provisions. A few species communicate by stridulation:rubbing body parts together to make sounds.Members of this family are typically detrivores (feeding on decomposingorganic matter); also occasionally coprophagous.Table XVI: Family Geotrupidae from cadavers in the Sonian Forest.Species # specimens Location Datestercorarius (Linnaeus) 122JZHJZHOR419/05/1019/05/1005/08/1013/08/10CollectionreferenceZO1.4ZO2.1ZO4.3ZO5.6, ZO5.11, ZO5.123.1.5. FAMILY HISTERIDAEHisterids are small beetles, rarely over 10 mm in length, commonly known asclown beetles. Their body form is typically rounded or ovoid. Histerids will hideunder the cadaver in the soil during the day and come out at night to feed.Both adults and larvae feed on maggots. Some species have developed anextensive specialization. For example, Hister helluo only occurs upon alderwhen the larvae of the alder leaf beetle (Agelastica alni, familyChrysomelidae) host and feed on the leafs.Order ColeopteraSuborder PolyphagaInfraorder StaphyliniformiaSuperfamily HisteroideaFamily Histeridae GyllenhaalSubfamily HisterinaeMargarinotus MarseulTable XVII: Family Histeridae from cadavers in the Sonian Forest.Species # specimens Location Datestriola (Sahlberg) 6ORAG19/05/1016/06/10CollectionreferenceZO1.1ZO3.1ASubfamily SaprininaeSpecies # specimens Location DateCollectionreferenceHypocaccus Thomsonrugifrons Paykull 1 AG 16/06/2010 ZO3.1BSaprinus Erichsonsemistriatus (Scriba) 26ORJZH19/05/201019/05/2010ZO1.1ZO2.3P a g e | 55

Order ColeopteraSuborder PolyphagaInfraorder StaphyliniformiaSuperfamily StaphylinoideaFamily Silphidae LatreilleSubfamily NicrophorinaeNicrophorus Fabricius3.1.6. FAMILY SILPHIDAESilphids are also knows as burying beetles. They are large, striking beetles,colored black with often a red or orange marking on the elytra. Silphid adultsmainly consume maggots; the larvae feed on decaying organic material.Silphids are considered to be important to forensic entomologists: since theyare found on cadavers, they can be used to help estimating a post morteminterval (PMI) (CARVALHO et al., 2000).Members of the genus Nicrophorus are unusual among the Coleoptera inexhibiting biparental care of their offspring.Table XVIII: Family Silphidae from cadavers in the Sonian Forest.Species # specimens Location Datehumator (Gleditsch) 24JZH05/08/1013/08/10CollectionreferenceZO4.1ZO5.3investigator Zetterstedt 1 JZH 13/08/10 ZO5.5BSubfamily SilphinaeNecrodes LeachOiceoptoma Leachvespilloides Herbst 10JZHJZH19/05/1013/08/10Species # specimens Location Datelittoralis (Linnaeus) 11thoracicum Linnaeus 5ORJZHJZH4JZH19/05/1013/08/1019/05/1005/08/1013/08/10ZO2.2ZO5.5ACollectionreferenceZO1.3ZO5.8ZO2.4ZO4.4ZO5.14Thanatophilus Leachrugosus (Linnaeus) 1 OR 19/05/10 ZO1.7sinuatus (Fabricius) 3 JZH 13/08/10 ZO5.13.1.7. FAMILY STAPHYLINIDAEStaphylinids, rove beetles, are a very large family with over 46.000 species inthousands of genera. They are primarily distinguished by their short elytra,leaving more than half of the abdomen exposed. The family Staphylinidae isvery old, with fossils from 200 million years old.One can expect that such a large family exposes great variation. Indeed,there is considerable variation among the species concerning size, form andhabitat. Sizes range from 1 to 35 mm. The form generally is elongated, withsome staphylinids having an ovoid shape. Members of the family are knownfrom every type of (coleopteran) habitat. Most staphylinids are predators ofinvertebrates, mostly insects.P a g e | 56

Order ColeopteraSuborder PolyphagaInfraorder StaphyliniformiaSuperfamily StaphylinoideaFamily Staphylinidae LatreilleSubfamily AleocharinaeTable XIX: Family Staphylinidae from cadavers in the Sonian Forest.Species # specimens Location DateOxypoda MannerheimSubfamily OxytelinaeCollectionreferencesp. 1 AG 16/06/10 ZO3.4Species # specimens Location DateCollectionreferenceAnotylus Thomsonsculpturatus (Gravenhorst) 1 OR 19/05/10 ZO1.2Subfamily StaphylininaeSpecies # specimens Location DateCollectionreferenceCreophilus Samouellemaxillosus (Linnaeus) 2 OR 19/05/10 ZO1.5Philonthus Stephenspolitus (Linnaeus) 2 JZH 19/05/10 ZO2.8succicola Thomson 1 JZH 19/05/10 ZO2.7Atenuicornis Rey 2 JZH 19/05/10 ZO2.7B3.2. MEERDAAL FOREST3.2.1. GENERALDuring all collecting moments (August 27 2003 to March 31 2006), a total of 205Coleoptera were captured by pitfall traps and hand capturing. The pitfallsresulted in a great number of Geotrupidae, Carabidae and some big sizedStaphylinidae. All of the collected Coleoptera were identified to species level.An overview of the number of specimens and the number of species perfamily is given in Table XX below.The column BE / NL in Table XX represents the number of known species perfamily in Belgium and The Netherlands. A total of 4163 species of coleopteransare recorded in Netherlands, spread over 96 families (VORST, 2010).Table XX: Overview of Coleoptera in the Meerdaal Forest, found upon cadavers of pig.The right column shows the number of species known in Belgium (BE) and TheNetherlands (NL), based on (1) LUC CRÊVECOEUR (pers. comm., 2011), (2) MARC DELBOL (pers.comm., 2011), (3) BELGIAN SPECIES LIST (2011) and (4) VORST (2010).Family # specimens # species BE / NLBuprestidae 1 1 23 (3) / 28 (4)Cantharidae 1 1 51 (3) / 50 (4)Carabidae 2 2 390 (3) / 372 (4)Cerambycidae 3 3 127 (3) / 88 (4)Chrysomelidae 3 1 334 (1) / 315 (4)Cleridae 9 4 12 (3) / 12 (4)Curculionidae 4 3 677 (2) / 539 (4)P a g e | 57

Dermestidae 8 2 23 (3) / 22 (4)Elateridae 14 7 73 (3) / 76 (4)Geotrupidae 4 1 8 (3) / 7 (4)Histeridae 44 6 62 (3) / 64 (4)Melyridae 2 2 11 (3) / 28 (4)Nitidulidae 15 3 92 (3) / 87 (4)Ptiliidae 1 1 45 (3) / 59 (4)Scarabaeidae 6 3 87 (3) / 93 (4)Scraptiidae 6 3 15 (3) / 15 (4)Silphidae 66 8 21 (3) / 21 (4)Staphylinidae 13 5 1060 (1) / 1057 (4)Tenebrionidae 4 2 41 (1) / 47 (4)TOTAL 205 57 2944 / 2980Laboulbeniales on carrion beetlesAll of these specimens have been screened for the presence ofLaboulbeniales; no infections were found.Explanation of results:The families are placed in alphabetical order, as well as the subfamilies withineach family, the genera within each (sub)family and the species within eachgenus.An overview of the collected families is given. Per family the number ofspecimens, of species, and of species known from Belgium / The Netherlands ismentioned. For each family an overview is given on the collected species. Foreach species the number of specimens and the collecting period ismentioned, as well as the specific collecting area.For all families that were not yet discussed in 3.1. SONIAN FOREST an introduction isgiven.3.2.2. FAMILY BUPRESTIDAEBuprestidae are a family of beetles, referred to as jewel beetles. Theiridescence common to these beetles is due to physical iridescence in whichmicroscopic texture in the cuticle reflects specific frequencies of light inparticular directions. Adult buprestids are often found on plants and trees. Thelarvae dig tunnels in the wood. Therefore, the larvae have a strongly enlargedand broadened prothorax, enabling them to maneuver in their tunnels. Jewelbeetles are mostly found on beech, birch, oak, bramble bushes, wiillows androses.Table XXI: Family Buprestidae from cadavers in the Meerdaal Forest.Order ColeopteraSuborder PolyphagaInfraorder ElateriformiaSuperfamily BuprestoideaFamily Buprestidae LEACHSubfamily AgrinilinaeCollectionSpecies # specimens Location DatereferenceAgrilus Curtisangustulus (Illiger) 1 Cadaver 5 2004 NICC2P a g e | 58

Order ColeopteraSuborder PolyphagaInfraorder ElateriformiaSuperfamily ElateroideaFamily Cantharidae ImhoffSubfamily CantharinaeCantharis Linnaeus3.2.3. FAMILY CANTHA RIDAESoldier beetles are highly desired by gardeners as biological control agents ofa number of pest insects. The larvae feed on soft bodied insects, most ofwhich are pests (caterpillars, grasshoppers, aphids, …).The adults are predators of aphids. They can also feed on nectar and pollen;for that reason cantharids are minor pollinators.Table XXII: Family Cantharidae from cadavers in the Meerdaal Forest.Species # specimens Location DateCollectionreferencesp. 1 Cadaver 6 13/05/04 C6 13.5BJ3.2.4. FAMILY CARABIDAETable XXIII: Family Carabidae from cadavers in the Meerdaal Forest.Order ColeopteraSuborder AdephagaSuperfamily CaraboideaFamily Carabidae LatreilleSubfamily CicindelinaeSpecies # specimens Location DateCollectionreferenceCicindela Linnaeuscampestris Linnaeus 1 Cadaver 1 29/04/04 C1 29.IV.04 1Subfamily TrechinaeSpecies # specimens Location DateCollectionreferenceAsaphidion des Gozisflavipes (Linnaeus) 1 Cadaver 6 19/05/04 C6 19.5.04BJ3.2.5. FAMILY CERAMBYCIDAEThe members of the Cerambycidae represent a great variety, with bodylengths ranging from 2,5 mm (Cyrtinus sp.) to more than 17 cm (Titanusgiganteus). They are referred to as longhorn(ed) beetles, as they possessextremely long antennae. Some species, however, have quite short antennae.There are only few truly defining characteristics for the family as a whole;therefore, the family is taxonomically difficult and the relationships betweenthe different lineages are barely understood.P a g e | 59

Table XXIV: Family Cerambycidae from cadavers in the Meerdaal Forest.Order ColeopteraSuborder PolyphagaInfraorder CucujiformiaSuperfamily ChrysomeloideaFamily Cerambycidae LatreilleSubfamily CerambycinaeSpecies # specimens Location DateCollectionreferenceClytus Laichartingarietis (Linnaeus) 1 Cadaver 4 13/05/04 C4 13.V.04 2Pyrrhidium Fairmairesanguineum (Linnaeus) 1 Cadaver 3 19/05/04 C3 19.5.4BJ 1Subfamily LepturinaeSpecies # specimens Location DateCollectionreferenceStenurella Villiersmelanura (Linnaeus) 1 Cadaver 5 19/07/04 C5 19.7.04 13.2.6. FAMILY CHRYSOMELIDAEThe family of leaf beetles is one of the largest phytophagous – plant-eating –Coleoptera. The tarsal formula is 5-5-5, but appears to be 4-4-4. Both adultsand larvae feed on different plant tissues. Many species are pests ofagriculture, e.g. the Colorado potato beetle (Leptinotarsa decemlineata) andthe common asparagus beetle (Crioceris asparagi).Table XXV: Family Chrysomelidae from cadavers in the Meerdaal Forest.Order ColeopteraSuborder PolyphagaInfraorder CucujiformiaSuperfamily ChrysomeloideaFamily Chrysomelidae LatreilleSubfamily CriocerinaeCollectionSpecies # specimens Location DatereferenceLilioceris Reittermerdigea (Linnaeus) 3Cadaver 2Cadaver 7Cadaver 729/04/0426/04/0405/05/04C2 29.IV.04C7 26.IV.04NICC4Order ColeopteraSuborder PolyphagaInfraorder CucujiformiaSuperfamily CleroideaFamily Cleridae LatreilleSubfamily KorynetinaeKorynetes HerbstP a g e | 603.2.7. FAMILY CLERIDAETable XXVI: Family Cleridae from cadavers in the Meerdaal Forest.Species # specimens Location Datecaeruleus (De Geer) 4Cadaver 4Cadaver 1019/05/0431/03/06CollectionreferenceC4 19.5.04BJC10 31.III.06

Necrobia Olivierruficollis (Fabricius) 1 Cadaver 4 04/11/03 C4 4.XI.03Brufipes (De Geer) 2Cadaver 1Cadaver 509/09/0304/11/03C1 9.IX.03AC5 4.XI.03violaceus (Linnaeus) 2 Cadaver 7 26/05/04 C7 26/05/04 33.2.8. FAMILY CURCULI ONIDAESnout beeltes encompass the largest animal family, with over 40.000 species.Characteristics are the distinctive long snout and geniculate antennae withsmall clubs.Curculionids feed on plants; most species are associated with a narrow rangeof hosts, in many cases a single species. The reproduction of these beetlesoccurs in one single leaf, from deposition of the eggs (in the midrib, exceptRhamphus, whose eggs are deposited in the leaf blade) to pupation of thelarvae and maturation.Table XXVII: Family Curculionidae from cadavers in the Meerdaal Forest.Order ColeopteraSuborder PolyphagaInfraorder CucujiformiaSuperfamily CurculionoideaFamily Curculionidae LatreilleSubfamily BrachycerinaeSpecies # specimens Location DateCollectionreferenceSitona Germarlineatus (Linnaeus) 1 Cadaver 2 16/04/04 C2 16.4.04Strophosoma Billbergcaptitatum (De Geer) 1 Cadaver 6 26/05/04 C6 26.5.4BJ 2melanogrammum (Forster) 2Cadaver 5Cadaver 6200426/05/04NICC2C6 26.5.4BJ 23.2.9. FAMILY DERMESTIDAEAdult skin beetles have round oval shaped bodies covered in scales or setae.The (usually) clubbed antennae fit into deep grooves.Dermestids have a wide variety of habitats and most species are scavengersof animal or plant material, such as:Animal cadavers;Feathers;Dead insects;Pollen;Nests of mammals, birds, bees or wasps;Egg cases of mantids (Dutch: bidsprinkhanen) (Thaumaglossa sp.);Pests of grain (Trogroderma sp.).Dermestids are important in forensic entomology, since the arrival of specificspecies to carrion occurs in a predictable succession. Dermestes maculatus isknown to arrive five to eleven days after death (RICHARDS & GOFF, 1997).Members of this family are also used in museums to clean animal skeletons, asthey eat all the flesh of skulls and bones.P a g e | 61

Table XXVIII: Family Dermestidae from cadavers in the Meerdaal Forest.Order ColeopteraSuborder PolyphagaInfraorder BostrichiformiaSuperfamily BostrichoideaFamily Dermestidae LatreilleSubfamily DermestinaeSpecies # specimens Location DateCollectionreferenceDermestes Linnaeushaemorrhoidalis Küster 5 Cadaver 1 2003 NICC XIVmurinus Linnaeus 3Cadaver 4Cadaver 629/04/0405/05/04C4 29.IV.04C6 5.V.043.2.10. FAMILY ELATERIDAEThe Elateridae (click beetles) constitute a cosmopolitan, diverse and speciesrichfamily of beetles. The larvae of some elaterids are important in agricultureas rhizophagous species feeding on underground parts of different plants, andin silvicultural contexts as saproxylic species feeding on wood decayingorganisms or as predators in woodland environments (MAJKA & JOHNSON, 2008).Much remains to be learned about the Elateridae. Diverse genera such asAmpedus, Limonius, Cardiophorus, and Dalopius are still in need of revisionarystudy, and phylogenetic study for most taxa is lacking.Table XXIX: Family Elateridae from cadavers in the Meerdaal Forest.Order ColeopteraSuborder PolyphagaInfraorder ElateriformiaSuperfamily ElateroideaFamily Elateridae LeachSubfamily ElaterinaeSpecies # specimens Location DateCollectionreferenceAgriotes Eschscholtzlineatus (Linnaeus) 2 Cadaver 1 26/04/04 C1 26.IV.04pallidulus (Illiger) 1 Cadaver 4 13/05/04 C4 13.V.04 4pilosellus (Schönherr) 2Cadaver 1Cadaver 429/04/0405/05/04C1 29.IV.04 2C4 5.V.04sputator (Linnaeus) 2 Cadaver 4 13/05/04 C4 13.V.04 3Subfamily DenticollinaeSpecies # specimens Location DateCollectionreferenceAnostirus Thomsonpurpureus (Poda) 1 Cadaver 3 26/04/04 C3 26.IV.04Athous Eschscholtzhaemorrhoidalis (Fabricius) 1 Cadaver 6 07/04/04 C6 7.4.4BJ Bvittatus (Fabricius) 5Cadaver 5Cadaver 613/05/0407/04/04C5 13/5BJ 1C6 7.4.4BJ BP a g e | 62

Order ColeopteraSuborder PolyphagaInfraorder ScarabaeiformiaSuperfamily ScarabaeoideaFamily Geotrupidae LatreilleGeotrupes Latreille3.2.11. FAMILY GEOTRUPIDAETable XXX: Family Geotrupidae from cadavers in the Meerdaal Forest.Species # specimens Location Datestercorarius (Linnaeus) 4Cadaver 5Cadaver 7Cadaver 8200413/05/0428/07/04CollectionreferenceNICC3C7 13.5BJ 2C8 28/7/04Order ColeopteraSuborder PolyphagaInfraorder StaphyliniformiaSuperfamily HisteroideaFamily Histeridae GyllenhaalSubfamily HisterinaeHister Linnaeus3.2.12. FAMILY HISTERIDAETable XXXI: Family Histeridae from cadavers in the Meerdaal Forest.Species # specimens Location DateMargarinotus (MARSEUL)unicolor Linnaeus 5purpurascens (Herbst) 2striola (Sahlberg) 3Cadaver 2Cadaver 7Cadaver 8Cadaver 8Cadaver 2Cadaver 4Cadaver 2Cadaver 804/08/0413/05/0404/08/0428/07/0404/08/0416/04/0404/08/0404/08/04CollectionreferenceC2 4.8.4BJC7 13.V.04C7 4.8.04BJC8 28.VII.04 A2C2 4.8.4BJC4 16.4.04C2 28/7/04C8 4.8.04BJ Aventralis (Marseul) 1 Cadaver 2 04/08/04 C2 4.8.4BJSubfamily SaprininaeSpecies # specimens Location DateCollectionreferenceHypocaccus Thomsondimidiatus dimidiatus (Illiger) 3 Cadaver 8 04/08/04 C8 4.8.04BJ ASaprinus Erichsonsemistriatus (Scriba) 30Cadaver 1Cadaver 1Cadaver 6Cadaver 8Cadaver 827/08/0309/09/0307/04/0404/08/0428/07/04C1 27.VIII.03C1 9.IX.03 CC6 7.4.4BJ DC8 4.8.04BJ AC8 28.VII.04 A13.2.13. FAMILY MELYRIDAESoft-winged flower beetles possess soft wing-covers, which are posteriorlybroader than towards the anterior. Both the head and pronotum are ratherwide. Members of this family are variously colored; the body is often coveredwith fine hairs.P a g e | 63

Adults prey on other flower-visiting insects, or may feed on pollen. The larvaeare carnivorous, or scavenge for dead animal material.Table XXXII: Family Melyridae from cadavers in the Meerdaal Forest.Order ColeopteraSuborder PolyphagaInfraorder CucujiformiaSuperfamily CleroideaFamily Melyridae LeachSubfamily DasytinaeSpecies # specimens Location DateCollectionreferenceDasytes Paykullcaeruleus (De Geer) 1 Cadaver 5 05/05/04 C5 5.V.04 2Subfamily MalachinaeSpecies # specimens Location DateCollectionreferenceMalachius Fabriciusbipustulatus (Linnaeus) 1 Cadaver 5 2004 NICC23.2.14. FAMILY NITIDULIDAESap beetles are small (2 to 3 mm in length) and colored black or brown. Somespecies have red spots on the elytra. Nitidulids belong to the Coleoptera thatare mostly found on flowers. However, the different species live in quitedifferent habitats and therefore maintain different lifestyles (DE OUDE, 1999).Being such a divergent group, Nitidulidae exhibit one of the most diversefeeding strategies among Coleoptera. They feed on flowers, but also ondecaying vegetation, carrion, dung, fungi and insects.Table XXXIII: Family Nitidulidae from cadavers in the Meerdaal Forest.Order ColeopteraSuborder PolyphagaInfraorder CucujiformiaSuperfamily CucujoideaFamily Nitidulidae LatreilleSubfamily MeligethinaeSpecies # specimens Location DateCollectionreferenceMeligethes Stephens(cf.) aeneus (Fabricius) 1 Cadaver 6 20/04/04 C6 20.4.04Subfamily NitidulinaeOmosita Erichsoncarinulatus Förster 12Cadaver 5Cadaver 519/05/0419/05/04Species # specimens Location Datecolon (Linnaeus) 2Cadaver 1Cadaver 809/09/0304/08/04C5 19.5.4BJ 1C5 19.5.4BJ 3CollectionreferenceC1 9.IX.03 BC8 4.8.4BJ CP a g e | 64

Order ColeopteraSuborder PolyphagaInfraorder StaphyliniformiqSuperfamily StaphylinoideaFamily Ptiliidae HeerSubfamily PtiliinaePtiliolum Flach3.2.15. FAMILY PTILIIDAEThe family of feather-winged beetles is a highly diverse yet poorly known groupof minute beetles with narrow and feather-like hindwings. Adult ptiliids areextremely small, ranging from 0,4 to 1,3 mm in length; the largest tropicalgenera may reach 2,0 mm in length. The members are only reliably identifiedon the basis of the female genital (CUPPEN & VORST, 2002). Compared to theadults, eggs of feather-winged beetles are very large. Due to this, only oneegg at a time is developed and laid. Some genera are known to beparthenogenetic (Acrotrichis, Bambara, Ptiliopycna).Currently there are 630 described species in about 85 genera, but there still is alot of work to do: large numbers of specimens in collections are waiting to bedescribed; the true number of species is consequently highly underestimated.Both the adults and larvae are found together in diverse habitats includingmoist leaf litter, mammal nests, rotting cacti, ant and termite colonies, deadtrees, sand, and others containing rotting organic material. Some species feedon the spores of particular fungi.Table XXXIV: Family Ptiliidae from cadavers in the Meerdaal Forest.Species # specimens Location DateCollectionreferencesp. 1 Cadaver 1 19/07/04 C1 19.7.4BJ3.2.16. FAMILY SCARABAEIDAEScarabs (Dutch: bladsprietkevers) have stout-shaped bodies, many of themwith bright metallic colors. They possess distinctive, clubbed antennaecomposed af specific plates. Those „lamellae‟ can be compressed into a ballor unfolded in order to sense odors. The front legs of many species arebroadened and adapted for digging.The scarabid family comprises over 30.000 species. The larvae, called grubs,are sluggish, c-shaped, pale yellow or white, with a well-developed head andthoracic legs. There are two feeding strategies:The larvae feed on roots, sap and decaying wood while the adults eatleaves and flowers;Both the larvae and adults feed on carrion, dung, skin and feathers.Most adults are nocturnal, although some genera are only active during theday. The grubs mostly live underground or under debris; they are neverexposed to sunlight.P a g e | 65

Table XXXV: Family Scarabaeidae from cadavers in the Meerdaal Forest.Order ColeopteraSuborder PolyphagaInfraorder ScarabaeiformiaSuperfamily ScarabaeoideaFamily Scarabaeidae LatreilleSubfamily AphodiinaeSpecies # specimens Location DateCollectionreferenceAphodius Illigersp. 1 Cadaver 7 26/04/04 C7 26.IV.04sp. 1 Cadaver 3 18/09/04 Cochon 3 18/09 Coléosp. 4 Cadaver 5 14/10/03 C5 14.X.2003Order ColeopteraSuborder PolyphagaInfraorder CucjiformiaSuperfamily TenebionoideaFamily Scraptiidae MulsantSubfamily AnaspidinaeAnaspis Geoffroy3.2.17. FAMILY SCRAPTIIDAEThe family Scraptiidae represents a group of Coleoptera with uncertainaffinities (LESAGE, 1991). Species of this family have previously been placed infamilies as Morbillidae, Melandryidae or Anthicidae; they are now consideredto be a closely related but separate family.Adults are often found on flowers; the larvae occur in decaying wood or in leaflitter.Table XXXVI: Family Scraptiidae from cadavers in the Meerdaal Forest.Species # specimens Location DateCollectionreferencesp. 1 Cadaver 5 19/05/04 C5 19.5.4BJ 2sp. 4 Cadaver 5 26/05/04 C5 26.5.04BJ 1sp. 1 Cadaver 6 19/05/04 C6 19.5.04BJOrder ColeopteraSuborder PolyphagaInfraorder StaphyliniformiaSuperfamily StaphylinoideaFamily Silphidae LatreilleSubfamily NicrophorinaeNicrophorus Fabricius3.2.18. FAMILY SILPHIDAETable XXXVII: Family Silphidae from cadavers in the Meerdaal Forest.Species # specimens Location Datehumator (Gleditsch) 8Cadaver 6Cadaver 8Cadaver 9Cadaver 1107/04/0404/08/0410/10/0531/03/06CollectionreferenceC6 7.4.4BJ AC8 4.8.4BJ BC9 10.X.2005C11 31/III/06investigator Zetterstedt 1 Cadaver 8 04/08/04 C8 4.VIII.04P a g e | 66

Subfamily SilphinaeNecrodes Leachvespilloides Herbst 2Cadaver 4Cadaver 529/09/0319/07/04Species # specimens Location Datelittoralis (Linnaeus) 38Cadaver 3Cadaver 4Cadaver 4Cadaver 7Cadaver 809/03/0408/10/0307/07/0426/05/0404/08/04C4 29.IX.03C5 19.7.04 2CollectionreferenceC3 09/03 Larves ColéoC4 8.X.03 larvesC4 7.7.04BJC7 26/05/04 4C8 4.8.04BJ BOiceoptoma Leachthoracicum Linnaeus 1 Cadaver 6 07/04/04 C6 7.4.4BJ CThanatophilus Leachrugosus (Linnaeus) 2sinuatus (Fabricius) 14Cadaver 3Cadaver 7Cadaver 4Cadaver 5Cadaver 5Cadaver 7Cadaver 719/05/0426/05/0413/05/0413/05/0416/04/0413/05/0426/05/04C3 19.5.4BJ 2C7 26/05/04 2C4 13.V.04 1C5 13/5BJ 2C5 16.4.04C7 13.5BJ 1C7 26/05/04 1 + 23.2.19. FAMILY STAPHYLINIDAETable XXXVIII: Family Staphylinidae from cadavers in the Meerdaal Forest.Order ColeopteraSuborder PolyphagaInfraorder StaphyliniformiaSuperfamily StaphylinoideaFamily Staphylinidae LatreilleSubfamily OmaliinaeOmalium GravenhorstSpecies # specimens Location DateSubfamily Oxytelinaerivulare (Paykull) 7Cadaver 1 + 4Cadaver 9200318/11/05Species # specimens Location DateCollectionreferenceNICCXIIC9 18.XI.05CollectionreferenceAnotylus Thomsonrugosus (Fabricius) 1 Cadaver 4 14/10/03 C4* 14.X.2003Subfamily PaederinaeSpecies # specimens Location DateCollectionreferenceLathrobium GravenhorstLathrobium s. str. 1 Cadaver 7 26/04/04 C7 26.IV.04Subfamily StaphylininaeSpecies # specimens Location DateCollectionreferenceP a g e | 67

Creophilus Samouellemaxillosus (Linnaeus) 3Cadaver 4Cadaver 7Cadaver 826/09/0326/05/0404/08/04C4 26.IX.03C7 26/05/04 1C8 4.8.4BJ APhilonthus Stephenstenuicornis Rey 1 Cadaver 3 26/04/04 C3 26.IV.043.2.20. FAMILY TENEBRIONIDAEThis family displays a broad range of variability, in both shape and size. Manymembers have black elytra. Typical is that the tarsi of the hind pair have foursegments, while the tarsi of fore and mid legs possess five segments (tarsalformula: 5-5-4). They eat both fresh and decaying vegetation. Majorpredators include birds, rodents, sunspiders and lizards.Both the adults and larvae feed on dead organic material and carrion. A wellknownspecies is the mealworm beetle (Tenebrio molitor), which is a pest inrepositories of flour and grain. The larvae of this species, the so-calledmealworms, are used as a food source for reptile, fish and birds.Table XXXIX: Family Tenebrionidae from cadavers in the Meerdaal Forest.Order ColeopteraSuborder PolyphagaInfraorder CucjiformiaSuperfamily TenebionoideaFamily Tenebrionidae LatreilleSubfamily LagriinaeSpecies # specimens Location DateCollectionreferenceLagria Fabriciushirta (Linnaeus) 1 Cadaver 5 2004 NICC2Nalassus Mulsantlaevioctostriatus (Goeze) 3Cadaver 5Cadaver 1205/05/042005C5 5.V.04 1NICC1P a g e | 68

4. DISCUSSION4.1. METHODSAlthough forensic entomology is used to aid murder investigations, only fewrecent literature results on observing cadavers is available. However, theknowledge obtained from this kind of research (in combination with dataconcerning the development of the different species) is necessary in order toestimate the (minimum) post mortem interval (PMI) and consequently the timeof death (ASISTM, 2009).Forensic entomology is generally a field concentrating on Diptera. Manycoleopterans also have forensic applications, but are often neglected byforensic entomology researchers (MIDGLEY, 2007). It is important to realize thatthe collection of Coleoptera is both semi-qualitative and semi-quantitative,since they run away with the least interference (VAN WIELINK, 2004). Also theweather has some great influence on decomposition and thus the populationsof insects. In this research, for both the experiments in the Sonian Forest andthe Meerdaal Forest no details concerning the weather have beenconsidered.4.2. IDENTIFICATION OF BEETLESThe identification of a few specimens took place only to genus level. This holdsfor Anaspis (Scraptiidae), Aphodius (Scarabaeidae), Cantharis (Cantharidae),Oxypoda (Staphylinidae) and Ptiliolum (Ptiliidae). Identification to species levelrequires the fullest understanding of the respective genera, including specificshapes of the male reproductive system, minor differences between tarsalsegments, … For example, the genus Aphodius comprises 86 species in 27subgenera, classified according the shape of the hind tarsi, the shape of thehead and the morphology of the male reproductive system.One of the problems for forensic investigators is the accurate identification ofthe larvae collected from a corpse. For this research, the identification ofSilphidae larvae was based on the identification key in KLAUSNITZER (1997).However, according to this identification key some characteristics of thelarvae corresponded with Necrodes, other with Thanatophilus. Even more, thewhole table of Silphidae larvae in KLAUSNITZER (1997) is problematic (pers.comm. HANS HUIJBREGTS, 2010). The larvae were finally determined as Necrodes(littoralis), based on their size. (The studied larvae were larger than mostThanatophilus adults.)The identification of Thanatophilus species is not evident. Thanatophilusrugosus is recognizable by its wrinkled elytra. Both T. dispar and T. sinuatus aredifficult to distinguish. Since T. dispar is rare (since 1960 only two observations inThe Netherlands; no observations found in Belgium), we could suggest ourspecimens to be T. sinuatus. Not the identification key (SCHILTHUIZEN &VALLENDUUK, 1998) but the species-specific shape of the seventh tergite (i.e. withsemicircular notch = T. sinuatus) was conclusive.P a g e | 69

4.3. LABOULBENIALES OF CARRION BEETLESThe discussion regarding Laboulbeniales of carrion beetles is not that simple.No specific research on Laboulbeniales of carrion beetles has been done sofar. Still, there are some reports of Laboulbeniales on carrion beetles. MAJEWSKI(2003) mentions seven species:Asaphomyces tubanticus on Catops coracinus, C. fuscus, C. westii,Nemadus colonoides, Sciodrepoides fumatus, S. watsoni (Catopidae): 9records;Dimeromyces balazucii on Scaphidema metallicum (Tenebrionidae): 1record;Ecteinomyces trichopterophilus on Acrotrichis and Baeocrara (Ptiliidae): 4records;Laboulbenia notiophili on Nothiophilus (Carabidae): 1 record on beetlecollected upon cadavers; accidental;Laboulbenia pseudomasei on Patrobus and Pterostichus (Carabidae): 1record on beetle collected upon cadavers; accidental;Rhachomyces philonthinus on Philonthus and Spatulonthus (Staphylinidae):4 records;Siemaszkoa valida on Ptenidium nitidum (Ptiliidae) : 1 record.The hosts of Asaphomyces tubanticus (Middelh. & Boelens) Scheloske, allmembers of the Catopidae (Coleoptera), occur in cadavers (and also often infungal fruiting bodies) (MAJEWSKI, 2003). Consequently, this species is until nowconsidered the only species systematically occurring on carrion beetles. A.tubanticus has already been observed in Belgium, on Catops fuscus, C.longulus, C. marginicollis and C. nigricans (RAMMELOO, 1986; DE KESEL &RAMMELOO, 1992; DE KESEL, 1997). In the current research, however, no Catopsspecies have been found.Both Dimeromyces balazucii W. Rossi & Cesari and Siemaszkoa valida T.Majewski have not yet been recorded in Belgium.In this work, no species of Laboulbeniales have been recorded on theexamined insects. Based on the current results (3.1. SONIAN FOREST and 3.2.MEERDAAL FOREST) and on SCHELOSKE (1969), DE KESEL & HAGHEBAERT (1991), DE KESEL(1997) and MAJEWSKI (2003) a parasite-host list for Laboulbeniales on carrionbeetles in Belgium can be drawn (cfr. Table XL below). Only the hosts inSCHELOSKE (1969) and MAJEWSKI (2003) that were also observed in the currentresearch were selected. Thereafter the species of Labulbeniales on those hostsalready present in Belgium were excluded using DE KESEL & HAGHEBAERT (1991)and DE KESEL (1997).Table XL: Parasite-host list for Laboulbeniales on carrion beetles in Belgium, based onthe results of the current research and on SCHELOSKE (1969), DE KESEL & HAGHEBAERT (1991),DE KESEL (1997) and MAJEWSKI (2003). Species in bold have not yet been recorded inBelgium.Parasite HostEuzodiomyces lathrobii Thaxt.Peyritschiella dubia (Thaxt.) I.I. Tav.Rhachomyces pilosellus (C.P. Robin) Thaxt.Lathrobium brunnipes, fovulum, elongatum,geminum, longulumPhilonthus politusLathrobium fulvipenne, geminumP a g e | 70

Peyritschiella princeps (Thaxt.) I.I. Tav.Peyritschiella protea Thaxt.Rhadinomyces cristatus (Thaxt.)Rhadinomyces pallidus Thaxt.Symplectromyces vulgaris (Thaxt.) Thaxt.Philonthus politusAnotylus rugosusLathrobium brunnipes, castaneipenne, fulvipenne,geminumLathrobium brunnipesPhilonthus sp.Lathrobium brunnipes has been recorded several times in Belgium (C RÊVECOEURet al., 2004), also parasitized ones: infections with Euzodiomyces lathrobii andRhadinomyces cristatus (D E K ESEL & H AGHEBAERT , 1991; D E K ESEL , 1997). However,no Lathrobium brunnipes specimens infected with Rhadinomyces pallidushave yet been found in Belgium.Important to mention is that cadavers are ephemeral substrates (cfr. 1.2.2. T HE„ ROTTING ‟ OF A CADAVER ). The time for coleopterans to arrive at the cadaver,develop and form new generations is not long enough in order to form stablepopulations.The CDI (cfr. 1.2.2.3. The early decomposition) of the late decomposition stagedisplays increasing soil carbon and nutrients compared to the earlydecomposition stage. This CDI composition thus changes during the differentstages of the „rotting‟ of a cadaver. This effect may exacerbate the inability todevelop stable beetle populations. The species of beetles in the studied areasbelong to different phenology and generation groups, which means that thespecies composition is able to change during the different stages ofdecomposition. Species characteristic of the bloat stage are replaced byother species in the early decomposition and even others in the latedecomposition.A number of these coleopterans has also been observed in plant remains,rotting fruiting bodies of fungi, animal and human feces, decomposing wood,all ephemeral substrates. This is indeed the case for the species ofLaboulbeniales on carrion beetles reported by M AJEWSKI (2003).Carrion beetles have all kind of different habitats (see above), includinghibernation sites, suggesting the difficulty for Laboulbeniales to adapt tocarrion beetles, since Laboulbeniales have their own environmentalpreferences (DE KESEL, 1996).4.4. SPECIES WORTH MENTIONINGThe carrion beetle Necrodes littoralis has been suggested to occur upon acadaver for only a short time (VAN WIELINK, 2004). However, observations at theMeerdaal Forest are inconsistent with this hypothesis. Necrodes littoralis hasbeen recorded upon cadaver 4 in October 2003 (19 larvae) and July 2004 (1adult specimen). According to SCHILTHUIZEN & VALLENDUUK (1998) this speciesoccurs on cadavers of rabbit, dog, goat, deer, sheep, fish and chicken.The current research adds cadavers of pig to that list, regarding the records inthe Meerdaal Forest. The adults of Necrodes littoralis are nocturnal. Collectionof coleopterans happened during daylight, which can be the explanation ofthe fact that only one adult specimen was found upon cadaver 4.P a g e | 71

The knowledge about the preferences of carrion beetles is limited. However,the suggestion that the species of the genus Nicrophorus can breed only in thepresence of rodent carrion (MELIS et al., 2004) is rejected since a total of 24representatives of Nicrophorus are collected in both the Sonian and MeerdaalForest (N. humator, N. investigator, N. vespilloides). Our results are confirmedby VAN WIELINK (2004), who mentions Nicrophorus upon cadavers of fox anddeer.The following observed species are not (yet) present in the BELGIAN SPECIES LISTdatabase (2011):Pyrrhidium sanguineum (Cerambycidae Meerdaal Forest, C3 19.5.4BJ 1)Dermestes haemorrhoidales (Dermestidae Meerdaal Forest, NICC XIV)Dasytes caeruleus (Melyridae Meerdaal Forest, C5 5.V.04 2)Pyrrhidium sanguineum and Dasytes caeruleus, however, have already beenobserved in Belgium by Hugo Raemdonck (unpublished data). Pyrrhidiumsanguineum is even suggested to be a common species in Belgium (MUYLAERT,1984). Dermestes haemorrhoidales is known in seven of the twelve provincesin The Netherlands, also those bordering Belgium; it has also been frequentlyobserved in De Kaaistoep (VORST, 2010; pers. comm. PAUL VAN WIELINK, 2011). Itis unlikely that this species would be new for the Belgian beetle fauna; morelikely is the suggestion that the BELGIAN SPECIES LIST (2011) is underprovided, evenfar from complete.26 specimens of Saprinus semistriatus (Histeridae) have been observed oncadavers of deer in the Sonian Forest, 30 specimens on cadavers of pigs in theMeerdaal Forest. Consequently the suggestion that S. semistriatus is verycommon on all cadavers and habitats (SCHILTHUIZEN & VALLENDUUK, 1998;TROUKENS, 2005) is confirmed.Representatives of Buprestidae, Cantharidae, Cerambycidae, Chrysomelidae,Curculionidae, Elateridae, Scraptiidae – 7,5% of all specimens collected at thecadavers – were most likely randomly present. The other species are reportedto be specifically adapted to carrion, to consume any kind of decayingmaterial (adults and/or larvae) or to be carnivorous.4.5. DIFFERENCES BETWEEN SONIAN FOREST AND MEERDAALFORESTA total of 79 species from 19 coleopteran families were found: 22 in the SonianForest and 57 in the Meerdaal Forest. Although the total of specimens in bothforests was nearly the same, the number of represented families in theMeerdaal Forest was significantly higher (cfr. Table XLI below).P a g e | 72

Table XLI: Comparison between Sonian Forest and Meerdaal Forest.Sonian Forest (deer)Meerdaal Forest (pig)Family # specimens # species # specimens # speciesBuprestidae - - 1 1Cantharidae - - 1 1Carabidae 22 4 1 1Cerambycidae - - 3 3Chrysomelidae - - 3 1Cleridae 1 1 9 4Curculionidae - - 4 3Dermestidae - - 8 2Elateridae - - 14 7Geotrupidae 122 1 4 1Histeridae 33 3 44 6Melyridae - - 2 2Nitidulidae - - 15 3Ptiliidae - - 1 1Scarabaeidae - - 6 3Scraptiidae - - 6 3Silphidae 37 7 66 8Staphylinidae 9 6 13 5Tenebrionidae - - 4 2TOTAL 224 22 205 57Notably more Geotrupes specimens have been recorded in the Sonian Forest.However, when recorded that much in the Sonian Forest, one should expectthem to also be present in large numbers in the Meerdaal Forest, since theytypically feed on decomposing organic matter.Also noteworthy is the difference in specimens of carabids: 22 in the SonianForest against only 1 in the Meerdaal Forest.However, in the Meerdaal Forest only the Coleoptera with forensic applicabilitywere collected: many Carabus violaceus and Geotrupes species wereobserved but not collected (YVES BRAET, pers. comm., 2011). Therefore, thenumber of specimens collected in each forest cannot be compared since theprotocols were different. It is suggested to replicate this research, focusing onthe following issues:(1) A single protocol should be used in both the Sonian Forest and MeerdaalForest;(2) Three plots should be studied in each forest, with all plots in the samevegetation type: a plot with deer cadaver, a plot with pig cadaver and acontrol plot;(3) Regarding the influence during the weather (cfr. 4.1. WEATHER), allobservations should be done in the same season, under similar weatherconditions (temperature, humidity), at the same time in the course of aday.Adapting Table XLI following Table XII (in 1.1. FORENSIC ENTOMOLOGY) results in atable only comprising carrion beetles, thus with forensic applicability:P a g e | 73

Table XLII: Comparison of carrion beetle families between Sonian Forest and MeerdaalForest.Sonian Forest (deer)Meerdaal Forest (pig)Family # specimens # species # specimens # speciesCleridae 1 1 9 4Dermestidae - - 8 2Histeridae 33 3 44 6Nitidulidae - - 15 3Scarabaeidae - - 6 3Silphidae 37 7 66 8Staphylinidae 9 6 13 5TOTAL 80 17 161 31No Trogidae have been recorded, although those beetles have beendescribed as being frequently present in the proximity of cadavers. DUCHATELET (1986) estimates the number of Western European species on twelve.Four species are present in Belgium, only one of which in the locality of Brussels(TROUKENS, 2007).Since all Coleoptera with forensic significance were collected in both theSonian Forest and Meerdaal Forest, it is no problem to compare the results ofthe two forests with each other. One should not forget, however, that thereare also other factors to be taken into account: soil, forest composition,season, temperature, humidity, ...Reducing the original table of Coleoptera emphasizes even more thedifferences between the two collecting areas. Half of the families with forensicimportance present in the Meerdaal Forest have not been recorded at all inthe Sonian Forest.A very important factor determining insect succession is the geographicalregion where the cadaver is found (ASISTM, 2009). Both the Sonian Forest andthe Meerdaal Forest are remnants of the ancient Silva Carbonaria. However,the soil in the Sonian Forest has some typical characteristics.Soil evolution processes, dating from the time that the climate in these regionswas similar to that in Siberia or northern Canada nowadays, ended some10.000 years ago, along with the ending of the Weichselian glaciation. The soilin the Sonian Forest has never been disturbed by man, resulting in apreservation of the unique soil features. These features include tongue-shapedspots at a depth of 30 to 120 cm. Soil studies have shown that these ferruginousspots are very old bursts, the soil in between being very compact (DE MEYER &LANGOHR, 1984; SANDERS et al., 1985). This means that the root system of treescan only grow superficially, in the humus layer, having both physiological andphysical consequences. All minerals and water available is absorbed by thetrees (physiological root competition). In addition, this superficial root carpetcauses a (physical) lack of space for the evolution of other root systems.The upper 30 cm of the soil in the Sonian Forest displays a dehydration andreduction of all biological processes. In the parts with beech forest, the uppersoil layer, consisting of decomposing leaves, is very thick, since beech leavesP a g e | 74

decompose slowly. The humus layer, only 3 to 5 cm thick, lies beneath andconsists of organic material, with lots of roots running through (LANGOHR &CUYCKENS, 1985). This thin, neutral humus layer indicates the poor soil fauna, assuggested by the results of this research (3.1.1. GENERAL, Table XIII).The Sonian Forest consists for almost 80% of beeches; in the Meerdaal Forest,this number is 31% (1.3. DESCRIPTION OF COLLECTING SITES). Beeches allow a smallamount of light to penetrate to the bottom and therefore, beech forest faunais poor in species. The Meerdaal Forest is a mixed forest, offering a variedfauna (and flora) (VANAUTGAERDEN, 2005).The differences in Coleoptera composition between both forests, will also bedependent on the cadavers themselves, as suggested by EL-KADY (1999) andVAN WIELINK (2004): cadaver species (body composition, e.g. fat ratio, musclemass) and size can have effects on decomposition rate, species compositionand insect succession.Comparing the results of the Sonian Forest with a study in De Kaaistoep (cfr.Part IV, 1.1. NATURAL LANDSCAPE DE KAAISTOEP) leads to the comparison of insectcomposition upon the same cadaver species (deer) in different habitats. Theconclusion of vegetation and soil type affecting the species of insects in theneighborhood of the cadavers, as suggested by the results of the currentresearch and VAN WIELINK (2004), is confirmed.P a g e | 75

5. CONCLUSION AND SUGGESTIONSNo species of Laboulbeniales have been recorded in this research. Havingtheir own restricted environmental preferences, Laboulbeniales may havedifficulties to adapt to carrion beetles, that instead have all kind of differenthabitats. More research is needed to confirm this hypothesis.Until now, Asaphomyces tubanticus is considered the only species ofLaboulbeniales systematically occurring on carrion beetles, only parasitizingmembers of the Catopidae. Based on the current results, there is a potentialfor finding Rhadinomyces pallidus on Lathrobium brunnipes in Belgium. Thehosts are present, also infected ones, parasitized by others than Rhadinomycespallidus; up to now the latter have not yet been recorded.Both Dimeromyces balazucii and Siemaszkoa valida, also previously collectedon carrion beetles, have not yet been recorded in Belgium.Our research has resulted in the following secondary conclusions andsuggestions:Carrion has long been known to entomologists as rich sources for insectspecies. Surprisingly few general studies of the faunal succession onindividual cadavers have been made. Yet, faunal inventory data aredesirably linked to the ecological coherence of the system studied.Cadavers have a significant ecological role.In the current experimental design there are too many uncertainties andinequalities in order to compare carrion beetle fauna. It is suggested toperform a follow-up research, in which three plots will be studied in both theSonian and Meerdaal Forest, taking the effects of the weather intoaccount.The suggestion of Necrodes littoralis occuring upon a cadaver for only ashort time is rejected. Also the hypothesis that species of the genusNicrophorus can breed only in the presence of rodent carrion is rejected.P a g e | 76

PART IVPRELIMINARY CHECKLISTOF LABOULBENIALESIN„DE KAAISTOEP‟P a g e | 77

1. INTRODUCTION1.1. NATURAL LANDSCAPE DE KAAISTOEP1.1.1. DESCRIPTION OF THE SI TEThe natural landscape De Kaaistoep lies immediately west of the urban areaof Tilburg, province Noord-Brabant, in the south of The Netherlands. It belongsto the TWM Gronden BV (Tilburgsche Waterleiding Maatschappij). Since 1994,the agricultural area has been transformed into a more natural landscape(VAN WIELINK, 1999).Figure XIII: The study area in the western part of De Kaaistoep, bounded by the A58motorway. Numbers 1-4 indicate ponds. © Topografische Dienst.The western part of De Kaaistoep, the actual research site, consists of opengrassland on poor sandy soil interrupted by straight rows of deciduous treesand shrubs. Four artificial small ponds were dug in 1994 and a big one ofabout 1 ha (Prikven) in 1998 (cfr. Figure XIII). Other measures were the plantingof indigenous trees, the removal of exotic ones, such as the American blackcherry (Prunus serotina), and mowing of the grasslands in autumn. In thewinter of 2005/2006, the canalized brook at the eastern side (Oude Leij) waspartially restored.P a g e | 781.1.2. BI ODI VERSITY IN DE KAAISTOEPSince 1995, the presence of all kind of organisms, plants, fungi and animals, inDe Kaaistoep has been studied. Considering all plants and animals that havebeen found in De Kaaistoep and identified, there is a total of nearly 7.000species (VAN WIELINK, 2009), of which 75% are animals. De Kaaistoep can beconsidered a hotspot in The Netherlands.

1.1.2.1. Plants and fungi in De KaaistoepThe number of species of plants and fungi found and identified in DeKaaistoep is presented in Table XLIII below.Total (species)Tracheophytes 423 35Bryophyta 156 7Algae 93 -Fungi * 940 54Lichens 70 2TOTAL FLORA 1.682 98* Only macrofungi have been considered.Table XLIII: Overview of plants and fungi in De Kaaistoep.PlantsRed List (species)1.1.2.2. Animals in De KaaistoepThe number of animals in De Kaaistoep is very high, although large groups arenot – or hardly – considered. De Kaaistoep has more than 5.000 identifiedspecies, of which almost 90% are insects (cfr. Table XLIV).In De Kaaistoep, intensive entomological research has been done and is stillgoing on. Work has to be done concerning mites, harvestmen, crustaceans,collembolans, annelids and mollusks.In The Netherlands, some 1.200 species of arachnids, 1.000 species of crustaceans, 196 species ofcollembolans, 180 species of annelids and 200 species of mollusks are known.Total (species)Mammals 27 4Birds 180 31Reptiles 1 1Amphibians 8 2Fish 14 -Insects ± 4.500 43Spiders 250 -Crustaceans 10 1Centipedes + millipedes 17 -Collembolans 2 -Annelids 2 -Mollusks 17 -TOTAL FAUNA 5.028 82Table XLIV: Overview of animals in De Kaaistoep.AnimalsRed List (species)1.2. LABOULBENIALES IN THE NETHERLANDS1.2.1. CONTRIBUTIONS OF MIDDELHOEKOnly few researchers contributed to the knowledge of Laboulbeniales fromThe Netherlands. The first observations were made by Prof. Dr. de Meyere(1904, unpublished) and the first paper was from BOEDIJN (1923). He focusedmainly on the development of Stigmatomyces baerii Karsten (cfr. 1.2.2.CONTRIBUTIONS OF BOEDIJN).P a g e | 79

In the 40s, Middelhoek studied and described different species ofLaboulbeniales in The Netherlands (MIDDELHOEK 1941, 1942, 1943a, 1943b,1943c, 1943d, 1945, 1947a, 1947b, 1949). An overview of all species ofLaboulbeniales known in The Netherlands is given in Table XLV.Table XLV: Laboulbeniales reported from The Netherlands (based on MIDDELHOEK, 1943a, 1943b, 1947,1949).Species Host Location YearAsaphomyces tubanticus (Middelh. & Boelens)ScheloskeCatops nigricans Enschede 1946Cantharomyces orientalis Speg. Catops nigricans Enschede 1946Corethromyces stilici Thaxt.Eumonoicomyces sp.Euzodiomyces lathrobii Thaxt.Stilicus rufipesStilicus rufipesOxytelus inustusLathrobium elongatumLathrobium geminumLathrobium laevipenneHengeloNieuw Zuid-BevelandZuid-LimburgOost-KapelleLonnekerSchin op Geul194119411939194019381943Haplomyces texanus Thaxt. Bledius fracticornis Denekamp 1943Idiomyces peyritschii Thaxt. Deleaster dichrous Vollenhoven 1941Laboulbenia barbara Middelh. & BoelensPhilonthus punctusPhilonthus punctusEysdenZuid-BevelandLaboulbenia benjaminii Balazuc ex Santam. Badister bipustulatus Oost-Kapelle 1943Laboulbenia cristata Thaxt.Paederus ripariusPaederus ripariusPaederus ripariusHengeloNieuw St. Joosland-19431939193819411937Laboulbenia elegans Thaxt. Harpalus aeneus Zuid-Limburg 1936Laboulbenia filifera Thaxt. Harpalus aeneus - -Laboulbenia flagellata Peyr.Laboulbenia pedicellata Thaxt.Laboulbenia subterranea Thaxt.Laboulbenia vulgaris Peyr.Acupalpus flavicollisAgonum fuliginosumAgonum marginatumAgonum moestumHarpalus aeneusPseudophonus griseusBembidion lunulatumBembidion ustulatumDyschirius sp.Dyschirius sp.Stilicus orbiculatusStilicus rufipesAsaphidion flavipesAtheta xanthopusBembidion assimileBembidion minimumBembidion normannumBembidion testaceumBembidion ustulatumDyschirius globosusDyschirius salinusDyschirius salinusDeventerOost-KapelleHilversumOost-KapelleOverijsselBaarnWalcherenDenekampDenekampLutterzandHengeloNieuw Zuid-BevelandLeidenHouthemBevelandNieuw St. JooslandArendskerkeBunde JulDenekampNieuw St. JooslandNieuw St. JooslandNieuw St. Joosland1947194319431943194719471943193819381939194119411938194319431941194519321939194019411943P a g e | 80

Mimeomyces zeelandicus Middelh. & Boelens Heterothops binotata Nieuw St. Joosland 1942Dyschirius luedersi Nieuw St. Joosland 1941Misgomyces dyschirii Thaxt.Dyschirius politusLochem 1942Monoicomyces californicus Thaxt. Oxytelus sculpturatus - 1939Atheta amicula Nieuw St. Joosland 1942Atheta gagatinaBeesterzwaag 1938Monoicomyces homalotae Thaxt.Atheta triangulum Nieuw St. Joosland 1941Atheta xanthopusHouthem 1943Monoicomyces nigrescens Thaxt. Atheta luteipes Oost-Kapelle 1942Philonthus albipesMiddelburg 1940Peyritschiella furcifera (Thaxt.) I.I. Tav.Philonthus rectangulus Nieuw St. Joosland 1942Philonthus cephalotus Driene 1945Peyritschiella princeps (Thaxt.) I.I. Tav.Philonthus sordidusHengelo 1941Oxytelus rugosusBaarn 1936Peyritschiella protea Thaxt.Oxytelus rugosusAbcoude 1942Philonthus albipesMiddelburg 1940Philonthus cephalotus Ipenrode 1934Peyritschiella vulgata (Thaxt.) I.I. Tav.Philonthus cephalotus Hengelo 1940Philontus sordidusHengelo 1943Philonthus cruentatusPhilonthus fimetariusPhilonthus marginatusRhachomyces philonthinus Thaxt. Philonthus marginatusPhilonthus marginatusPhilonthus variansPhilonthus variansOost-KapelleHengeloBemelenGulpenValkenburgHengeloAmsterdam1941194119431943194319411943Rhadinomyces pallidus Thaxt. Lathrobium elongatum Severen 1942Quedius mesomelinus Driene 1940Quedius mesomelinus Borghaaren 1943Symplectromyces vulgaris (Thaxt.) Thaxt.Quedius mesomelinus Grot Sint-Pieter 1943Quedius mesomelinus Maastricht 1943Teratomyces philonthi Thaxt. Philonthus trossulus Beesterzwaag 1938Middelhoek described several species of Laboulbeniales that were new for TheNetherlands. He also described three species new for science: Laboulbeniabarbara Middelh. & Boelens on Philonthus punctus (Gravenhorst),Mimeomyces zeelandicus Middelh. & Boelens on Heterothops binotata(Gravenhorst) and Barbariella tubantica Middelh. & Boelens, currentlyAsaphomyces tubanticus (Middelh. & Boelens) Scheloske, on Catops nigricans(Spence)(MIDDELHOEK, 1943a, 1949; INDEX FUNGORUM, 2010).1.2.2. CONTRIBUTIONS OF BOEDIJNBoedijn studied species of Laboulbeniales on Fannia (Homalomyia) canicularis(Linnaeus). He suggested that the fungus represented in his drawings isStigmatomyces baerii H. Karst. (1869) (BOEDIJN, 1923). However, the host of thisfungus, Fannia canicularis, is everywhere else parasitized by another species ofLaboulbeniales, i.e. Stigmatomyces ceratophorus Whisler, currentlyFanniomyces ceratophorus (Whisler) T. Majewski (INDEX FUNGORUM, 2010).P a g e | 81

2. MATERIALS & METHODS2.1. HOSTS2.1.1. COLLECTION OF HOSTSInsect species were collected from fall 1995 until November 2010 by the insectresearch group of the Koninklijke Nederlandse Natuurhistorische Vereniging(KNNV), division Tilburg. All collecting sites were situated in De Kaaistoep(Tilburg, The Netherlands, N51°33‟ – E5°01‟). Some characteristics of DeKaaistoep are discussed in 1.1.1. DESCRIPTION OF THE SITE.Several sampling methods were used: pitfall traps, window traps, bands andrings, malaise traps and by hand.2.1.1.1. Pitfall trapsFrom April 8, 200 until May 22, 2001, pitfall traps were functioning around twooaks in the western part of De Kaaistoep. The pitfall traps were placed in threecircles of four pitfalls each. One circle as close as possible to the base of thestem, the second circle at 1,5-2 m distance and the third about 6 m away fromthe stem.All pitfall traps were made of 1,5 L cups, placed in PVC tubes, of 11 cm wideand 15 cm deep. They were filled with 4% formaldehyde and a few drops ofdetergent. Hardboard plates covered the pitfalls at a height of at least 2 cmabove the opening. The pitfalls were emptied every two weeks.Occasionally, a pitfall trap was damaged by vehicles or lifted by moles.2.1.1.2. Window trapsWindow traps were used to capture flying insects. Transparent acrylate screensmeasuring 1 x 2 m each were placed 1,25 to 2,25 m above the soil. A PVCgutter, 20 cm wide and 7 cm deep, sampled the falling insects. Water with 4%formaldehyde and a few drops of detergent was used as conservation liquid.The gutter was emptied by a tube with a tap, on which a piece of nylonstocking was bound. The liquid was reused as many times as possible. Insectswere collected from both sides of the window trap. The window traps wereinspected and emptied every week.Storm winds were able to break the window to pieces. Especially the upperscreen was prone to wind damage.2.1.1.3. Bands and ringsBands, made of brown paper, were attached to oaks. After some experiments,a simple strip of packing paper of about 60 cm wide was chosen. The paperwas longitudinally rumpled and wrapped around the stem. Insects hiding inand underneath the paper were easily collected by carefully opening andshedding the paper bit by bit over a white plastic tray. These bands wereattached to the trees about 1,60 m to 7 m above the soil. The bands wereinspected every 6-8 weeks. Sometimes, they were destroyed by strong windsand even jackdaws (Corvus monedula). If wet, the bands could not beinspected; this happened only occasionally.P a g e | 82

Two kinds of pipes were used as stem embracing rings. One was made ofrather thick rubber (inner width 3 cm), cut longitudinally and attached to thestem of the tree. The open side was directed outwards and the side thatcontacted the tree was kitted. A T-pipe with a bottle containing 70% ethanolwas attached to an opening and served as a collecting device. The other ringwas made in the same way but a smaller pipe was used (1 cm diameter) andthe open side was turned towards the stem.2.1.1.4. Malaise trap (VAN ZUIJLEN et al., 1990)The Malaise trap consisted of a black tent with fine meshes (mesh size ± 1 mm).The tent was placed in this way that the highest ridge (1,9 m) with thecollecting device pointed as much as possible to the sun. The openings, eachwith a surface of about 2 m 2 , were pointed to south-(south)east and north-(north)west. The collecting device contained a 70% ethanol solution.Malaise traps work with very simple principles: all insects fly along the bothsides of the fine meshed dark net; they creep, then run or fly to the highestpoint. Also in the ridge, they force to get to the highest and brightest point, inthe south, and end up in the collecting device (OWEN, 1983).The Swedish entomologist Rene Malaise introduced this trap in 1937.2.1.1.5. Light trapsAs a light source four lamps of 500 Watt each (Philips ML and Osram HWL, colortemperature 3700 and 4100 K) were used. Two lamps were at the top cornersand two in the middle of a vertical white polyester screen of 3,5 m wide and1,9 m high. The lights were always switched on at nightfall. Basically all insects(except Diptera) were identified on the screen and if necessary collectedmanually and either killed with ethyl acetate or directly put into 70% alcoholfor storage.2.1.2. IDENTIFICATI ON OF HOS TSIdentification of hosts was done at the Natuurmuseum Brabant up to specieslevel or at least genus level, using appropriate determining keys:BOEKEN (1987): Identification of Carabidae;FREUDE et al. (1964, 1974): Identification of Staphylinidae;Any name changes and updates on taxonomy concerning Coleoptera werereviewed using VORST (2010).2.2. LABOULBENIALESP a g e | 832.2.1. SCREENING FOR AND IMB EDDING OF LABOULBENIALESWhen infected hosts were found in the collection of De Kaaistoep, they weretransferred to separate vials (Eppendorfer or similar, with 70% ethanol) andbrought to the National Botanic Garden of Belgium for preparation andmounting.Thalli were removed from the hosts and mounted in permanent microscopeslides. As a result, two collections were formed: a collection of insect hosts (in70% ethanol) and a permanent collection of Laboulbeniales slides. Everysingle host specimen received a unique number and is stored separately. Asmany slides as possible were made from each infected host. Each slide hasbeen labeled with the host‟s number, followed by a unique character (a, b, c).

Screening of the insects was done with a stereomicroscope at 50x. Thalli wereremoved and mounted on permanent slides following the protocol fromBENJAMIN (1971) and DE KESEL (1998). The latter uses the AraGly embeddingmedium, composed of:5 mL lactic acid;60 mL aq.dist;35 g purified Arabic gum;30 mL glycerin;5 g phenol chrystals;0,1 g cotton blue.Upon each slide, a thin layer medium was placed. The thalli were positioned inthe medium, and given some time (minutes) to dry. Hereafter, a drop of theAraGly medium was placed upon the thalli, followed by the cover slips. Thisway, the briefly fixed-dried Laboulbeniales remained better in place whilefinishing the slide. The microscope slide collection (numbers ending with –a) isdeposited at the Herbarium of the National Botanic Garden of Belgium;numbers ending with –b and –c are kept at respectively the Herbarium ofNatuurmuseum Brabant (Tilburg, The Netherlands) and the private collection ofthe author (Chantemerle-lès-Grignan, France). All infected insects are kept atNatuurmuseum Brabant (Tilburg, The Netherlands).Photographs of intact specimens were taken some 24 hours after mountingusing an Olympus BX51 light microscope with digital camera and AnalySIS Fiveimaging software (Soft Imaging System GmbH). Measurements were takenfrom calibrated photographs of adult thalli; i.e. thallus length, receptaclelength, perithecium length and width and length of inner/outer appendages.2.2.2. IDENTIFICATI ON OF LABOULBENIALESPreparation and identification of the specimens was done by André De Kesel.Identification of the thalli was done up to species level, using appropriateidentification keys and/or original descriptions:DAINAT et al. (1974) : Identification of Stigmatomyces;DE KESEL (1998): Identification of Laboulbenia;DE KESEL (2002): Identification of Rhachomyces;MAJEWSKI (1994);SANTAMARIA (1998): Identification of Laboulbenia;SANTAMARIA (2003): Identification of Haplomyces and Hesperomyces;THAXTER (1896): Identification of Haplomyces.2.3. PRESENTING RESULTSBoth a parasite-host and a host-parasite list are given. In the parasite-host listall hosts are recorded under the parasite. These have been arranged inalphabetical order. In the host-parasite list all recorded hosts are arranged ina systematic order. The nomenclature of hosts follows VORST (2010)(Carabidae, Staphylinidae, Coccinellidae).P a g e | 84

The descriptions of the taxa are based on DAINAT et al. (1974), MAJEWSKI (1994)and DE KESEL (1997); measurements were taken from the specimens of thecurrent study. Values between round brackets are extremes given by MAJEWSKI(1994) and DE KESEL (1997). Some values are presented between squarebrackets, representing exceptional or abnormal measurements compared toother studied thalli of the concerning species. Additional characteristics andremarks are added with reference to the specimens of the current study.Species marked with (*) were cited by MIDDELHOEK (1943).P a g e | 85

3. RESULTS3.1. PARASITE-HOST LISTA Total of nine species of Laboulbeniales were found on 10 different hosts. Allhosts were Coleoptera, except Drosophila subobscura (Diptera,Drosophilidae). Six laboulbenialean species are new for The Netherlands.Fungus Host Collection referenceLaboulbenia calathi Calathus melanocephalus Haelewaters 3a, 3b, 3cLaboulbenia eubradycelli Bradycellus harpalinus Haelewaters 8a, 8b, 8cBradycellus verbasciHaelewaters 7a, 7b, 7cLaboulbenia pedicellata (*) Bembidion guttula Haelewaters 12a, 12bLaboulbenia vulgaris (*) Bembidion properans Haelewaters 4a, 4b, 4cHaplomyces texanus (*) Bledius gallicus Haelewaters 6a, 6b, 6c, 6dHesperomyces virescens Harmonia axyridis Haelewaters 1a, 1b, 1cRhachomyces lasiophorus Anthracus consputus Haelewaters 2a, 2bStichomyces conosomatis Sepedophilus nigripennis Haelewaters 5a, 5bStigmatomyces majewskii Drosophila subobscura Haelewaters 9a, 9b, 9cHaelewaters 10a, 10b, 10c3.2. HOST- PARASITE LISTHostDipteraDrosophilidae*DrosophilinaeDrosophila subobscura CollinColeopteraCarabidae*TrechinaeBembidion guttula (Fabricius)Bembidion properans (Stephens)*HarpalinaeAnthracus consputus (Duftschmid)Bradycellus harpalinus (Serville)Bradycellus verbasci (Duftschmid)Calathus melanocephalus (Linnaeus)ColeopteraStaphylinidae*TachyporinaeSepedophilus nigripennis (Stephens)*OxytelinaeBledius gallicus (Gravenhorst)ColeopteraCoccinellidae*CoccinellinaeHarmonia axyridis (Pallas)FungusStigmatomyces majewskii H.L. Dainat, Manier &BalazucLaboulbenia pedicellata Thaxt.Laboulbenia vulgaris Peyr.Rhachomyces lasiophorus (Thaxt.) Thaxt.Laboulbenia eubradycelli HuldénLaboulbenia eubradycelli HuldénLaboulbenia calathi T. MajewskiStichomyces conosomatis Thaxt.Haplomyces texanus Thaxt.Hesperomyces virescens Thaxt.P a g e | 86

4. DESCRIPTION OF THE SPECIESAND DISCUSSIONS4.1. LABOULBENIA CALATHI T. MAJEWSKIFigure XIV: Thallus of Laboulbenia calathi (from Calathus melanocephalus, specimen 3a, thalli 2, 3and 4). Scale bar = 100 µm. Picture by Danny Haelewaters (2010).Thallus pigmented, dark olive-brownish, (230-)200-330(-420) µm long.Receptacle 128-230(-300) µm long, cells I and II up to two times longer thanbroad, cell II to two times longer than cell I, cells III and IV isodiametric or up to1,5 times longer than broad, cell V small, oval. Insertion cell dark, constricted;outer appendage up to 175(260) µm long, always simple, composed ofelongated cells; inner appendage composed of a small basal cell and twobranches not exceeding the top of the perithecium or a little longer,terminated in young thalli by clusters of antheridia; in older thalli someantheridia may proliferate into short branchlets. Stalk cell of perithecium moreor less elongated, perithecium (95-)97-134(-140) x (35-)38-60(-73) µm, ovate orelongated, distinctly darkened, nearly free, with subapical blackening, the topdirected upwards, with slightly protruding posterior lips. Ascospores two-celled,(53-)(-63) x (3-)(-4) µm.Studied material [Coleoptera, Carabidae, Harpalinae]:Calathus melanocephalus (Linnaeus):NETHERLANDS Tilburg, Kaaistoep. Pitfall trap 1, 7-14.x.2010.Haelewaters 3a, 3b, 3c.Specificity and geographical distribution:So far on species of the genus Calathus Bonelli in Poland (MAJEWSKI, 1994) andBelgium (DE KESEL, 1997).Remarks:Some thalli display an irregular, rough surface upon the receptacle, mainlycells I and II. The older thalli show more irregularities than the young,immature thalli.One thallus has a receptacle of 128 µm long. The shortest receptaclerecorded by MAJEWSKI (1994) and DE KESEL (1997) measures 140 µm.Laboulbenia calathi is new for the mycoflora of The Netherlands.P a g e | 87

Figure XV: Detail of thallus of Laboulbenia calathi (from Calathus melanocephalus, specimen 3a,thallus 1), showing the striation on cell I and granulation. Scale bar = 20 µm. Picture by DannyHaelewaters (2010).Discussion:Cells I and II of some, not all adult thalli of Laboulbenia calathi show somestriation (on cell I) and granulation, which has not yet been described for thisspecies. This may suggest that L. calathi, as other species of Laboulbenia, hasa certain amount of variability.4.2. LABOULBENIA EUBRADYCELLI HULDÉNFigure XVI: Thallus of Laboulbenia eubradycelli (from Bradycellus harpalinus, specimen 8a, thallus 2).Scale bar = 100 µm. Picture by Danny Haelewaters (2010).P a g e | 88Thallus (162-)294-372(-460) µm long, yellow-brownish, perithecium andandrostichum darkened, except cell V. Receptacle (95-)213-286(-365) µmlong, cell I slender, elongated, distally yellowish pigmented, cell II broader in itsdistal part, up to eight times longer than broad, septum II/VI oblique, muchlonger than septum II/III, cells III and IV about 1,5 times longer than wide,darkened, cell IV distinctly cuneiform, cell V small, obtriangular. Insertion celldark, thick, constricted; outer appendage up to 252 [420] µm long, simple,composed of elongated cells; inner appendage short, composed of a small

asal cell and two short branches terminated by one or two antheridia. Stalkcell of perithecium flattened, situated obliquely, or elongated, perithecium(75-)95-136(-145) x (32-)40-55(-78) µm, about 2/3 free, ovate, slightlyasymmetrical, with a more convex posterior margin, distinct subapical spotsand prominent rounded posterior lips. Ostiolum mostly abaxially oriented; theperithecium than is asymmetrical.Studied material [Coleoptera, Carabidae, Harpalinae]:Bradycellus harpalinus (Serville):Tilburg TWM, Kaaistoep-west, 128.8-394.6. Licht, 31.viii.08.Haelewaters 8a, 8b, 8c.Bradycellus verbasci (Duftschmid):Tilburg TWM, Kaaistoep-west, 128.8-394.6. Licht, 6.viii.08.Haelewaters 7a, 7b, 7c.Specificity and geographical distribution:So far on species of the genus Bradycellus Erichson and TrichocellusGanglbauer in many European countries, on Madeira Island, and in Mexico(MAJEWSKI, 1994). Type on Bradycellus caucasicus (Chaudoir) in Finland.Remarks:DE KESEL (1997) mentions that thallus length varies depending on the speciesof host. Thalli of of Bradycellus harpalinus and B. verbasci (180-420 µm) differsignificantly from those of B. ruficollis and Trichocellus placidus (160-200 µm).Laboulbenia eubradycelli is new for the mycoflora of The Netherlands.4.3. LABOULBENIA PEDICELLATA THAXT.Figure XVII: Thallus of Laboulbenia pedicellata (from Bembidion guttula, specimen 12a, thallus 2).Scale bar = 100 µm. Picture by Danny Haelewaters (2010).Thallus (110-)542-572(-590)[830] µm long, yellow-brownish, perithecium andupper receptacle darkened. Receptacle (80-)447-477(-500)[725] µm long,stout or slender, variable in length; cell I cuniform, at least 2 times longer thanbroad, cell II stout, always longer than cell I, 2-10 times longer than broad orslender, cylindrical, often narrowed and nearly hyaline in its upper half, cell IIIP a g e | 89

isodiametric or slightly elongate, rarely up to 2 times longer than broad, cells IVand V similar in length, cell IV isodiametric, cell V usually narrower, septum IV/Vis either vertical or slightly oblique. Insertion cell slightly constricted, dark; basalcell of outer appendage externally inflated, subtending a ramified branch, itsbranchlets numerous, hyaline, the posterior branchlet (the primary axis) withblack septum and external blackening in lower part, fragile; basal cell of innerappendage smaller than the outer one, subtending several ramifiedbranchlets directed upwards; some of the branchlets may proliferate fromantheridia visible in very young thalli on short inner branchlets; branchlets inmature thalli usually do not exceed the top of the perithecium; only theprimary branch of the outer appendage – if preserved – is distinctly longer.Stalk cell of perithecium isodiametric or slightly elongated, perithecium [50-](80-)125-130(-140) x [28-](35-)61-78(-80) µm, ovate, about 2/3-3/4 free, withsubapical darkening and somewhat protruding posterior lips. Spores (40-)(-52)x (2-)(-5) µm.Studied material [Coleoptera, Carabidae, Trechinae]:Bembidion guttula (Fabricius):Tilburg TWM, Kaaistoep-west, 128.8-394.6. Licht, 4.vii.09.Haelewaters 12a, 12b.Specificity and geographical distribution:On many species of the genus Bembidion Latreille in a broad sense, and onrepresentatives of the genus Dyschirius Bonelli, Pogonus Dejean andPterostichus Bonelli. Type on Bembidion sp., USA.Remarks:One thallus shows a swollen cell VII.Discussion:One thallus of Laboulbenia pedicellata displays a swollen cell VII, which canbe added to the other known characteristics of variability of L. pedicellata(MAJEWSKI, 1994), i.e. the length of cells I + II, narrowing of cell II, color andsculpture of the receptacle and appendage structure. According to HULDÉN(1983), in the typical long forms, cell II is constricted in its upper part. However,the specimens in this study do not display any constriction of cell II in the longforms. These observations fit in the morphological variation in L. pedicellata assuggested by TAVARES (1985), MAJEWSKI (1994) and DE KESEL (1997). Anomaliesoccur, such as compartmentation in cell II.HULDÉN (1985, ref. in DE KESEL, 1997) distinguishes five groups within Laboulbeniapedicellata, based on the shape of cell II, the perithecium and the size of thespores. Compared to the characteristics described by Huldén, the specimensof L. pedicellata of De Kaaistoep belong to morph-group I. This morph-grouphas – until now – only been described for specimens attached to the femur ofthe forelegs, while the specimens of this research were taken from thepronotum and prosternum. Moreover, this morph-group was not yet observedon species on Bembidion guttula.P a g e | 90

4.4. LABOULBENIA VULGARIS PEYR.Figure XVIII: Thallus of Laboulbenia vulgaris (from Bembidion properans, specimen 4c, thallus 1).Scale bar = 50 µm. Picture by Danny Haelewaters (2010).Thallus (180-)199-215(-480) µm long, yellow-brownish, more or less darkened formost of its length, often olivaceous. Receptacle stout, wedge-shaped orelongated, (120-)135-156(-380) µm long; cell I 2-3 times longer than broad, cellII sometimes flattened or isodiametric, but usually elongated, up to four timeslonger than broad and often thinner in the middle part, cells III and IVisodiametric or elongated especially in slender thalli, usually of the same size,septum IV/V does not make contact with cell III, cell V obtriangular, half aslong as cell IV but often longer, less often distinctly shorter. Insertion cell dark,slightly constricted, outer appendage up to 240 µm long, simple or – moreoften – divided on the third cell, seldom on the second one, the lower twosepta are darkened, slightly constricted, the cells of the outer appendageelongated except the two or three basal cells which are shortened in manythalli; inner appendage consisting of small basal cell and two short antheridialbranchlets, antheridia not proliferating, the basal septum of each branchletoften darkened. Stalk cell of perithecium flattened or isodiametric, rarelyelongated; perithecium (100-)111(-160) x (35-)41-49(-70) µm, half free, ovate,straight or slightly bent outwards and protruding, rounded posterior lips.Ascospores two-celled, (45-)(-58) x (2,5-)(-3,5) µm.Studied material [Coleoptera, Carabidae, Trechinae]:Bembidion properans (Stephens):NETHERLANDS, Tilburg, Kaaistoep. Pitfall trap 1, 8-15.iv.2010.Haelewaters 4a, 4b, 4c.Specificity and geographical distribution:Laboulbenia vulgaris is widespread in Europe, Africa and Asia, it is also found inboth Americas. Laboulbenia vulgaris occurs most often on species of thegenus Bembidion Latreille in a broad sense, less often on other carabids: onTrechus Schellenberg and related genera, on Trechoblemus Ganglbauer,Deltomerus Motschulsky and exceptionally Nebria Latreille.Discussion:Laboulbenia vulgaris is morphologically very variable. This had led toconfusions and a relatively high number of synonyms. This variation primarilyP a g e | 91

occurs in the lower receptacle (cell I and cell II), the outer appendage andthe degree of darkening. Damage of the appendage followed byregeneration leads to aberrant thalli. In addition, complete thalli are ratherscarce.4.5. HAPLOMYCES TEXANUS THAXT.Figure XIX: Thallus of Haplomyces texanus (from Bledius gallicus, specimen 6b, thallus 1). Scale bar= 50 µm. Picture by Danny Haelewaters (2010).P a g e | 92Thallus (240-)279-667 µm, clear brownish-yellow, venter of perithecium andantheridium amber-yellow. Receptacle (30-)84-104 µm long, cell I ofreceptacle 2-2,5 times longer than broad, narrow, cell II nearly isodiametric,blackened to a greater or lesser extent, cell III slightly elongated, blackenedwith the exception of the anterior margin, rarely cells II and III withoutblackening; cell III supporting a compound antheridium which is 34-38(-70) µmlong, 1,5-2 times longer than broad. Stalk cell of perithecium elongated,hyaline, 62-265 µm long; secondary stalk cell and basal cells more or lesselongated, perithecium (100-)152-333 x (45-)54-83(-85) µm, with inflated venter,tapering gradually to a blunt apex with minute teeth.Studied material [Coleoptera, Staphylinidae, Oxytelinae]:Bledius gallicus (Gravenhorst):Tilburg TWM, Kaaistoep-west, 128.8-394.6. Licht, 24.vii.08.Haelewaters 6a, 6b, 6c, 6d.Specificity and geographical distribution:Only on species of the genus Bledius Samouelle in several European countriesand in the USA where this species was described (type on Bledius rubiginosusErichson).Discussion:Haplomyces texanus displays distinctive variation in the blackening of cell II.Specimens attached to the abdomen and femur of the second leg of Blediusgallicus have a clearly blackened cell II. Specimens attached to thepronotum of Bledius gallicus on the other hand have a hyaline cell II is hyaline;these specimens show a darker ring between cells I and II, which has neverbeen described before suggesting a position specific characteristic ofHaplomyces texanus.

4.6. HESPEROMYCES VIRESCENS THAXT.Figure XX: Thallus of Hesperomyces virescens (from Harmonia axyridis, specimen 1a, thallus 1).Scale bar = 50 µm. Picture by Danny Haelewaters (2010).P a g e | 93Thallus (210-)274-286(-375) µm, hyaline to yellowish. Receptacle (60-)76-88 µmlong, with cell I triangular to rhomboidal, longer than broad, cell II slightlylonger than broad, rhomboidal to trapezoidal, cell III slightly shorter than cell II,almost isodiametric, slightly inflated dorsally. Primary appendage (48-)50-53(-58) µm long, consisting of (3-)4 superposed cells. Basal cell 1,5 times longerthan broad, longer than each of the remaining cells of the appendage, thirdand fourth cells bearing one and two antheridia respectively. Antheridia withoutwardly curved efferent necks. Stalk cell of perithecium elongated, slightlybroadened distally; perithecium (140-)181-201(-253) x (50-)52-54(-81) µm,asymmetric, fusiform, broadest near the middle, and then gradually taperingtowards a short, broad, indistinct neck, and a subacute, lobulated,asymmetrical apex. Septa between the horizontal tiers of wall cells marked byconstrictions. The perithecial tip area includes two short lower lobes, twounicellular elongated upper lobes and two prominent lips surrounding theostiole; the lower lobes are minute whereas the upper lobes are finger-like andusually curved outwards; the tips of the upper lobes exceed the perithecialapex height. Ascospores two-celled, (55-)(-70) µm long.Studied material [Coleoptera, Coccinellidae, Coccinellinae]:Harmonia axyridis (Pallas):Tilburg TWM, Kaaistoep-west, 128.8-394.6. Licht, 6.viii.08.Haelewaters 1a, 1b, 1c.Harmonia axyridis (Pallas):Tilburg TWM, Kaaistoep-west, 128.8-394.6. Licht, 31.viii.08.Haelewaters 11a, 11b, 11c.Specificity and geographical distribution:On Coccinellidae of the genus Adalia Mulsant, Chilocorus Leach, HarmoniaMulsant, Hippodamia Dejean, Propylea Mulsant, Psyllobora Dejean, in severalEuropean countries, and in Africa, Asia, America and Oceania (De Kesel,1997).Remarks:Hesperomyces virescens is new for the mycoflora of The Netherlands.

Discussion:Two species of the genus Hesperomyces occur in Europe, i.e. H. virescens andthe much less common H. coccinelloides (SANTAMARÍA, 2003). The specimensfound in De Kaaistoep belong to H. virescens, they were found on Harmoniaaxyridis, an invasive coccinelid species.H. axyridis is native to northeastern Asia, including Taiwan, China, Korea,Japan, southern Siberia, Ryukyu Islands and Bonin Islands, and Australia(MANNIX, 2001). It was identified as a biocontrol agent for aphids and scaleinsects, and has been introduced in the United States and Europe. H. axyridis isa generalistic species, less demanding for environmental factors, therefore itcauses a threat to all native species and biodiversity (ADRIAENS & GYSELS, 2002).For the time being, nothing is known about the range and worldwidedistribution of Hesperomyces virescens on Harmonia axyridis.4.7. RHACHOMYCES LASIOPHORUS (THAXT.) THAXT.Figure XXI: Thallus of Rhachomyces lasiophorus (from Anthracus consputus, specimen 2a, thallus 1).Scale bar = 100 µm. Picture by Danny Haelewaters (2010).Thallus (180-)202(-220)[480] µm long, usually straight, pale brownish,appendages dark brown. Receptacle axis below the perithecium (60-)78(-290) µm long, consisting of 8-18 cells which are isodiametric, darkened in thelower part, slightly widening distally; no proliferating branches visible.Appendages of type A brown-black, up to 221 [300] µm long, but in maturethalli often broken off, only in the lower part of the receptaculum; type Bappendages up to 112 µm long, abundantly along the whole receptacle axisand around the stalk cell of the perithecium (cell VI), consist of 4-5 cells whichare nearly equal in length, the distal cell is somewhat longer and slightlyswollen in the lower part; type C appendages sparsely dispersed on the mainaxis, forming a small group near the base of the perithecial stalk cell, up to 30P a g e | 94

µm long, two-celled. Stalk cell of perithecium short, perithecium (110-)(-140) x(45-)(-60) µm, elliptical or long ovate, constricted and darkened subapically.Studied material [Coleoptera, Carabidae, Harpalinae]:Anthracus consputus (Duftschmid):Tilburg TWM, Kaaistoep-west, 128.8-394.6. Licht, 6.viii.08.Haelewaters 2a, 2b.Specificity and geographical distribution:Mainly on species of the genus Acupalpus Latreille, also on Agonum Bonelli,Atranus Leconte, Badister Schellenberg, Baudia Ragusa and Dromius Bonelli inEurope (France, Italy, Germany, Poland), Asia (Korea) and North America(USA) (MAJEWSKI, 1994). Type on Atranus pubescens Dejean, USA.Remarks:Rhachomyces lasiophorus is new for the mycoflora of The Netherlands.Discussion:Anthracus consputus in this study is host for Rhachomyces lasiophorus. R.lasiophorus only parasitizes Acupalpus species; it is the only representative ofthe genus Rhachomyces parasitising Acupalpus species (MAJEWSKI, 1994).Anthracus is a subgenus of Acupalpus (pers. comm. PAUL VAN WIELINK, 2010).Concerning to DE KESEL (1997), thalli of Rhachomyces lasiophorus occur onmesothorax, metathorax and legs. In this study, specimens of R. lasiophorushave been collected from the elytra. The hosts of R. lasiophorus are typical forbanks of stagnant water, swamps and marshes (DESENDER et al., 1995), acommon habitat at De Kaaistoep.4.8. STICHOMYCES CONOSOMATIS THAXT.Figure XXII: Thallus of Stichomyces conosomatis (from Sepedophilus nigripennis, specimen 5b, thallus1). Scale bar = 50 µm. Picture by Danny Haelewaters (2010).Thallus (195-)263-295(-315) µm long, brown-yellowish, body of the peritheciumand appendage darkened. Receptacle 67-95(-120) µm long, cell I about twotimes longer than broad, all superposed cells of the receptacle-appendageaxis isodiametric or slightly elongated; cell II giving rise to one or twoperithecia, cell III to perithecia or – seldom – to antheridial branchlets, or sterile.Appendage axis consisting of 1-4 usually elongated cells, gradually smallertowards the end of the axis; the distal cell continuing into antheridial or sterileprimary branch, the other axis cells separating corner cells; antheridialP a g e | 95

anches (15-)34-38(-40) µm long, terminated by 2-5 antheridia which mayproliferate into short branchlets. Perithecium one per thallus, less frequently 2-4on both sides of the receptacle; stalk cell as well as secondary stalk cell andone of the basal cells elongated, perithecium (75-)126-130(-152) x (30-)40-43(-45) µm, slender, with slightly differentiated neck tapering to narrow, distal partof stalk cell and secondary stalk cell hyaline, rounded apex. Ascospores twocelled,(32-)(-45) x (2-)(-3) µm.Studied material [Coleoptera, Staphylinidae, Tachyporinae]:Sepedophilus nigripennis (Stephens):Tilburg, TW Kaaistoep. Potval, 13-27.1.2001.Haelewaters 5a, 5b.Specificity and geographical distribution:On species of the genus Sepedophilus Gistel in USA, Algeria, Great Britain,Belgium, Poland, Spain and Japan.Remarks:Stichomyces conosomatis is new for the mycoflora of The Netherlands.Discussion:Most thalli of Stichomyces conosomatis have only one perithecium and none,one or two perithecial primordia. Both TAVARES (1985) and MAJEWSKI (1994)describe the development of secondary perithecia upon the cell above cell II.Since cell III basically never bears perithecia (cfr. Part I, 1.3.2. THE RECEPTACLE),this cell should be considered as cell II‟. The thalli studied in this research haveonly perithecia(l primordial) upon cell II, consistent with the Belgian material(DE KESEL, 1997).4.9. STIGMATOMYCES MAJEWSKII H.L. DAINAT, MANIER &BALAZUCFigure XXIII: Thallus of Stigmatomyces majewskii (from Drosophila subobscura, specimen 10a, thallus1). Scale bar = 100 µm. Picture by Danny Haelewaters (2010).P a g e | 96Thallus (320-)250-258(-372) µm long, hyaline to brownish-yellow. Receptacle133-148 µm long, slender, with cell II always shorter than the cell I; cell I long,narrower at the base, remarkably divided in two: granular protoplasmaticmass in the upper part, optically empty hyaline mass in the lower part.Appendage (41-)51-76 µm long, its axis consisting of four cells, of which the first

one is sterile; seven large antheridia, biseriate (2x3 + 1 at the top). Cellularsupport of appendage very long, swollen below the insertion of theappendage; this swelling and the basal cell of the axis of the appendagecolored brown. Perithecium having two parts: middle part and neck: middlepart protruded, yellow-brownish, 86-153 µm, neck large, almost square, mostlyas long as the rest of the perithecium, conical tip, with four regular lips, 78-163µm.Studied material [Diptera, Drosophilidae]:Drosophila subobscura Collin:NETHERLANDS, Tilburg, Kaaistoep-West. Beer trap, Brand, 19-26.viii.2008.Haelewaters 9a, 9b, 9c.Drosophila subobscura Collin:NETHERLANDS, Tilburg, Kaaistoep-West. Beer trap, Brand, 19-26.viii.2008.Haelewaters 10a, 10b, 10c, 10d.Specificity and geographical distribution:On species of the genus Drosophila Fallèn: on Drosophila obscura Fallèn andDrosophila subobscura Collin in France (DAINAT et al., 1974), on Drosophilaobscura Fallèn and Drosophila rufifrons Loew in Austria (ERHARD, 2001).Remarks:Two giant species were recorded in the current research, one thallus of 491µm long; another thallus was 643 µm long.Stigmatomyces majewskii is new for the mycoflora of The Netherlands.Discussion:Stigmatomyces majewskii was distinguished from closely related S.entomophilus based on the following features:Perithecia of S. entomophilus with a neck that is much longer as the restof the perithecium;Apendages of S. entomophilus brown and consisting of three cells.Cell I is not remarkably divided in two. In some thalli, the granularprotoplasmatic mass in cell I is abundantly present, much more than thehyaline mass. Stigmatomyces majewskii displays morphological variation, assuggested before by ERHARD (2002) (based on the number of antheridia).P a g e | 97

5. CONCLUSION AND SUGGESTIONSIn The Netherlands a total of 35 species of Laboulbeniales have beenrecorded up to now, starting with the very first observation in the 1940s. Thehosts represent 5 families, 11 subfamilies, 24 genera and 57 species. Until now,no Dutch insect collections have been screened by mycologists in search forLaboulbeniales, except (partly) the insect collection at the NatuurmuseumBrabant (Tilburg). There is no doubt that a systematic study of insect collectionsof The Netherlands will result in a considerable increase of the number of bothhosts and parasites.Since 1995, the presence of all kind of organisms in De Kaaistoep has beenintensively studied. Our short prospection has resulted in nine species ofLaboulbeniales, six of which are new for The Netherlands: Laboulbenia calathi,Laboulbenia eubradycelli, Hesperomyces virescens, Rhachomyceslasiophorus, Stichomyces conosomatis and Stigmatomyces majewskii. Thelatter is rare; it has been reported just twice, i.e. from France (type, DAINAT etal., 1974) and Austria (ERHARD, 2001).Yet, other species will be discovered in De Kaaistoep, since the observednumber of species in an area depends on the diversity of habitats in that areaand perhaps even more the intensity of searching for new species. In DeKaaistoep, more research should be done in order to find Laboulbeniales:capturing of insects and screening for Laboulbeniales. However, these firstresults confirm the entomological and mycological value of the naturelandscape De Kaaistoep. They also emphasize the persistent need forinventory.P a g e | 98

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