Revising the Freshwater Thelohania to Astathelohania gen. et comb. nov., and Description of Two New Species

Crayfish are common hosts of microsporidian parasites, prominently from the genus Thelohania. Thelohania is a polyphyletic genus, with multiple genetically distinct lineages found from freshwater and marine environments. Researchers have been calling for a revision of this group for over a decade. We provide evidence that crayfish-infecting freshwater Thelohania are genetically and phylogenetically distinct from the marine Thelohania (Clade V/Glugeida), whilst also describing two new species that give further support to the taxonomic revision. We propose that the freshwater Thelohania should be transferred to their own genus, Astathelohania gen. et comb. nov., in a new family (Astathelohaniidae n. fam.). This results in the revision of Thelohania contejeani (Astathelohania contejeani), Thelohania montirivulorum (Astathelohania montirivulorum), and Thelohania parastaci (Astathelohania parastaci). We also describe two novel muscle-infecting Astathelohania species, A. virili n. sp. and A. rusti n. sp., from North American crayfishes (Faxonius sp.). We used histological, molecular, and ultrastructural data to formally describe the novel isolates. Our data suggest that the Astathelohania are genetically distinct from other known microsporidian genera, outside any described family, and that their SSU rRNA gene sequence diversity follows their host species and native geographic location. The range of this genus currently includes North America, Europe, and Australia.


Histopathology
For histopathological screening, crayfish were dissected to obtain antennal gland, eye, gill, gonad, gut, heart, hepatopancreas, muscle, and nerve tissue. These tissues were preserved in Davidson's Freshwater Fixative (35.5% tap water, 31% 95%-ethanol, 22% formaldehyde, 11.5% glacial acetic acid) for 24-48 h and then moved to 70% ethanol. The tissues were wax-embedded, sectioned (3-4 µm), mounted on glass slides, and stained with hematoxylin and alcoholic eosin as specified in Bojko et al. [5]. Histology slides were screened using a Leica DM500 microscope. Biopsies of the antennal gland, gill, hepatopancreas, and muscle tissue were also fixed in 96% molecular grade ethanol for molecular diagnostics and a third biopsy of the same tissues placed into 2.5% glutaraldehyde in a 0.1% sodium cacodylate buffer for transmission electron microscopy (TEM).
Resin infiltrated samples were cured for 72 h at 60 • C before semi-thick sections (500 nm) were stained with toluidine blue. Ultra-thin sections were collected on carbon coated Formvar 100 mesh grid (EMS, Hatfield, PA, USA). Sections were stained with 2% aqueous uranyl acetate and lead citrate (EMS, Hatfield, PA, USA). Sections were viewed with an FEI Teenai G2 Spirit Twin TEM (FEI Corp., Hillsboro, OR, USA) and digital images were captured with a Gatan UltraScan 2k × 2k camera and Digital Micrograph software (Gatan Inc., Pleasanton, CA, USA). All morphology measurements were acquired from TEM images and ImageJ software [31].

Molecular Diagnostics
Microsporidia-infected muscle tissue underwent DNA extraction using Qiagen's DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. Extracted DNA was used in a Promega 'Flexi-Tag' PCR (4Promega, Madison, WI, USA) consisting of 2.5 mM MgCl 2 , 1 mM dNTPs, 0.25 µL Promega Taq polymerase, 10 µL buffer, 1 µM forward primer V1F (5 -CACCAGGTTGATTCTGCCTGAC-3 ), 1 µM reverse primer MC3r (5 -GATAACGACGGGCGGTGTGTACAA-3 ) in a 50 µL reaction volume [32]. The thermocycler conditions for the reaction consisted of an initial denature at 94 • C for five minutes followed by 35 cycles of 94 • C-55 • C-72 • C, with each temperature held for one minute, and a final extension period at 72 • C for seven minutes. The resulting amplicons were visualized using gel electrophoresis on a 1.5% agarose gel. The microsporidia-specific amplicon size was~1100 bp. The bands were excised from the gel and extracted using Qiagen's gel extraction kit (Qiagen, Hilden, Germany). The amplicons were sent for sequencing using Eurofins Genomics (eurofinsgenomics.com; accessed on 20 January 2022) for both forward and reverse orientation.

Phylogenetics and Genetic Comparisons
A maximum-likelihood (ML) phylogenetic tree was constructed for representative species from across the Microsporidia (n = 150), including all available Thelohania isolates and those sequenced in this study. Sequences were downloaded from NCBI, or provided by authors, and aligned using MAFFT in CIPRES [33], resulting in 2519 bp comparable columns (including gaps). The alignment was uploaded to the IQtree server [34] for ML tree construction, resulting in a tree inferred from 1000 bootstrap replicates and based on the evolutionary model: GTR+F+I+G4, according to Bayesian information criterion (BIC The sequence demarcation tool v.1.2. [35] was used to compare the genetic similarity of the rRNA (SSU) gene for all available freshwater Thelohania isolates, along with T. butleri (marine; Glugeida), other genera in the 'orphan lineage' (Hamiltosporidium, Neoflabelliforma, Areospora), and the new isolates sequenced in this study.

Pathology, Ultrastructure, and Development for Microsporidiosis in Faxonius virilis
One of the four F. virilis specimens exhibited signs of microsporidiosis in the form of white muscle tissue, visible through the ventral cuticle of the abdomen ( Figure 1A-C). This individual had a loss of righting response and subsequent decline in physiological condition in captivity. The remaining three F. virilis individuals did not exhibit clear signs of gross pathology. Histological screening of all individuals revealed microsporidian spores developing within sporophorous vesicles (SPV) within the sarcolemma of host skeletal and heart muscle fibers ( Figure 1D-I). Multiple developmental stages were observed during histological screening.
The developmental pattern for the microsporidium-infecting F. virilis occurred within the sarcolemma of the muscle fibers, with various stages of spore development occurring within close proximity to one another ( Figure 2A). The development began with a binucleate meront in direct contact with the host cytoplasm and often proximally associated with host muscle fibers ( Figure 2B,C). SPVs (8.1 ± 0.7 µm in diameter; n = 10, SD) were observed to house developing meronts, which divided into up to eight early sporonts ( Figure 2D). During sporogony, a sporogonial plasmodium, which is presumably formed from the merging of binucleate counterparts and subsequent meiosis (not observed), divides into up to eight uninucleate sporoblasts via rosette-like division ( Figure 2E,F). Dense bodies created by aggregations of granules were observed within the SPVs in the early stages of sporogony prior to the formation of individual sporoblasts ( Figure 2E). As the sporonts developed into sporoblasts, electron-dense organelles began to develop ( Figure 2F). Microtubular-like (73 ± 10 nm in diameter; n = 10, SD) and tubular-like (241 ± 26 nm in diameter; n = 10, SD) structures were observable within the episporontal space, which became more numerous as the development of the sporoblasts progressed ( Figures 2G and 3A). Sporoblasts were characterized by a thick electron-dense plasmalemma and the early development of the organelles, including the polar filament and anchoring disc ( Figure 3B-D).
bly formed from the merging of binucleate counterparts and subsequent meiosis (not observed), divides into up to eight uninucleate sporoblasts via rosette-like division ( Figure  2E,F). Dense bodies created by aggregations of granules were observed within the SPVs in the early stages of sporogony prior to the formation of individual sporoblasts ( Figure  2E). As the sporonts developed into sporoblasts, electron-dense organelles began to develop ( Figure 2F). Microtubular-like (73 ± 10 nm in diameter; n = 10, SD) and tubular-like (241 ± 26 nm in diameter; n = 10, SD) structures were observable within the episporontal space, which became more numerous as the development of the sporoblasts progressed ( Figures 2G and 3A). Sporoblasts were characterized by a thick electron-dense plasmalemma and the early development of the organelles, including the polar filament and anchoring disc ( Figure 3B-D).

Figure 1. Gross pathology and histopathology of microsporidian infections in Faxonius virilis and
Faxonius rusticus: (A) muscle tissue of infected crayfish is white and visible through the ventral cuticle of the abdomen (black arrow); (B) a transverse section of the abdomen reveals white muscle tissue (black arrow) presumably due to infection; (C) during dissection, white muscle tissue throughout the body cavity was white (black arrow) from the infection; (D) heart tissue with groups of developing spores; (E) a higher magnification of (D) of one cluster of developing spores in the heart tissue (HT) and the evident sporophorous vesicles containing the spores (black arrow); (F) Figure 1. Gross pathology and histopathology of microsporidian infections in Faxonius virilis and Faxonius rusticus: (A) muscle tissue of infected crayfish is white and visible through the ventral cuticle of the abdomen (black arrow); (B) a transverse section of the abdomen reveals white muscle tissue (black arrow) presumably due to infection; (C) during dissection, white muscle tissue throughout the body cavity was white (black arrow) from the infection; (D) heart tissue with groups of developing spores; (E) a higher magnification of (D) of one cluster of developing spores in the heart tissue (HT) and the evident sporophorous vesicles containing the spores (black arrow); (F) abdominal muscle tissue exhibiting a heavy microsporidian infection; (G) high magnification image of a cluster of spores (black arrow) developing within the heart tissue and the production of granulomas (white arrow); (H) microsporidian spores (white arrow) developing within the muscle stalk of the eye (black arrow); (I) an immune response to the microsporidian infection in the abdominal muscle resulting in the production of several granulomas (black arrow).

of 19
abdominal muscle tissue exhibiting a heavy microsporidian infection; (G) high magnification image of a cluster of spores (black arrow) developing within the heart tissue and the production of granulomas (white arrow); (H) microsporidian spores (white arrow) developing within the muscle stalk of the eye (black arrow); (I) an immune response to the microsporidian infection in the abdominal muscle resulting in the production of several granulomas (black arrow). All mature spores observed were uninucleate. Uninucleate mature spores were contained within SPVs and were oval in shape, with a wider posterior end ( Figure 3E). Mature spores were 3.4 ± 0.1 μm (n = 7, SD) in length and 2.0 ± 0.3 μm (n = 10, SD) in width, with 16-17 coils of the polar filament (118 ± 3 nm in diameter; n = 10, SD) arranged in two or three layers ( Figure 3F). The mature spore ultrastructure included an anchoring disc, bilaminar polarplast, a coiled polar filament, and a posterior vacuole ( Figure 3G). The spore wall was composed of an electron-lucent endospore (82 ± 12 nm; n = 10, SD) and an electron-dense exospore (25 ± 3 nm; n = 10, SD), which thinned at the apex of the spore above the anchoring disc ( Figure 3H).

Pathology, Ultrastructure, and Development for Microsporidiosis in Faxonius rusticus
Two F. rusticus specimens exhibited signs of microsporidiosis, with white muscle tissue visible through the ventral cuticle of the abdomen. Upon dissection, white musculature was seen throughout the body cavity of the specimen ( Figure 1A-C). Histological screening of the infected F. rusticus individuals revealed microsporidian spores developing within SPVs within the sarcolemma of the hosts' skeletal and heart muscle fibers ( Figure 1D-I).
Multiple developmental stages were observed during our histological screening, which were observed in greater detail using TEM.
The development of the novel microsporidium occurred within the sarcolemma of the host muscle fibers and various developmental stages were visible in close proximity to one another within individual SPVs ( Figure 4A). Mature spores were not found to be dimorphic, and all observed spores were uninucleate. The development of this microsporidium began with large binucleate meronts developing in direct contact with host cytoplasm ( Figure 4B). Meronts were not contained within an SPV and had a simple plasmalemma.     Merogony included the development of an SPV (5.2 ± 0.6 µm in diameter; n = 10, SD), which appeared to develop from the plasmalemma ( Figure 4C). The binucleate meront progressed into a rosette-shaped plasmodium, which divided to form eight uninucleate sporoblasts ( Figure 4D,E). Microtubular-like (70 ± 9 nm in diameter; n = 10, SD) and tubular-like structures (244 ± 32 nm in diameter; n = 10, SD) were abundant within the episporontal space at this stage of development. As the sporoblasts continued to develop, their plasmalemma thickened and became more electron dense. They developed organelles, beginning with the polar filament ( Figure 4F,G). As the sporoblast progressed into a mature spore, a thick, electron-lucent endospore became apparent ( Figure 4H).
The ultrastructure of a mature spore included an anchoring disc, a bilaminar polarplast, a posterior vacuole, and a polar filament, which coiled 13-14 times (141 ± 14 nm in diameter; n = 10, SD) ( Figure 4I-K). The mature spores were uninucleate and oval, with a wider posterior end. The spores were 3.2 ± 0.5 um (n = 10, SD) in length and 1.7 ± 0.3 um (n = 10, SD) in width with a spore wall composed of an electron-lucent endospore (57 ± 18 nm; n = 10, SD) and electron-dense exospore (25 ± 6 nm; n = 10, SD), which thinned at the apex of the spore above the anchoring disc ( Figure 4J,K). Table 2 provides morphological information for the two new species, and provides a comparison to other related species, following a table provided by Moodie et al. [9].

Genetic Similarity and Phylogenetic Placement of the Novel Microsporidians
The four microsporidian SSU sequence isolates from F. virilis were identical to one another, as were the two isolates from F. rusticus ( Figure 5); however, the novel isolates from each host were genetically distinct (98% coverage; 83.71% similarity; e-value: 0.0). A 775 bp sequence from the novel microsporidium-infecting F. virilis (OM630068) showed 84.79% similarity to a T. contejeani isolate (MF344630: 97% coverage; e-value: 0.0) from Austropotamobius pallipes in Italy. Similarly, a 735 bp sequence from the novel microsporidium-infecting F. rusticus (OM630067) was 87.36% similar to the same T. contejeani isolate (MF344630: 96% coverage; e-value: 0.0). Our sequence demarcation plot highlights the genetically distinct freshwater Thelohania species based on the geographic location from which the isolates were found ( Figure 5).

Figure 5.
A similarity matrix reflecting the percent similarity between different Astathelohania (=Thelohania) rRNA (SSU) gene isolates. The key provides a color scheme that reflects the similarity between isolates (blue/low to red/high). The figure was designed using the sequence demarcation Tool v1.2 [35].
Phylogenetic analysis revealed that our novel microsporidia grouped in an 'orphan' lineage at the base of Clades IV and V, along with the other freshwater Thelohania isolates from Europe and Australia (bootstrap: 100%), revealing a genetic similarity between species from specific continental ranges (Figures 6 and 7). Grouping below our microsporidia and the existing freshwater Thelohania are the genera Hamiltosporidium and Neoflabelliforma ( Figure 6). The phylogenetic analysis also revealed that freshwater Thelohania and marine ('true') Thelohania spp. are genetically distinct, with T. butleri branching separately in Clade V ( Figure 6). A sequence demarcation plot of the SSU rRNA gene of all isolates found in the 'orphan' lineage, and also comparing T. butleri, emphasizes the genetic dissimilarity between freshwater Thelohania and marine ('true') Thelohania with <75% similarity ( Figure 5). Therefore, we propose the freshwater members of the genus Thelohania Figure 5. A similarity matrix reflecting the percent similarity between different Astathelohania (=Thelohania) rRNA (SSU) gene isolates. The key provides a color scheme that reflects the similarity between isolates (blue/low to red/high). The figure was designed using the sequence demarcation Tool v1.2 [35].
Phylogenetic analysis revealed that our novel microsporidia grouped in an 'orphan' lineage at the base of Clades IV and V, along with the other freshwater Thelohania isolates from Europe and Australia (bootstrap: 100%), revealing a genetic similarity between species from specific continental ranges (Figures 6 and 7). Grouping below our microsporidia and the existing freshwater Thelohania are the genera Hamiltosporidium and Neoflabelliforma ( Figure 6). The phylogenetic analysis also revealed that freshwater Thelohania and marine ('true') Thelohania spp. are genetically distinct, with T. butleri branching separately in Clade V ( Figure 6). A sequence demarcation plot of the SSU rRNA gene of all isolates found in the 'orphan' lineage, and also comparing T. butleri, emphasizes the genetic dissimilarity between freshwater Thelohania and marine ('true') Thelohania with <75% similarity ( Figure 5). Therefore, we propose the freshwater members of the genus Thelohania be relocated to a new genus, Astathelohania gen. et comb. nov., based on genetic and phylogenetic dissimilarity of the 18S rRNA sequences. The novel microsporidia described here are named Astathelohania virili n. sp. and Astathelohania rusti n. sp., and the species T. contejeani, T. montirivulorum, and T. parastaci, are revised to become members of this genus. 12 of be relocated to a new genus, Astathelohania gen. et comb. nov., based on genetic and ph logenetic dissimilarity of the 18S rRNA sequences. The novel microsporidia describ here are named Astathelohania virili n. sp. and Astathelohania rusti n. sp., and the species contejeani, T. montirivulorum, and T. parastaci, are revised to become members of this genu Figure 6. A maximum-likelihood phylogenetic tree of all crayfish-infecting, freshwater Theloha isolates as well as wide-scale Microsporidia representation of each existing clade. The annotat maps demonstrate the distinct crayfish-infecting Thelohania species present per continent. The i lates sequenced in this study are denoted on the tree using the host (Faxonius sp.) and the mic sporidian isolate number. Two isolates are present for a novel microsporidian species from F. ru cus (i18 and i24), and four isolates were sequenced from F. virilis (i60, i98, i55, i53). The tree w constructed using MAFFT aligned rRNA (SSU) gene sequences followed by IQtree [34]. The t was annotated in FigTree v.1.4.4. Figure 6. A maximum-likelihood phylogenetic tree of all crayfish-infecting, freshwater Thelohania isolates as well as wide-scale Microsporidia representation of each existing clade. The annotated maps demonstrate the distinct crayfish-infecting Thelohania species present per continent. The isolates sequenced in this study are denoted on the tree using the host (Faxonius sp.) and the microsporidian isolate number. Two isolates are present for a novel microsporidian species from F. rusticus (i18 and i24), and four isolates were sequenced from F. virilis (i60, i98, i55, i53). The tree was constructed using MAFFT aligned rRNA (SSU) gene sequences followed by IQtree [34]. The tree was annotated in  Representative phylogenetic-inferred cladograms of native crayfish species ("Crayfish") from the families Cambaridae (native geography: North America), Astacidae (native geography: Europe and North America), Cambaroididae (native geography: China and Japan), and Parastacidae (native geography: South America, Madagascar, Australia, New Zealand), compared with microsporidian isolates from the freshwater Thelohania (now revised to Astathelohania) ("Parasites"). The accession numbers for the isolates are listed by the name of the species on each tree. The microsporidian cladogram was developed from the tree presented in Figure 6. For the "Crayfish" tree, cytochrome oxidase 1 DNA sequence data were aligned using MAFFT and constructed using IQtree [34]. The trees were drawn and annotated in Family description: Binucleate, uninucleate, and potentially dimorphic microsporidian parasites that develop within sporophorous vesicles in the muscle tissue of freshwater crustacean hosts. Spores are ellipsoidal, oval, or pear-shaped. Species considered to be members of this family should phylogenetically group with other members of this family using DNA, RNA, or amino acid sequence data, and clade with the type genus and species (Astathelohania virili).
Type genus and species: Astathelohania virili n. sp. Stratton, Reisinger, Behringer, Bojko 2022 Genus: Thelohania (freshwater) replaced by Astathelohania Stratton, Reisinger, Behringer, Bojko 2022 Astathelohania genus description: This genus should accommodate uninucleate or binucleate species that undergo merogony and sporogony in a sporophorous vesicle. Members of this genus infect freshwater Astacoidea Latreille, 1802 hosts (crayfish), which are globally present. Gene sequence data should be considered when determining the placement of a species into this genus and that data should be used to infer a phylogenetic analysis, showing clustering with other Astathelohania species, accounting for possible geographic sequence diversity observed in this study.

Astathelohania virili n. sp. Stratton, Reisinger, Behringer, Bojko 2022
Species description: The microsporidian parasite infects the muscle and heart tissue of F. virilis and undergoes merogony and sporogony in a sporophorous vesicle. The spores are uninucleate and include 16-17 coils of the polar filament. The spores are oval in shape with a wider posterior end and measure 3.4 ± 0.1 µm (SD) in length and 2.0 ± 0.3 µm (SD) in width. To be a candidate for this species, sequence similarity must be shared by comparison to available SSU sequence data for this isolate. Phylogenetically, the parasite must clade with the original sequence provided in this manuscript for Astathelohania virili. Species description: The microsporidian parasite infects the muscle and heart tissue of F. rusticus and undergoes merogony and sporogony in a sporophorous vesicle. The spores are uninucleate and include 13-14 coils of the polar filament. The spores are oval in shape with a wider posterior end and measure 3.2 ± 0.5 m (SD) in length and 1.7 ± 0.3 µm (SD) in width. To be a candidate for this species, sequence similarity must be shared by comparison to available SSU sequence data for this isolate. Phylogenetically, the parasite must clade with the original sequence provided in this manuscript for Astathelohania rusti.

Discussion
Crayfish can harbor a diverse suite of pathogens, and the freshwater Thelohania are a major group of crayfish-infecting microsporidia [4,5]. In this study, we present a taxonomic revision for freshwater Thelohania based on SSU rRNA sequence data and phylogenetics, proposing that crayfish-infecting, freshwater members of Thelohania, a polyphyletic genus, be transferred to the Astathelohania gen. et comb. nov., housed in the family Astahelohaniidae n. fam., making a clear distinction from the Clade V family, Thelohaniidae, which now houses marine and terrestrial Thelohania spp. In addition, we describe two new species of Astathelohania, Astathelohania virili n. sp. and Astathelohania rusti n. sp., from two crayfish hosts in North America, using histopathology, ultrastructure, intracellular development, and SSU phylogenetics.
As genetic data become increasingly available for microsporidia, it has become clearer that traditional data (e.g., phenotypic, ecological, developmental) alone are unable to delineate accurate phylogenies-a combination of these data are required to currently identify species and their taxonomy, evident by several recent species revisions [40,41].
For some of the first microsporidian genera described, such as the Nosema, it has proven vital to incorporate genetic data as part of a revision [41]. Several studies have called for a taxonomic revision of the polyphyletic genus Thelohania since it has become increasingly apparent that the marine Thelohania and the freshwater Thelohania are not closely related genetically and are in fact clades apart [7,15,21]. Other studies have begun to revise the polyphyletic genus by placing terrestrial Thelohania species into more appropriate genera based on genetic, phylogenetic, developmental, and ecological data [23,24]. Our study provides further evidence to support taxonomic revision through the discovery of two new species in this 'orphan lineage'.
Based on our phylogenetic analysis, freshwater Thelohania branch outside of both Clades IV and V, and importantly branch together in a well-supported group separate from the marine T. butleri (Clade V), the only 'true' Thelohania species with genetic data available ( Figure 6). Our taxonomic revision is further supported by several recent studies [15,29]. The sequence demarcation plot we provide illustrates the dissimilarity between marine and freshwater Thelohania, based on the SSU rRNA gene ( Figure 5).
Further, the family Thelohaniidae remains polyphyletic and also requires taxonomic revision [42]. Many of the genera and species assigned to this family have undergone recent revision on the basis of genetic dissimilarity [23,24,[43][44][45]. Our phylogenetic tree further highlights the need for the novel Astathelohania genus to be placed into a new family (Astathelohaniidae n. fam.) considering that all crayfish-infecting, freshwater Thelohania do not fall into the same clade as any genetically validated members of the family Thelohaniidae ( Figure 6) [39].
Therefore, we propose a revision in which the crayfish-infecting, freshwater members of the Thelohania are distinguished and relocated to the Astathelohania n. gen. and Astathelohaniidae n. fam. This new genus and family are named for the Thelohania, maintaining their important historic connotations, but additionally represent the freshwater crayfish hosts of this genetically distinct lineage, helping to maintain the historic genus and family names that once represented these species for over a century of published literature.

Two Novel Crayfish Parasites in the USA
To date, seven microsporidia have been formally described from crayfish hosts, but none of these are known from the crayfish genus Faxonius [5]. The genus Faxonius is the third most species-rich genus of crayfish in the world behind Procambarus and Cambarus, yet little is known about the pathogens this group harbors [4,46]. Astathelohania virili n. sp. and A. rusti n. sp. are the first formally described microsporidia found to infect members of the genus Faxonius. Both crayfish hosts, F. virilis and F. rusticus, have invasive ranges throughout North America, but these novel parasites were found in the native range of each host. Further research should examine whether these novel parasites are found in the invaded ranges of the crayfish hosts.
There have been reports of two suspected T. contejeani infection within North America and an unofficial T. cambari species reported [17][18][19]. These reports were all based on the observation of octosporous development and spore size. However, the size range of spores for the suspected T. contejeani infections overlap with both our spore size ranges and the range described for A. contejeani and A. parastaci (Table 2) [10,15,17,18]. We also now know of many microsporidian groups that undergo octosporous development within an SPV outside of Thelohania [15,47]. Therefore, until these infections are rediscovered, and genetic data become available, we cannot say whether these reports are accurate. Similarly, the unofficial species T. cambari was placed in the genus based on spore size and observation of octosporous development [19]. The spores were much larger in size than our Astathelohania species but do overlap with the size range reported for binucleate spores of A. montirivulorum and A. parastaci (Table 2) [9,10,19]. Genetic and ultrastructural data must become available before T. cambari can be formally recognized.

Host-Parasite Co-Evolution
The discovery of these novel parasites allowed us to examine the possibility of hostparasite co-evolution of crayfish hosts and Astathelohania microsporidia. Phylogenetic studies of the superfamily Astacoidea illustrate that the divergence of families and genera are geographically affiliated [46,48]. Families in the Northern (Cambaroididae, Astacidae, and Cambaridae) and Southern (Parastacidae) hemispheres diverged over 265 mya [49]. The family Cambaridae is the youngest yet most diverse crayfish lineage, undergoing diversification and radiation approximately 90 mya [50].
The diversity observed within the Astathelohania genus may also represent a geographic split. The microsporidia A. montirivulorum and A. parastaci are only known from the Australian crayfish C. destructor in the family Parastacidae [9,10]. Astathelohania contejeani has been found to infect three members of the family Astacidae which include A. pallipes, Astacus astacus, and P. leniusculus, and all isolates were discovered in Europe [13][14][15]. Finally, A. virili and A. rusti infect two members of the North American family Cambaridae. Our sequence demarcation plot highlights that isolates discovered in the oldest host family (Parastacidae) are least similar to isolates from the youngest family (Cambaridae) (Figures 5-7). There is little genetic variation between European isolates since they are all the same microsporidian species; however, two strains of A. contejeani have been described and are evident in the phylogenetic tree ( Figures 5 and 6) [13]. In Australia, the Astathelohania (A. montirivulorum and A. parastaci) infect the same host species and are 93% similar to one another [9,10]. In North America, A. virili and A. rusti show considerable genetic variation which may be because North American crayfishes are a significantly more diverse group compared to crayfishes in the families Astacidae and Parastacidae. Therefore, if there is a host-parasite co-evolution it would make sense that their parasites would also be more genetically diverse.

Conclusions
It is a vital taxonomic step to separate the crayfish-infecting, freshwater Thelohania into their own distinct genus, avoiding polyphyly in ongoing taxonomic studies concerning the 'true' marine Thelohania. Here, we have provided a description of the Astathelohania n. gen., in the family Astathelohaniidae n. fam., to provide valuable systematic distinction for this lineage. This has resulted in three species of Thelohania being revised and the addition of two new species. The two new species we describe provide a North American perspective of Astathelohania diversity, which is now viewed as a globally diverse genus. We see wellsupported groups in our phylogeny, which combine all suggested Astathelohania species with 100% bootstrap support, as well as splitting the various genera based on geography and host diversity.