Insects and their Laboulbeniales (Ascomycota, Fungi) of Lake Eustis and Emeralda Marsh Conservation Area: A case study on urbanization and diversity

Abstract A rapid biodiversity assessment of insects and associated Laboulbeniales fungi was conducted over the course of five nights in August, 2018, at two central Florida lakes: Lake Eustis and the nearby protected and restored National Natural Landmark, Emeralda Marsh Conservation Area (EMCA), which encompasses a portion of Lake Griffin. Lake Eustis was surveyed for Laboulbeniales in 1897 by mycologist Dr. Roland Thaxter but has not since been investigated. Because Lake Eustis has been urbanized, with the lake perimeter almost entirely altered by human development, the site offers a look into Laboulbeniales diversity across a 121‐year timeline, before and after human development. By surveying Lake Eustis and EMCA, a modern case study comparison of Laboulbeniales and insect diversity between a developed and a protected and restored system is made. A total of 4022 insects were collected during the rapid assessment. Overall, insect abundance was greater at EMCA, with 3001 insects collected, than 1021 insects collected from Eustis. Although family‐level insect richness was comparable between sites, with 55 families present at EMCA and 56 at Eustis, 529 out of 3001 (17.6%) of the insects collected at EMCA were hosts to parasitic Laboulbeniales fungi, whereas only 2 out of 1021 (0.19%) collected from Eustis were infected. A total of 16 species of Laboulbeniales found at EMCA compared with only one at Eustis. The current number of Laboulbeniales species documented at Eustis was incredibly depauperate compared with the 26 species and two varieties recorded by Thaxter in 1897. These findings suggest the possibility of utilizing Laboulbeniales as indicators of ecosystem health, and future research should investigate this question further. A figure displaying host–parasite records and a species list of Laboulbeniales are presented. Finally, updated occurrence records for species of Ceratomyces and Hydrophilomyces are provided.


| Insect diversity
Assessing biodiversity of insects and fungi presents challenges as they are two most diverse groups of eukaryotes and also suffering a paucity of trained taxonomists. Knowledge of insects and fungi can be described as highly uneven, with representative members, often those associated with agriculture, industry, or disease, receiving vastly more attention than other groups (Ainsworth et al., 2018;Kim, 1993). In both groups, millions of species remain undescribed (Grimaldi & Engel, 2004;Hawksworth & Lücking, 2017).
Insect diversity studies have yielded a range of estimates for global and site-specific studies, with a number of researchers trying their hand at different techniques and methods in order to arrive at sound estimates. Insects are so diverse that researchers do not even agree on estimates of currently described species. Estimates range from 750,000 (Wilson, 1992) to 1.4 million (Hammond, 1992). Based on work by Gaston (1991) and Resh and Cardé (2003), Grimaldi and Engel (2004) endorse the estimate of 925,000 for currently named species. Although this discrepancy may seem surprising, it is also understandable, given the lack of sufficient incentives for researchers to spend time scouring old literature, synonymizing, cataloging, and producing monographs.
Regarding estimates of living species, both described and undescribed, estimates of insect richness are even more variable. The lowest estimate is about 2 million species (Grimaldi & Engel, 2004), and the largest is a staggering 30 million tropical insect species (Erwin, 1982). Erwin's estimate was based onfogging techniques on tree canopies in neotropical forests, upon which extrapolations were made for total insect diversity. Erwin recorded trees as having unique species of insects in their canopies and used the total tropical tree diversity of approximately 50,000 species to extrapolate. Most researchers now agree this estimate is much too high largely because the assumption that the insects found in tree canopies would be highly host-specific is likely erroneous (Grimaldi & Engel, 2004). Grimaldi and Engel endorse Gaston's (1991) estimate of about 5 million total living insect species. This estimate was based on surveying collections held by systematists around the world. Despite this method having some potential shortcomings, for example, collection biases of individual collectors and the presence of unexamined or unknown duplicates held across collections, the authors believe it is currently the most accurate estimate of global insect diversity. If this estimation is accepted, then the aforementioned figure of 925,000 named insects would represent 20% of extant insect diversity.
In a quickly changing climate, there is increasing evidence of mass declines of insects and, therefore, a pressing need to monitor insect biodiversity at local and regional scales (Kim & Byrne, 2006).
Comparable data have not been recovered for fungi as fungal conservation is in its early stages (Mueller, 2017). However, there is indication of declines in fungal species richness in response to human disturbances, including but not limited to nutrient loading, mass tree die-off due to introduced pathogens, acidification, and habitat loss (Arnolds, 1991;Treu et al., 2014). Biodiversity studies usually focus on vertebrate animals and vascular plants, whereas those focused on invertebrates and fungi are rare (Fiesler & Drake, 2016). Despite being ubiquitous and essential components of the biosphere, macroinvertebrates such as insects remain underserved with respect to their risk assessment and conservation status. As of 2006, <0.1%

of described insects had been assessed for inclusion in the Red
List maintained by the International Union for the Conservation of Nature (IUCN) (Rodrigues et al., 2006).
Because of the staggering diversity and abundance of insects, there exists feasibility concerns when designing biodiversity studies.
Rapid biodiversity assessments of insects are frequently employed in order to glean broad but manageable data sets that can be, albeit tentatively, extrapolated as a flexible measure of communities and populations (Ward and Larivière, 2004). There is currently a concerted effort by entomologists to establish optimal sampling methods for assessing insect biodiversity by taxa, population, assemblage, community, habitat, and region (Brown, 1997;Hughes et al., 2000;Kim, 1993;Ward and Larivière, 2004). Many scientists agree that establishing protected areas is the most effective way to protect multikingdom species diversity, particularly when considering understudied, vulnerable, and uncharismatic groups, which includes many insects and fungi (Hughes et al., 2000).

| Fungal diversity
The state of knowledge of fungi is substantially behind that of insects. A widely cited estimate of global fungal diversity is upward of 1.5 million (Hawksworth, 1991). Mycologists generally agree this is a conservative estimate, in part because it was based primarily on extrapolations from fungus-to-plant ratios in temperate regions and did not give due consideration to the hyperdiverse realm of insect-associated fungi, such as Laboulbeniales, or account for tropical species diversity (Hawksworth, 1991;Hawksworth & Lücking, 2017). The highest estimate of fungal diversity is currently 6 million, which was put forward by Taylor et al. (2014). The most recent estimate (Hawksworth & Lücking, 2017) of extant fungi is 2.2 to 3.8 million, and the updated fungus-to-plant ratio for temperate zones is 8:1. Of that, ~138,000 species have been described (Hibbett et al., 2016;Kirk, 2019). With only ~6% of the lower estimation being known to science, the remaining task is tremendous.
Unlike many plant and animal groups, fungi do not broadly enjoy the benefits of being well-studied and clearly understood. New species are most likely to be discovered by investigating relatively understudied habitats and microhabitats, including insect bodies, lichen-dwelling fungi, and cryptic species, through environmental (eDNA) sequencing (Hawksworth & Lücking, 2017) and within natural history collections (Wijayawardene et al., 2020).
In addition to fungi being relatively poorly studied, the often sporadic, ephemeral, and unpredictable appearance of fruiting bodies complicates obtaining baseline data on factors such as occurrence and abundance and has constrained our ability to provide clear objective assessments of fungi overtime. The complex biotic and abiotic forces leading to a species even producing a fruiting body remains unknown in many cases and likely involves the combination and interactions of degree days, soil temperature, precipitation volume, vegetation patterns, and so forth (Mihail et al., 2007). Although some fungi, for example, some species of morels, can be reliably found in the same place at more or less the same time every year, other species, such as Ionomidotis sp. (personal observation) or Hericium bembedjaense (Jumbam et al., 2019) may be seen once in a given location and then not again for years, if ever. Although substantial efforts have recently been made in fungal conservation, this field remains in its early stages (Mueller, 2017). According to the State of the World's Fungi (Ainsworth et al., 2018), only 56 species of fungi have been evaluated for placement on the IUCN Red List, of which 43 ended up being included. Comparatively, 25,452 species of plants and 68,054 species of animals have been evaluated. It is therefore imperative that fungi receive increased attention, concern, and action.

| Laboulbeniales
Laboulbeniales (Ascomycota, Fungi) are microscopic obligate parasites on arthropods, primarily occurring on insects. Laboulbeniales are considered the most diverse lineage of insect-associated fungi, with ~2325 described species in 145 genera, but current estimates indicate there are at least 40,000 species awaiting description Kirk, 2019;Weir & Hammond, 1997).
The impact of Laboulbeniales fungi on their insect hosts are not fully understood, and basic studies of their biology are still limited (Haelewaters et al., 2021). Only a handful of scientists in the world specialize in the study of Laboulbeniales, and yet because of the size, diversity, and uniqueness of this lineage, it is undoubtedly a cradle of novel taxonomic and ecological information.
One early and prolific researcher of Laboulbeniales was Dr. Roland Thaxter. During his career at Harvard University between 1891 and 1932, Thaxter described >1000 species of Laboulbeniales and made substantial contributions to our understanding of their development and general biology. Most of his life's work on Laboulbeniales is contained within a five-volume set of his Contribution towards a Monograph of the Laboulbeniaceae (1896Laboulbeniaceae ( , 1908Laboulbeniaceae ( , 1924Laboulbeniaceae ( , 1926Laboulbeniaceae ( , 1931, a tremendous contribution to mycology.

| Foundation of study
One of Thaxter's collection sites for Laboulbeniales was in Eustis, a small central Florida city on the east shore of Lake Eustis. Beginning in the early 1800s, colonial Europeans forcefully established Eustis on Seminole land (Preserving Eustis History, n.d.). The city was named after General Abraham Eustis, who was known for his role in wars against the Seminole people (Preserving Eustis History, n.d.).
The numerous connected waterways in the region allowed for Eustis to become a hub for steamboats, and the construction of the railroad that connected many Floridian towns in 1880 led to an increase in settlement from <500 in 1900 to 21,300 in present day Eustis.
According to his travel records, Thaxter was in Eustis during the very early days of the city, from September 25th to October 10th, 1897 (Pfister, 1982), before most of the present urbanization. Because the methods employed by Thaxter during this trip are unpublished and unrecorded, it is unclear precisely how much time was spent collecting or the precise locations from which he collected (D. H. Pfister, personal communication Eustis has changed considerably over the last 100 years, with a majority of the lake perimeter being cleared for housing and other human infrastructure (Google Earth, n.d.). In addition, since Thaxter's visit in 1897, no research has been published dedicated to Laboulbeniales in Eustis, or even from the state of Florida.
The overall goal of this study was to conduct a biodiversity assessment over time, as well as between two habitats. By returning to Eustis and attempting to re-collect species recorded by Thaxter, the goal was to provide insights into shifts in biodiversity of Laboulbeniales and their associated insects since 1897.

Because Eustis is now impacted by urbanization, Emeralda Marsh
Conservation Area (EMCA), was also sampled as a control. EMCA includes a portion of Lake Griffin and surrounding habitat and is located ~14 km from the east shore of Lake Eustis. Due to its designation as a National Natural Landmark since 1974 and subsequent and ongoing restoration efforts, EMCA provides a closer approximation of the habitat in which Thaxter sampled ( Figure 1). Two biodiversity assessments were made: one over time (between Eustis in 1897 and Eustis in 2018) and one between habitats (between Eustis and EMCA in 2018). The working hypothesis for this study was that EMCA, the restored site, would contain greater insect and fungal diversity than Eustis, the unprotected and unrestored site. I further hypothesized EMCA would be more likely to harbor species recorded by Thaxter than Eustis. These data provide a baseline for understanding how urban development around lake systems may affect biodiversity of insects and their accompanying Laboulbeniales parasites, and to begin to explore if and how Laboulbeniales may serve as a proxy for biodiversity and an indicator for ecosystem health.

| Site description
Lake Eustis and EMCA (including Lake Griffin) are part of the Central Valley Region (Region 7508) of Florida ( Figure 1). Lakes in this subtropical region are categorized by being large, shallow, and eutrophic (Lake County Water Atlas, Emeralda Marsh, n.d.). Lake Eustis and Lake Griffin are part of the Ocklawaha Chain of Lakes, which includes a total of 10 connected lakes. The headwaters of this chain is Lake Apopka, which is fed by a natural spring and by rain. Lake Griffin is the most downstream of the 10 lakes and Lake Eustis is directly upstream from Lake Griffin. Lake Griffin empties northward into the Ocklawaha River, which ultimately connects to the St. Johns River (St. Johns River Water Management District, Lake Apopka Basin, n.d.) The surface area of Lake Griffin is ~38 km 2 , and that of Lake Eustis is~31 km 2 . The average depth of Lake Griffin is ~2 m, and that of Lake Eustis is ~3 m. The bottom of the Lake Griffin is composed of soft organic matter measuring an average of 1.7 m thickness (Fulton et al., 2015). Equivalent measurements for Lake Eustis were not available. Over the past ~150 years, the Ocklawaha Chain of Lakes have experienced a barrage of human manipulations including, draining, dredging, levying, agricultural conversion, waste dumping, and nutrient loading. In the 1950s, the eastern area of Lake Griffin was levied and drained and converted into agricultural land and muck farms.
These farms became an external source of nutrient loading into Lake Griffin (Fulton et al., 2015).
As of 2004, pervious and impervious percentages of the Lake Griffin Basin was 65% and 35%, respectively, whereas the basin containing Lake Eustis (Burrell Basin) was 50% pervious/impervious (Fulton et al., 2004). Surface area coverage by emergent and floating-leaved vegetation decreased from ~50% in the 1940s to <2% in the 1970s (Fulton et al., 2015). Similar data and exact mapping of wetland and vegetation loss are not available for Lake Eustis; however, mention is made in an issue of Engineering News and American Railway Journal (1884)  Griffin has been hypereutrophic (Fulton, 2015). In the early 1990s, Restoration activities have been extensive at Lake Griffin but comparable efforts have not been made at Lake Eustis (Fulton et al., 2015). Likely as a result of these activities, total phosphorus (TP), chlorophyll-a, and total nitrogen (TN) have been decreasing with statistical significance in Lake Griffin between 1994 and 2012.
Comparatively, Lake Eustis (as well as other lakes in the chain) have not seen significant changes in TP, but did have significant decreases in chlorophyll-a and TN. Overall, the environmental improvements F I G U R E seen in Lake Eustis were smaller in magnitude than in Lake Griffin.
The relatively moderate improvements in Lake Eustis may be due to the upstream restoration efforts at Lake Apopka, whereas Lake Griffin is likely benefiting from the extensive restoration efforts at EMCA in addition to the upstream efforts (Fulton et al., 2015).

| Insect collection
A rapid biodiversity assessment was conducted over five days,

| Fungal collection
All collected insects were scanned for infections of Laboulbeniales under a Nikon stereomicroscope at 20-40×. Presence/absence data were recorded for each insect. All insect specimens were identified family level or lower, using Marshall (2006) and are housed at

| Data and data analyses
Abundance, species richness, and species diversity (Simpson's and Shannon-Weiner, H') were calculated for both insects and Laboulbeniales. Comparisons are presented from the two time periods for collections in Eustis (1897 and 2018) as well as between Eustis and EMCA. As this was a case study resulting from a single collection event at each site, actual statistical comparisons could not made as it would have been psuedoreplication.

| RE SULTS
A total of 4022 insects were collected during the rapid assessment ( Figure 2

| Ceratomyces Thaxt
The genus Ceratomyces was established by Thaxter (1892) and currently contains 21 species. Very few publications contain information on Ceratomyces since Thaxter's contributions (Bernardi et al., 2014;Goldmann & Weir, 2018;Santamaria, 1999;Shen et al., 2009;Tavares, 1985). Because relatively few contributions have been made to this genus, it is of value to update occurrence records for all species of Ceratomyces found during this study. In addition to the data obtained here additional occurrence and range extension data are published for the first time from the collection of Dr. Richard K.
Benjamin, which is currently housed in the mycological herbarium at SUNY-ESF. Further information about these collections, such as host data and precise locality, can be accessed through mycoportal.org.  (Figure 5e).

Remarks: This species has not been formally recorded in North
America since its original publication by Thaxter (1908), where he reported the type from Brazil, and a specimen from Eustis, FL. This species was also reported from Argentina (Tavares, 1985;Thaxter, 1931 (Figure 5d).

| D ISCUSS I ON
Despite being relatively understudied compared with other groups of fungi, the Laboulbeniales possess certain qualities that lend themselves toward being a model group for studies in ecosystem health. For one, these fungi are reasonably visually detectable ectoparasites, which form 3-dimensional parenchymatous structures, referred to as thalli (Blackwell et al., 2020;Haelewaters et al., 2021;Weir & Hammond, 1997). Additionally, the fungi persist intact on the host for decades if the host is collected and preserved either in by pinning or stored in ethanol. Therefore, entomological collections serve as a tremendous repository for these organisms, allowing researchers to utilize an alternative source for novel taxa, host lists, or habitat associations (Haelewaters et al., 2021;Kaishian & Weir, 2018). Similarly, previous systematic and spatial work by entomologists already exist and can be utilized for the study of this group. Furthermore, as discussed before, some fungi, particularly those that form fleshy sporocarps, may only fruit during extremely narrow and/or sporadic windows of time.
Because Laboulbeniales lack a known asexual stage and possess F I G U R E 6 (a) Hydrophilomyces hamatus; (b) a cluster of Hydrophilomyces gracilis (a) (b) F I G U R E 7 Insect family richness, abundance, and infection at Lake Eustis spores that are only briefly viable in the environment (Cottrell & Riddick, 2012;De Kesel, 1995), the presence of their fruiting body in the environment is more reliable and consistent indicator of species presence than many other groups of fungi. This study expanded upon studies by Andersen and Skorping (1991) and  (Cottrell & Riddick, 2012;De Kesel, 1993, 1995Nalepa & Weir, 2007;Richards & Smith, 1955;Scheloske, 1969;Weir & Beakes, 1995  . No other species collected in this study are known to be occurring on invasive insects. For additional discussion of introduced species of Laboulbeniales see .
When considering Laboulbeniales, collection of insects offers a two-for-one assessment of insects and fungi, providing a more textured, multikingdom understanding of biodiversity at a given site. In this study, similar richness of insect families was found at both sites, whereas the presence of fungi was markedly different, with a 17.6% infection rate at EMCA and a 0.19% infection rate at Eustis. In this case, without attention paid to the fungal dimension, incorrect conclusions may be drawn about population trends or robustness, and overall biodiversity of these two habitats. A multikingdom assessment consolidates resources and effort, which increases the feasibility of conducting biodiversity monitoring. Making such work more feasible is attractive, given the mounting pressure of climate change and the related impacts on biodiversity. These findings also highlight that specialist organisms, such as the highly host-specific and obligately associated members of the Laboulbeniales, may be of particular risk for population decline, range restriction, loss of genetic diversity, extirpation, and total extinction in a changing climate (Thomas, 2000;Warren et al., 2001). For such organisms, establishing protected areas and carrying out focused monitoring protocols is of great importance (Chape et al., 2005).

ACK N OWLED G EM ENTS
Thank you to my field assistants Emma Kaishian and Will Stallings.

O PE N R E S E A RCH BA D G E S
This article has earned an Open Data badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://doi.org/10.5061/ dryad.47d7w m3f5.

DATA AVA I L A B I L I T Y S TAT E M E N T
• All insect and fungal material is available for loan upon request from SUNY-ESF.