Revision of Hemiquedius Casey (Staphylinidae, Staphylininae) and a review of beetles dependent on beavers and muskrats in North America

Abstract Based on newly discovered characters on the male genitalia, external morphology and an accumulation of ecological data, we revise the single member of the genus Hemiquedius. Two new species, H. infinitus Brunke & Smetana, sp. n. and H. castoris Brunke & Smetana, sp. n., from eastern North America are described, and H. ferox (LeConte), restricted to peninsular Florida, is re-described. Hemiquedius castoris is strongly associated with the microhabitats provided by nest materials of the North American beaver and muskrat. A key to the three species of Hemiquedius is provided and diagnostic characters are illustrated. We also review the beetles known to be obligate associates of beavers and muskrats, and discuss the potential role of these keystone vertebrates in beetle evolution and distribution. Based on nest-associated beetles and their closest living relatives, beaver and muskrat lodges may extend distributions northward by moderating winters, promote sympatric speciation and act as refugia against extinction of lineages on a broader timescale. Further research into these potential impacts by ecologists and evolutionary biologists is encouraged.


Introduction
The genus Hemiquedius (Fig. 1A) is endemic to North America and a member of the predaceous rove beetle subtribe Acylophorina (Staphylininae: Staphylinini) (Brunke et al. 2016). Currently, one widespread eastern species, Hemiquedius ferox (LeConte, 1878), is recognized that occurs from northern Ontario, south to eastern Texas, east to Florida and north to Nova Scotia (Smetana 1971a(Smetana , 1978(Smetana , 1990. It inhabits a wide variety of water-soaked decaying organic matter at the edges of forested and open wetlands and is consistently but sparingly collected by submerging this debris by 'treading' it under foot (see Smetana 1971a). Several specimens of Hemiquedius collected from beaver and muskrat houses were observed by Smetana (1971b) to differ from all others by their elytral setation but were not recognized as a separate species at that time due to limited material and a lack of other corroborating differences. Since then, an entire rove beetle assemblage has been discovered that is strongly associated with the nest materials of beaver and muskrat houses, including several species new to science (Campbell 1979, Hoebeke 1985, Smetana 1995. Most of these species were described relatively recently and were overlooked until beaver and muskrat nests were targeted for sampling. Here we re-assess the taxonomic status of the ecologically specialized population of Hemiquedius and other putative species based on morphological characters and CO1 barcode data. Although ecosystem engineering by beavers is well known to positively impact populations of wetland fauna such as fish, birds, reptiles and invertebrates (Rosell et al. 2005), the promotion of invertebrate speciation by beavers is not well understood. Based on the diversity of lineages that have evolved specialized associations with beaver lodge-building activity, their impact on speciation may be significant and widespread. To facilitate future investigation of this phenomenon, we review the beetle species that appear to specialize on the microhabitat provided by beaver and muskrat houses.

Material and methods.
Specimens were examined from the following institutions:

Microscopy, illustration, photography and mapping
All specimens were examined using a Nikon SMZ25 stereomicroscope. To allow for the routine dissection of the terminal abdominal segments (including the aedeagus), distilled water was applied directly to the tip of the abdomen using a fine paintbrush. As a precaution against DNA degradation, specimens examined in the present study were never subjected to high ambient humidity relaxing chambers or entirely submersed in water. Genitalia were washed with 70% alcohol and placed in glycerin for observation. Genitalia were placed in glycerin filled vials for long-term storage, which were pinned with their respective specimen.
Measurements were performed using the live measurement module in NIS Elements BR v4.5. Measurements were taken as listed below, but only proportional (HW/ HL, PW/PL, EW/EL, ESut/PL, PW/HW) and forebody measurements were stated directly in descriptions due to variability in body size. Total body length is generally difficult to measure accurately in Staphylinidae due to the contractile nature of the abdomen. Abbreviations for measurements are as follows:

HL
Head Length, at middle, from the anterior margin of frons to the nuchal ridge.

HW
Head Width, the greatest width, including the eyes.

PL
Pronotum Length, at middle.

EL
Elytral Length, greatest length taken from level of the anterior most large, lateral macroseta to apex of elytra. Its length approximates the length of the elytra not covered by the pronotum and therefore contributing to the forebody length.
Line illustrations were performed in Adobe Illustrator CS6 based on photographs. Photomontage was accomplished using a motorized Nikon SMZ25 microscope and NIS Elements BR v4.5. Photos were processed in Adobe Photoshop CS6. Distribution maps were created using SimpleMappr (Shorthouse 2010). The graph of pronotum length versus width was created in Microsoft Excel and then modified in Adobe Illustrator. Regression lines were added in Excel but for qualitative purposes only.

Molecular data
Extraction, amplification and sequencing of the barcoding fragment of CO1 was performed by the Biodiversity Institute of Ontario (BIO) (Guelph, Ontario, Canada). Sequences were uploaded to Barcode of Life Datasystems v4 (BOLD) (http://www.boldsystems.org) and those sequences deemed to be barcode compliant by BOLD were assigned BINs (Barcode Index Numbers, Ratnasingham and Hebert 2013) as tentative species hypotheses. Using the Taxon-ID tree tool in the workbench of BOLD, barcodes with BINs were visualized in a neighbor-joining tree using the BOLD aligner and Kimura-2 Parameter distances. The barcodes are available in BOLD as the published dataset DS-HEMIQUED. Novel sequences were uploaded to GenBank under the accession numbers: MF966147-MF966149.

Results
A revision of Hemiquedius ferox resulted in the recognition of three species: H. infinitus Brunke and Smetana, sp. n. and H. castoris Brunke and Smetana, sp. n., from eastern North America and H. ferox (LeConte), restricted to peninsular Florida. Although H. infinitus and H. castoris occur sympatrically, H. castoris is strongly associated with the nest material of North American beaver and muskrat, while H. infinitus occurs outside of this microhabitat, along the margins of various wetlands. A total of three DNA barcodes >500bp (barcode compliant) and two incomplete barcodes (176 and 306 bp) were generated from available dried specimens. An additional 3 specimens of H. infinitus were processed but failed to provide sequences, likely due to inadequate preservation. Hemiquedius infinitus and H. castoris were represented by 3 and 2 sequences, respectively, and both species were represented by barcodes >500bp that were assigned BINs. Their process IDs are given in the material examined section under the corresponding species. Although two BINs were identified by BOLD (BOLD:ABW6323 and BOLD:ACL9384), sequences did not cluster by the morphology and ecology-based species concepts proposed herein (tree not shown here). However, an OTU (operational taxonomic unit) cluster analysis of both compliant and incomplete barcode fragments in BOLD suggested only a single OTU (average distance = 0.94%, maximum distance = 2.78%). It is likely that, with additional barcode compliant sequences in the future, the two existing BINs will be synonymized into one. Molecular data were unavailable for H. ferox as DNA from three dried specimens failed to amplify.

1
Elytral disc with fine setation laterally ( Elytral disc without fine setation (Fig. 1C); scutellum usually with only fragments of shallow microsculpture, never entirely covered (single specimen seen with only margins missing microsculpture); collected from a wide variety of wetland edge microhabitats but not known from beaver or muskrat houses; aedeagus as in Fig. 2A Male sternite VIII without or with slight emargination (Fig. 1E); median lobe with obtuse apex (Fig. 2C); paramere distinctly constricted at base (Fig. 2C, G); pronotum slightly wider than long (Fig. 3B); broadly distributed across eastern North America, not known from peninsular Florida (Fig. 4)  Diagnosis. Hemiquedius can be readily recognized as a member of the subtribe Acylophorina by its elongate, non-lobed and cylindrical mid and hind tarsomeres, and the empodial setae of the hind tarsus, which is distinctly longer than that of the foretarsus. Within the subtribe, Hemiquedius has a unique habitus ( Fig. 1A) but is also distinguished by a combination of: foretibia without distinct spines; at least sutural half of elytron lacking regular, evenly distributed setae; antennae non-geniculate; pronotum roughly parallel-sided.  Diagnosis. Hemiquedius ferox can be distinguished by the distinct emargination of male sternite VIII (Fig. 1D), acute apex of the median lobe ( Fig. 2A) and very slightly elongate pronotum (Fig. 3C). It is also the only species known from peninsular Florida. Coloration: body dark brown, pronotum sometimes moderately paler, dark reddish brown, abdomen with strong iridescence; palpi reddish brown; legs except coxae light reddish to reddish brown, paler than rest of body, coxae dark brown; antennae dark reddish brown, apical 1-2 segments slightly paler.
Head transverse, slightly more so in males, temples slightly smaller to slightly longer than eyes, middle of disc without punctures. Antennomeres elongate, antennomere 3 extremely elongate, segments decreasing in length to penultimate, which is slightly elongate.
Pronotum slightly longer than wide, weakly converging anteriad, disc without microsculpture, wider than head (Fig. 3C). Elytra broader and considerably shorter than pronotum at middle, fine, uniform setation restricted to epipleuron (as in Fig. 1C), disc with only a few sparse rows of setae, elytral disc without distinct microsculpture, scutellum at most with shallow fragments of meshed microsculpture, never entirely covered with a coarse mesh.
Abdominal tergites with pubescence moderately sparse, setae separated by far more than their diameter, especially sparse at middle of each disc.
Median lobe in lateral view narrowed to simple apex, which is deflexed ventrad at apical fifth (Fig. 2B); median lobe in parameral view with acute apex, expanded subapi- cally ( Fig. 2A); paramere in situ shorter than median lobe ( Fig. 2A), broad and scarcely constricted at base, with small median incision, peg setae arranged in a pair of crescentshaped fields (Fig. 2E); apical margin of male sternite VIII with distinct emargination (Fig. 1D); male tergite X triangular, with apex ranging from obtuse to slightly acute, not emarginate; male sternite IX with small but distinct and semi-circular emargination.
Female tergite X elongate triangular, with thin median extension that gradually extends from lateral margin in most specimens, shape overlapping with some specimens of H. infinitus.
Distribution. Figure 4. This species is currently known only from Dade and Volusia counties in peninsular Florida.
Bionomics. Specimens have been collected using FITs in hammock forests, 'pinelands', and one specimen came to a UV light. Two specimens were teneral (3.III to 28.IV, 15.XI to 24.II).
Comments. Hemiquedius ferox is distinguishable externally from other species of the genus by the slightly longer pronotum and the distinctly emarginate male sternite VIII.  Diagnosis. Hemiquedius castoris can be easily distinguished by the setose lateral portions of the elytral disc (Fig. 1B) and the coarse meshed microsculpture on the entire surface of the scutellum (Fig. 1B) Similar to Hemiquedius ferox and differing only in the following: palpi and antennae slightly darker, dark brown to dark reddish brown; antennae on average slightly thinner and shorter in appearance, in most specimens antennomere 8 and 9 slightly less elongate; head slightly more transverse in females rather than males; pronotum slightly wider than long; elytral disc with dense fine setae on lateral portion, scutellum with distinct, transverse meshed microsculpture on entire surface; punctures on abdominal tergites slightly denser; median lobe narrowed to shorter apex, length and shape of narrow apical portion highly variable (as in Fig. 2D); median lobe in parameral view with obtuse apex (as in Fig. 2C); paramere more strongly constricted at base (as in Fig. 2C); paired lobe of paramere elongate relative to that of H. infinitus (Fig. 2F); apical margin of male sternite VIII without or with barely discernable emargination (Fig. 1E); female tergite X in most specimens with median extension broader at base and more strongly constricted from lateral margin.

Hemiquedius castoris
Etymology. We describe this species in honor of Canada on its 150 th birthday. Like its national animal, the North American Beaver, Canada promotes a diverse community within its greater environment. The species epithet refers to the close association of this rove beetle with beaver lodges.
Distribution. Figure 4. This species is currently known only in Canada from northern Ontario to New Brunswick but very likely occurs broadly across eastern North America where beavers and muskrats occur. Its distribution includes both the boreal and deciduous forest regions.
Bionomics. All specimens with collecting data have been taken from the nest material within beaver or muskrat lodges, some of which were abandoned for several years. Teneral specimens have been collected in August and September.
Comments. Hemiquedius castoris is most similar to the sympatric H. infinitus but can be easily distinguished based on the fine setation on the elytral disc. The genitalia of these two species are extremely similar and only differ by the shape of the paramere (Fig. 2F vs. 2G). It is interesting to note that all boreal records of Hemiquedius represent H. castoris. Hemiquedius castoris is not likely to be sympatric with H. ferox as its host, the North American Beaver, does not occur naturally in peninsular Florida (Peck 2007).

Diagnosis.
Hemiquedius infinitus can be distinguished by a combination of the elytral disc without fine dense setae (Fig. 1C), pronotum with PW/PL ≥ 1.00 (Fig. 3B) and the indistinct or absent emargination of male sternite VIII (as in Fig. 1E). Hemiquedius infinitus and H. castoris cannot be distinguished by their CO1 barcodes.
Extremely similar to H. castoris and differing only in the following: elytral disc without fine dense setae (Fig. 1C); scutellum with shallow microsculpture in small fragments or with, at most, entire disc except margins with shallow meshed microsculpture; head not sexually dimorphic; pronotum slightly more elongate on average (Fig. 3B); paramere more strongly constricted at base forming a more rounded lobe (Fig. 2G).
Etymology. The species epithet means unbounded in Latin and refers to the occurrence of this species in a variety of wetland habitats, though not inside the lodges of beavers or muskrats.
Distribution. Figure 4. This species is currently known from a wide area of eastern North America: southern Ontario and Quebec, south to the Florida panhandle, west to Texas and north to northern Michigan. Its distribution corresponds well with that of the eastern deciduous forest. All data from Smetana (1971a) can technically be associated with this species except two specimens from peninsular Florida and two from Quebec, with setose elytra.
Bionomics. Specimens have been collected from a variety of wetland edge habitats ranging from open eutrophic ponds to shaded vernal forest pools.
Comments. Hemiquedius infinitus is most similar to H. castoris but can be easily distinguished based on the lack of fine dense setae on the elytral disc. The genitalia of these two species are extremely similar, variable and only differ by the shape of the paramere. At present H. infinitus is not known to be sympatric with H. ferox but may overlap with it in northern Florida.

Species and distributional limits in Hemiquedius
A taxonomic revision of Hemiquedius ferox revealed the existence of three distinct species: one known only from peninsular Florida and a pair of highly similar and sympatric eastern species that are thus far allopatric with the former. The eastern pair of species represent an unusual situation in Staphylinidae, where CO1 barcodes are not diagnostic and male genitalia are almost identical (contra Hendrich et al. 2014, von Beeren et al. 2016). However, non-overlapping differences in external morphology and ecology indicate that these two species are, possibly rather recently, reproductively isolated. Other examples of similar species that are not distinguish-able by CO1 barcodes exist in the Coleoptera and an example was discovered by Grebennikov et al. (2017), who explored the longhorn beetle (Cerambycidae) fauna of the Russian Far East. Two species, separable by external morphology and host plant received the same BIN and were not reciprocally monophyletic in neighborjoining trees (Grebennikov et al. 2017). An almost identical situation to Hemiquedius is known in the ground beetles (Carabidae) with Pterostichus castor Goulet & Bousquet, strictly associated with beaver lodges, and Pterostichus ebeninus Dejean occurring in various forested and unforested wetlands (Larochelle and Lariviere 2003). The species are at least partly sympatric in the northern U.S. and southern Ontario (Bousquet 2012). Male genitalia in these species are identical but there are consistent differences in external morphology, such as presence/absence of an iridescent sheen and shape of the pronotum (Goulet and Bousquet 1983). Copulation in Pterostichus castor was observed on the undersides of branches composing the surface of the lodge (Webster and DeMerchant 2012a). A preference for mating in or on the lodge surface may provide a mechanism for reproductive isolation and subsequent speciation in beaver-associated beetles.
The distribution of Hemiquedius castoris is certainly greater than that reported here. We expect this species to occur in at least Wisconsin to New England and across the maritime provinces of Canada. Beaver and muskrat lodges are poorly sampled and the presence of H. castoris in the boreal forest region makes it likely to be rather widely distributed across Canada. The North American Beaver and Muskrat do not occur in peninsular Florida (Banfield 1974) and H. castoris may be truly allopatric with H. ferox.

Obligate associations with beavers and muskrats in the Coleoptera
Thus far, eleven species of beetle are obligate associates of beaver and muskrat lodges in North America ( Table 1). Representatives of the Carabidae, Leiodidae and Staphylinidae are known but the evolution of this association has occurred most frequently in the staphylinids. Most of these associates are predators within the lodge but the most extreme of them, the leiodids, are strongly modified for life as adult and larval ectoparasites of beavers, feeding on dead skin and wound exudates (as reviewed in Peck 2006Peck , 2007. Beaver and muskrat lodges represent a stable and predictable microhabitat that is maintained year-round, and therefore are host to a diverse assemblage of obligate and, even more numerous, facultative associates among the beetles (e.g., Bousquet 1985, Smetana 1995, Webster and DeMerchant 2012b. Unlike the other species of the genus and nearly all other Acylophorina, H. castoris is distributed north to the boreal forest of northwestern Ontario, possibly due to milder overwintering conditions within its host's lodge. A similar situation is known for the carabid Atranus pubescens (Dejean), which is strongly associated with beaver lodges in the northern part of its range but occurs in caves and deep litter in the southern United States (Bousquet 1985). In North America, the Holarctic rove beetle genus Coprophilus is represented by the non-native (western Palaearctic) C. striatulus (Fabricius), C. sexualis Leech, limited to a small range in the Pacific Northwest, and the obligate beaver-associate Coprophilus castoris Campbell in the northeast (Campbell 1979, Hoebeke 1995. Coprophilus castoris appears to be much more closely related to the Palaearctic species rather than the other native Canadian species and may be a relict in North America, surviving in a specialized and moderated niche. Therefore, another potential impact of beavers and muskrat lodges on biodiversity may be in the form of a stable refugium against extinction on a broader time scale. Although beavers and muskrats undoubtedly have a positive impact on invertebrate diversity at multiple spatial and taxonomic levels, the mechanisms involved in these interactions remain unexplored and untested by ecologists and evolutionary biologists. We hope that this review draws attention to this interesting phenomenon and the diverse community supported by the beaver, a charismatic national symbol of Canada.  (Smetana 1971a)