Checklist of thallus-forming Laboulbeniomycetes from Belgium and the Netherlands, including Hesperomyces halyziae and Laboulbenia quarantenae spp. nov. Author Haelewaters, Danny Purdue University, West Lafayette, Indiana, United States of America & University of South Bohemia, Ceske Budejovice, Czech Republic & Ghent University, Ghent, Belgium https://orcid.org/0000-0002-6424-0834 danny.haelewaters@gmail.com Author Kesel, Andre De Meise Botanic Garden, Meise, Belgium https://orcid.org/0000-0002-5287-4637 text MycoKeys 2020 71 23 86 http://dx.doi.org/10.3897/mycokeys.71.53421 journal article http://dx.doi.org/10.3897/mycokeys.71.53421 1314-4049-71-23 8E50180F4871515AA7C2AB2EB0BA1197 Laboulbenia quarantenae De Kesel & Haelew. sp. nov. Figure 4 Diagnosis. Morphologically similar to Laboulbenia vulgaris Peyr., but the insertion cell is attached to the lower fifth of the posterior margin of the perithecial wall and the outer appendage is composed of 4-6(-8) branches resulting from successive dichotomies starting at the suprabasal cell , which is poorly pigmented or nearly hyaline. The LSU sequence shares 89.7-98.0% identity with other sequenced taxa of Laboulbenia , 97.4% with L. flagellata from Agonum nigrum , 97.5-98.0% with L. flagellata from Limodromus assimilis , 97.0-98.0% with L. flagellata from Agonum emarginatum / A. micans / Loricera pilicornis / Oxypselaphus obscurus , and 97.0-97.7% with L. vulgaris from Bembidion tetracolum / Ocys harpaloides . Unique molecular synapomorphies in the LSU at positions 503, 545. Types. Holotype : Belgium, Province Vlaams Brabant, Meise, Domein van Bouchout, 50.9267056N , 4.3220028E , 30 m a.s.l., 26 Apr. 2019, leg. A. De Kesel, rivulet-associated grassland, on Bembidion (Philochtus) biguttatum (Fabricius, 1779) ( Coleoptera , Carabidae ), ADK6448 (BR), slide BR5020212163329V (1 mature thallus, prothorax). Isotypes: ibid. , slides BR5020212162292V (2 mature thalli, right mesofemur), BR5020212161264V (6 mature thalli, right protibia), BR5020212166412V (5 immature thalli, mesothorax), BR5020212165385V (1 mature thallus, right protibia), and BR5020212164357V (1 mature thallus, right mesofemur). Paratype : Belgium, Province Vlaams-Brabant, Meise, Domein van Bouchout, 50.92745N , 4.323917E , 32 m a.s.l., 30 Apr. 2020, leg. A. De Kesel, rivulet-associated grassland, on B. (P.) biguttatum , ADK6523 (BR), slide BR5020195033527V (2 mature thalli, mesosternum). Etymology. From quarantena , which was used in 14th-15th century Venetian language for a forty-day isolation period. The new species was described during the 2020 quarantine period imposed to curb the spread of the COVID-19 virus. Description. Thallus 300-465 µm long from foot to perithecial tip; colored hyaline at the lower receptacular cells (I and II) and the inner appendage, otherwise pigmented light to dark brown; especially the upper receptacular cells (III, IV and V), cell VI, and the perithecium darkening with age. Cell I elongated, usually straight, 56-107 x 22-33 µm ; sometimes bent and then wider at the upper end. Cell II slender, mostly with parallel margins, longer than cell I, 73-160 x 29-40 µm , anterior margin shorter than posterior. Cells III and VI side by side, with septum II-III always much shorter than septum II-VI. Cell III with a narrow base, 29-43 µm long, widening upwards and then 22-29 µm wide at the apex. Cell VI more or less rectangular, 30-34 x 23-30 µm . Cell IV more or less rectangular, slightly broader than long, 20-32 x 25-30 µm . Cell V small, triangular, situated in the inner-upper corner of cell IV, 9-14 x 7-14 µm , as pigmented as surrounding cells. Insertion cell brownish black, flattened, barely marking a constriction on the posterior margin of the thallus, attached to the lower fifth of the posterior margin of the perithecial wall, 18-25 µm wide and 90-128 µm from the perithecial tip. Inner appendage hyaline, composed of 2-4(-6) short branches, rarely exceeding the perithecial tip, 88-150 µm long, resulting from successive dichotomies starting at the basal cell , the latter 9-14 x 6-12 µm . Antheridia short, flask-shaped, few in number, usually on the young inner appendage and arising laterally from its suprabasal cell . Outer appendage up to 250-335 µm long, extending beyond the perithecial tip, often entirely light brown, composed of 4-6(-8) branches, resulting from successive dichotomies starting at the suprabasal cell ; the basal cell longer than broad, 23-32 x 15-21 µm , almost entirely hyaline. Perithecium ellipsoid, venter only very slightly asymmetrical, anterior and posterior margins almost equally convex, 109-157 x 43-64 µm , length/width ratio 1.9-2.5, widest in the middle; perithecial tip asymmetrical, with prominent and rounded posterior margin; preostiolar spots black, in older thalli merging into a pre-apical ring, always with distinctly paler zone under the posterior spot. Ascospores two-celled, hyaline, 59-65 x 4.2-5.5 µm , with slime sheath. Material sequenced. Belgium, Province Vlaams Brabant, Meise, Domein van Bouchout, 50.9267056N , 4.3220028E , 30 m a.s.l., 26 Apr. 2019, leg. A. De Kesel, rivulet associated grassland, on Bembidion biguttatum ( Coleoptera , Carabidae ), ADK6448 (BR), isolate E13T12 (3 mature thalli, prothorax, LSU: MT371368). Hosts and distribution. Thus far only known on Bembidion biguttatum from the type locality in Belgium. Reported as Laboulbenia sp. nov. in De Weggheleire (2019) . Notes. Morphologically, L. quarantenae mostly resembles L. vulgaris Peyr., but it differs from it by the very low position of the insertion cell (regardless of the origin of the thallus), the successive dichotomous branching of the outer appendage, the poorly pigmented to nearly hyaline basal cell of the outer appendage, and the slender habitus. Although these characters may vary to some extent, eventually resulting in specimens that are morphologically close to L. vulgaris , our LSU phylogeny (Figure 2 ) shows that sequences of typical L. vulgaris obtained from Carabidae known to host L. vulgaris - Bembidion tetracolum Say, 1823 and Ocys harpaloides (Audinet-Serville, 1821) ( Santamaria et al. 1991 ; Majewski 1994 ; Haelewaters et al. 2019a ; De Kesel et al. 2020 )-fall in a monophyletic clade separated from L. quarantenae . The two isolates of L. vulgaris from B. tetracolum were collected in Belgium (isolate E10T2) and Latvia (isolate E11T6), from populations that are 1,550 km apart, but they were placed together among isolates from O. harpaloides (all from Belgium). Laboulbenia quarantenae , on the other hand, was collected between <1 and 21 km distance from where hosts of L. vulgaris were collected. Figure 2. Maximum clade creditability tree of Laboulbenia isolates reconstructed from an LSU dataset, with L. bruchii as outgroup. The topology is the result of Bayesian inference performed with BEAST. For each node, ML BS (≥ 65) and Bayesian pp (≥ 0.7) are presented above/below the branch leading to that node. Isolates are color-coded by host; L. quarantenae sp. nov. is highlighted with gray shading. Phylogenetically, L. quarantenae may be more closely related to L. flagellata than to L. vulgaris . Laboulb quarantenae and L. flagellata (sensu lato) were retrieved as sister taxa in our phylogeny, although no statistical support was retrieved for this sister relationship. Whereas species boundaries are evident based on our phylogeny, it goes without saying that both taxon sampling and sequence data need to be greatly expanded upon to resolve relationships among species of Laboulbenia . The new species is apparently very rare and was never found in combination with L. vulgaris , the more common parasite from Bembidion biguttatum in Belgium ( De Kesel 1998 ; De Kesel et al. 2020 ). Figure 3. Hesperomyces halyziae Haelew. & De Kesel from Halyzia sedecimguttata A mature thallus from slide D. Haelew. 955a, holotype B mature thallus from slide BR5020212156406V. Scale bar: 100 µm . In Europe, many species of Laboulbenia have been reported on Bembidion Latreille, 1802 ( Santamaria et al. 1991 ). Of those, L. pedicellata Thaxt. and L. vulgaris Peyr. are among the most reported ones. Bembidion biguttatum belongs to subfamily Trechinae . To our knowledge, this species is infected by either L. murmanica Hulden (S. Santamaria pers. comm.), L. pedicellata ( Scheloske 1969 ; Majewski 1994 ), or L. vulgaris ( Majewski 1994 ; De Kesel et al. 2020 ). Based on the position of its insertion cell as well as the morphology of both the outer appendage and the androstichum (cells II, IV, and V), L. quarantenae is fundamentally different from these three species. The outer appendage of L. quarantenae is reminiscent of the one from L. flagellata , which, however, is a more robust species reported from 80 genera of Carabidae belonging to Anthiinae , Brachininae , Elaphrinae , Harpalinae , Loricerinae , Nebriinae , and Patrobinae (but not Trechinae ) ( Santamaria et al. 1991 ; Santamaria 1998 ; Haelewaters et al. 2019a ). Figure 4. A-I Laboulbenia quarantena De Kesel & Haelew. from Bembidion biguttatum , specimen ADK6448: A mature thallus from prothorax, slide BR5020212163329V, holotype B mature thallus from prothorax with less pigmented perithecium C mature thallus from the right mesofemur D-F mature thalli from the right protibia G immature thallus from the prothorax H mature thallus from the right mesofemur I ascospores J-K laboulbenia vulgaris Peyr: J mature thallus from prothorax of Bembidion tetracolum , specimen ADK5557 K mature thallus from mesothorax of Ocys harpaloides , specimen ADK6353. One of the diagnostic characteristics of the new species-the positioning of the insertion cell -is shown in a mature thallus of L. quarantenae ( E ) and one of L. vulgaris ( J ). Scale bar: 100 µm . Bembidion biguttatum , the host for L. quarantenae , belongs to the subgenus Philochtus. Representatives of Laboulbenia reported from Bembidion subgenus Philochtus are few and include two species only: L. pedicellata and L. vulgaris . Two thalli of Laboulbenia "sp. similar to L. vulgaris " from Bembidion bruxellense Wesmael, 1835 [as B. rupestre (Linnaeus, 1767) are illustrated in Majewski (1994 : Pl. 53, Figs 1 , 2 ). Their morphology comes close to L. quarantenae but cell V is much larger and the insertion cell is not situated low enough along the posterior margin of the perithecial wall. Also L. parvula is reported on subgenus Philochtus in Santamaria et al. (1991) , but this species is much smaller (180-190 µm total length) compared to L. quarantenae , it has a deeply pigmented basal cell of the outer appendage, the inner and outer appendage each carry 4-8 very slender branches, and its perithecial tip is rather squarish. As we explore patterns of speciation of taxa in both Herpomycetales and Laboulbeniales using integrative taxonomy, we can start linking some of these patterns to morphological or life history traits. One candidate trait is the haustorium-a rhizoidal structure that penetrates the host's integument to make contact with the haemocoel, increasing surface area for nutrient uptake and providing holdfast. We hypothesize that - due to the invasive nature of their haustorium - Herpomycetales and haustorial Laboulbeniales , such as species of Hesperomyces , maintain close interactions with their hosts, possibly involving adaptations to the hosts' defense systems and leading to escape-and-radiate coevolution ( Ehrlich and Raven 1964 ). These developments result in an evolutionary arms race, with specialization and leading to speciation (One Host One Parasite model, Figure 1 ). While all 27 species of Herpomyces form multiple haustoria, not all Laboulbeniales penetrate their host. Recently, Tragust et al. (2016) presented evidence for four species of Laboulbeniales to be superficially attached to their host, and also L. flagellata and L. vulgaris do not seem to perforate their hosts. There are no strict developmental barriers for non-penetrating species and their ascospores may develop on multiple arthropods given that they co-occur in a given microhabitat, resulting in parasite species with more than one host (e.g., L. vulgaris in Figure 2 ), in contrast to the host-specific species of Hesperomyces . Undoubtedly, other factors come into play; more studies of speciation and species limits, specificity, host shifting, and transmission patterns are needed to test said hypothesis.