Laboulbeniales (Ascomycota) of the Boston Harbor Islands I: Species Parasitizing Coccinellidae and Staphylinidae, with Comments on Typification

Abstract 
 This paper, based on a recent comprehensive sampling of insects, is the first report of Laboulbeniales from the New England region since the 1930s. We present 7 new records of laboulbenialean parasites on Staphylinidae (rove beetles) and Coccinellidae (lady beetles) from the Boston Harbor Islands National Recreation Area. These are Clonophoromyces nipponicus Terada & I.I. Tav., Hesperomyces virescens Thaxt., Ilyomyces cf. mairei F. Picard, Laboulbenia philonthi Thaxt., Peyritschiella protea Thaxt., Stichomyces conosomatis Thaxt., and Teratomyces actobii Thaxt. One of these parasite species, C. nipponicus, has not been found previously outside of its type locality in Japan. Examination of Roland Thaxter's 1891–1932 slides led to the designation of lectotypes for L. philonthi, P. protea, S. conosomatis, and T. actobii. The following synonymy is established: Teratomyces brevicaulis Thaxt. = T. actobii. In addition, we discovered new localities for H. virescens (from Canada, Cuba, Guatemala, and Japan) and L. philonthi (from Canada, Grenada, Panama, Trinidad, and Venezuela).


Introduction
Existing museum collections provide host specimens from a wide range of collection locations and dates for the study of arthropod ectoparasites. Collections made in the context of an all taxa biodiversity inventory (ATBI) provide sample populations from host collections that should exhibit minimal bias in parasite distribution and diversity. Parasite density for obligate ectoparasites can be determined because they are fairly easily observed on the exoskeleton of a given host; these parasites persist on dead individuals, and thus, historical collections of infected hosts serve as records of species relationships and population dynamics through time. They also provide a measure of parasite abundances and host usage across a single habitat or system. In this paper, we present fungal parasites of 2 host families collected in a survey of all arthropods in an island system.

The Order Laboulbeniales
The Laboulbeniales form an order of fungi (phylum Ascomycota, class Laboulbeniomycetes) that obligately parasitize arthropod hosts. They are ectoparasites, that is, they occur externally on various parts of the insect. Phylogenetic analyses of DNA sequences indicate that the class Laboulbeniomycetes, which also includes the order Pyxidiophorales, is sister to the Sordariomycetes-a clade that includes saprotrophs and pathogens of plants, animals, and fungi, and that generally produce perithecia (Blackwell and Malloch 1989, Schoch et al. 2009, Weir and Blackwell 2001. Roland Thaxter, a professor at Harvard University (Cambridge, MA) from 1891 to 1932, undertook the first systematic study of the Laboulbeniales, and his 5 monographic volumes (1896,1908,1924,1926,1931) and many non-illustrated papers are still among the most-often cited papers in Laboulbeniales research. Despite Thaxter's intense local collecting activities, there is no evidence that he explored the sites now included in the Boston Harbor Islands (BHI) National Recreation Area.
The Laboulbeniales lack hyphae and, unique among related fungi, they exhibit determinate growth resulting in a well-defined thallus, which can be interpreted as a reduced hyphal system (Santamaría 1998, Tavares 1985. Thalli are often less than 300 µm in length, and consist of a receptacle with a perithecium or multiple perithecia and appendages bearing antheridia, which produce spermatia (some species in the genera Aporomyces, Laboulbenia, and Rickia, however, seem to lack antheridia [Rossi andSantamaria 2008, 2015;Thaxter 1926]). We currently do not know how these fungi reproduce. The thallus attaches to the host integument at the foot cell, and at least some species form haustoria-simple or branched rhizoidal structures that penetrate the host's integument to provide additional stability and surface area for nutrient uptake (Benjamin 1971;Gäumann and Dodge 1928;Thaxter 1896Thaxter , 1908. Presumably, haustoria penetrate the host's integument to make contact with the cavity (haemocoel) and draw nutrient material from it. Although only observed in some genera, it is thought that all Laboulbeniales produce haustoria whether simple and minute or well developed (Benjamin 1971).
Laboulbeniales species can be monoecious (with both male and female sexual organs on the same thallus) or dioecious (with separate female and male thalli), and they rely on host contact to disperse their sticky, 2-celled ascospores (De Kesel 1993. Most Laboulbeniales exhibit a high degree of host specificity, with a host spectrum ranging from a single species to several related or congeneric species (De Kesel 1996, Majewski 1994, Scheloske 1969, Tavares 1985, Thaxter 1896. Phylogenetically unrelated insects living together in the same microhabitat (e.g., in ant nests or saltmarshes; Blum 1924, De Kesel and may become infected by the same species of Laboulbeniales. This finding implies that the parasite's success is dependent on host characteristics as well as environmental features (De Kesel 1996).
Although some studies have suggested that insects infected with Laboulbeniales do not experience adverse effects on their fitness (Benjamin 1971, Scheloske 1969, Whisler 1968), negative effects on hosts have been reported (Báthori et al. 2015;Kamburov et al. 1967;Riddick 2006Riddick , 2010Strandberg and Tucker 1974). Laboulbeniales can alter reproductive behaviors of infected hosts, such as oviposition patterns (Strandberg and Tucker 1974), and cause injury to the host's appendages and abdominal tissues (Gemeno et al. 2004, Thaxter 1908. Some researchers have suggested that heavily infected hosts with high numbers of thalli on the head, eyes, antennae, mouthparts, legs, and/or elytra may no longer be able to detect food efficiently, mate, or behave as predators (Nalepa andWeir 2007, Scheloske 1969). Laboulbeniales have also been implicated in playing an influential role in the population dynamics of their hosts (Kamburov et al. 1967).
The family Coccinellidae includes species with aggregating behavior and overwintering patterns that are conducive to infection by Laboulbeniales (De Kesel 2011), and the family Staphylinidae includes the most commonly infected taxa in both the tropics and temperate regions (Weir and Hammond 1997).

Methods
The Harvard Museum of Comparative Zoology houses a collection of the Coccinellidae and Staphylinidae from the BHI that includes 1431 individuals representing more than 85 species. These specimens were collected for the ATBI from 13 islands using a variety of methods: litter sampling; pitfall, malaise, light, and bowl traps; and hand collecting (for details, see Rykken and Farrell 2013).
We examined pinned insects under dissecting microscopes at 10-50x magnification to record the diversity of the Laboulbeniales on their hosts. When we found infected insects, we removed individual fungal thalli from the host using Minuten Pins (BioQuip #1208SA, Rancho Dominguez, CA) inserted onto wooden rods. We removed thalli or groups of thalli at the foot and then mounted them in eosin dye in glycerol (1 g eosin powder per 2 ml 25% glycerol), PVA Mounting Medium (BioQuip #6371A), or Amann solution (Benjamin 1971). We placed a tiny amount of Hoyer's medium on each slide with the tip of a Minuten pin, positioned thalli in the Hoyer's, allowed the specimen to dry briefly, and placed a drop of the mounting medium on the cover slip, which was then dropped sideways onto the Hoyer's medium. In this way, the briefly fixed, dried thalli remained in place when we added the cover slip, which was ringed with transparent or white nail varnish. We viewed mounted specimens at 400-1000x magnification for identification using numerous relevant systematic and taxonomic sources (listed in the Literature Cited). Slides are deposited at the Farlow Herbarium (FH; Harvard University, Cambridge, MA).

Study site: Boston Harbor Islands
BHI consists of 34 islands and peninsulas in waters near Boston, MA. The islands range in size from 3 to 150 ha and lie as far as 20 km from shore. The islands support a variety of habitats, including marine and estuarine intertidal wetlands and freshwater marshes (Tiner et al. 2003). The vegetation of the islands reflects succession after varying levels of human disturbance, and individual island floras are heavily influenced by usage history (Elliman 2005). Although fungi have not been specifically targeted in surveys of the islands, the invertebrates of the BHI are well documented (Rykken and Farrell 2013).
An ATBI, initiated by the National Park Service in 2005, sought to document all species inhabiting the islands, with particular attention to Arthropoda. The documented diversity of the BHI (Rykken and Farrell 2013) likely captures the true diversity of the islands. We expected lower diversity relative to the mainland, as is commonly found in island ecosystems (Gillespie and Roderick 2002) because of the islands' geographic isolation, small size, and level of human disturbance (Davidson et al. 2011). Island areas and distances from the mainland are available at the National Park Service website (http://www.nps.gov/boha/).

Results
We screened 253 Coccinellidae, 27 (10.7%) of which were infected with Laboulbeniales, and 1178 Staphylinidae, 20 (1.7%) of which were infected. For an overview of the BHI sites with Laboulbeniales records, see Figure 1.
In the course of the study we also found new localities for Hesperomyces virescens Thaxt. (from Canada, Cuba, Guatemala, and Japan) and Laboulbenia philonthi Thaxt. (from Canada, Grenada, Panama, Trinidad, and Venezuela).
Remarks. The first and only record for North America, 8 thalli of I. cf. mairei were found on 1 specimen of S. clavicornis from Thompson Island (MCZ-ENT00601500), collected in 2007 (Haelewaters 2013). Stenus rove beetles are common in riparian habitats but only very rarely are reported with Laboulbeniales (Santamaría 2003). Prior studies reported the genus Ilyomyces on representatives of the subfamily Steninae (Coleoptera, Staphylinidae) from France (Picard 1917, Santamaría 2003, Spain (Santamaría 1992(Santamaría , 2003, and Indonesia (Weir 1995), with I. mairei previously only known in Europe (Picard 1917;Santamaría 1992Santamaría , 2003. Ilyomyces cf. mairei in North America is reported on Stenus clavicornis, a congener of its European hosts, S. aceris and S. elegans. Stenus clavicornis is native to Europe and has been known in North America since 1968 (Majka and Klimaszewski 2008). Ilyomyces species have been reported on several Stenus species in Europe, and this incidence at the BHI likely represents either unintended co-colonization of North American habitats by the staphylinid host and its fungal parasite; host pursuit, in which host-range expansions are followed by the parasite from the same origin populations; or host-shift events, although this would imply that the parasite was already present in the US on another (ecologically) similar host, which as yet has not been proven , Haelewaters 2015, Nicholls et al. 2010, Roy et al. 2011.
Distribution and hosts. So far reported in Europe from: Austria, Czech Republic, France, Germany, Great Britain, Greece, Italy, Latvia, Lithuania, the Netherlands, Poland, and Spain; as well as from Korea, Turkey, Argentina, Ecuador, Guatemala, Mexico, and the US , Majewski 2008, Santamaría et al. 1991 Remarks. Thaxter (1896Thaxter ( , 1908 collected this species only on the American continent, with records from the New England region, California, and Florida in the US; and from Mexico and Guatemala. Hence, the suggestion was made that L. philonthi "appears to be strictly American" (Thaxter 1908), which is not the case as exemplified by the more recent numerous records across 3 climate zones in Europe: subboreal (De Kesel and Krastina 2006), temperate , Majewski 2009, Santamaría et al. 1991, and mediterranean to subtropical (Santamaría et al. 1991). Between the time of Thaxter (1908) and this study, only 1 American collection has been made, in Ecuador (Proaño Castro and Rossi 2008).
Because Thaxter (1893Thaxter ( , 1896Thaxter ( , 1908 designated no type specimen, we decided to re-examine Thaxter's slides of L. philonthi, which are deposited at FH. This led to our selection of a slide to serve as lectotype. Study of Thaxter's material also revealed new, unpublished records of this species for Grenada, Trinidad, Panama, and Venezuela, and extends the total thallus length recognized for this species. In his original description, Thaxter (1893) stated that the total length from foot to perithecial tip was 290-360 µm. Thalli from Poland and the Iberian Peninsula measure up to about 550 µm (Majewski 1994) and 558 µm (Santamaría 1998). The Venezuelan material, however, provided us with the longest thalli observed so far, up to 830 µm in length (slide FH 00313498). Thaxter (1908) mentioned that L. philonthi is the common species on Philonthus spp. in temperate South America, which may be supported by the current new records.
Remarks. Among Thaxter's material, there is a series of 9 slides of S. conosomatis, labeled as having been collected in Belmont and Waverley, both in close proximity in eastern Massachusetts. Collections in this series were made in 1900 and 1901, and we assume that Thaxter had all 9 slides available when he made the description of S. conosomatis (Thaxter 1901). Because the slide that Thaxter indicated as holotype (FH 00313506, unpublished) is in unsatisfactory condition, we decided to designate a lectotype (= FH 00313505).
This species was commonly found on Sepedophilus littoreus (L., 1758) [as Conosoma pubescens; see Herman 2001 for the complex taxonomic history of this species] in the New England states (Thaxter 1901(Thaxter , 1931. This is the first published record from the US since Thaxter's contributions. Most thalli of S. conosomatis have only 1 perithecium and 0, 1, or 2 perithecial primordia. Both Tavares (1985) and Majewski (1994) described the development of secondary perithecia upon the cell above cell II. Since cell III never bears perithecia, this cell should be considered cell II'. Of the examined thalli on slide FH 00313423, 2 thalli have a perithecial primordium in addition to a normal (but broken) perithecium, both borne on cell II; in the other thallus, the third cell of the receptacle axis (cell II') gives rise to the single perithecium.  (Huggert andEriksson 2010, Santamaría et al. 1991), Algeria (Maire 1920), and Sri Lanka (Thaxter 1931)  Remarks. Of several available slides of collections from Kittery Point, ME, in agreement with the original description (Thaxter 1894), slide FH 00313507 is in good condition and contains a thallus with diagnostic characters. This specimen is designated above as the lectotype.

Teratomyces actobii
The genus Teratomyces Thaxt. consists of 10 species, 9 described by Thaxter (1893Thaxter ( , 1894Thaxter ( , 1896Thaxter ( , 1900Thaxter ( , 1901Thaxter ( , 1931 and 1 by Rossi (2010). Additionally, 2 collections of Teratomyces have been made that are undescribed (New Zealand [Hughes et al. 2004], Bolivia [Weir and Rossi 2001]). Identification of species is difficult in this genus. Species delimitation is based on characters such as receptacle structure and color, length of cell VI, number of perithecia, and length and color of appendage; these characters can vary among thalli from a single host specimen (Hughes et al. 2004). The extent of morphological variability is often subject to debate in its use in defining species limits, especially when "morpho-species" occur on a single host specimen.
Although our material from the BHI is immature, the receptacle structure and especially the typical variability in blackening make us believe that it belongs to T. actobii (Thaxter 1896). Thaxter (1931)  its blackening typically extends to the basal part of cell III. These characteristics separate it clearly from T. actobii. The distinction between the latter species and T. brevicaulis is based on the size of cell VI (stalk cell of perithecium) and the larger clavate appendage cells in T. brevicaulis. Confusion arises because both species are sometimes found on a single host specimen (Thaxter 1896). In all of the Thaxter slides we examined, the distinction between T. actobii and T. brevicaulis as described by Thaxter is clear except for 1 record on Erichsonius basalis Motschulsky, 1858 [as Actobius] from Sri Lanka (slide FH 00313508; see Thaxter 1931); these specimens combine features of T. actobii (the receptacle varies from hyaline to black) and of T. brevicaulis (having a short cell VI and slender perithecium of T. brevicaulis. For this reason, we consider T. actobii and T. brevicaulis to be synonyms thereby accepting considerable within-species morphological variability. Both names were introduced in the same publication (Thaxter 1894). We choose to use the T. actobii for this species over T. brevicaulis because this name has been more widely used. We were unable to find any position-relatedness for either of the two forms; both were removed from legs and abdomen.
Generating DNA-sequence data will be the final key to unlock the phylogenetic relationship among morpho-species in Teratomyces.

Discussion
These observations are the first to document the Laboulbeniales of the Boston Harbor Islands, and the first reports of Laboulbeniales on Coccinellidae and Staphylinidae in the New England area since the time of Roland Thaxter (1858-1932. The study of Laboulbeniales can bring new insight to the community dynamics of host-parasite interactions. Insects disperse with their parasites; thus, there is potential for infection of native hosts by infected introduced insect species. Although more research is needed, we hypothesize that host pursuit (Nicholls et al. 2010) followed by host shift is the mechanism at play in (the distribution of) Ilyomyces cf. mairei on the BHI. Helmus et al. (2014, references therein) demonstrated that the acceleration of global shipping has increased establishment rates of exotic species, including beetles. The rapid and far-reaching transport of these beetles provides increased opportunities for dispersal together with their associated fungi. In addition, Humans often actively transport and spread insects used or studied for biological control. The study of obligate and non-motile ectoparasites of insects, such as the Laboulbeniales, provides an avenue to document effects of human-mediated host dispersal on parasite dynamics and distributions. However, further research is needed to fully characterize the diversity of the Laboulbeniales. Advances in molecular methods for the Laboulbeniales will no doubt accelerate determinations of parasite identities and host relationships.
In this study, we documented new records and revised species ranges from a relatively well-sampled region. With more thorough documentation of species diversity, future studies should characterize the mechanisms governing the distribution of these fascinating fungal parasites. With respect to Laboulbeniales research, the BHI collection at the Harvard Museum of Comparative Zoology is still largely unexplored. Of the Laboulbeniales discussed in the present paper, some species were previously known from the US (H. virescens) or specifically from New England (L. philonthi, P. protea, S. conosomatis, T. actobii). Others had not been reported from North America (I. cf. mairei, C. nipponicus) and are new records for the continent. We found the Laboulbeniales on the predicted host genera. Bryoporus testaceus, Hippodamia tredicimpunctata tibialis, and Stenus clavicornis are new host species for C. nipponicus, H. virescens, and I. cf. mairei, respectively. Future work will include screening the Carabidae and other families in the BHI collection, as well as continued sampling on the Boston Harbor Islands.