﻿Rediscovery of Histiotusalienus Thomas, 1916 a century after its description (Chiroptera, Vespertilionidae): distribution extension and redescription

﻿Abstract Histiotus is a Neotropical genus of bat that currently includes 11 species. The systematics of Histiotus has been the focus of several studies over the last decades. However, no broad systematic revision has been made, and taxonomic issues such as synonymies, use of subspecies, and specimens that do not fit the description of valid species still persist, as pointed out by several authors. Histiotusalienus was described in 1916 and is known only by the holotype. Here we present a second record of H.alienus and an amended diagnosis of this species. We use qualitative, quantitative, and morphometric analyses based on data from 184 specimens of Histiotus and almost all valid species. Our amended diagnosis establishes the taxonomic limits of H.alienus, as well as a comprehensive comparison with congeners. We also explore new diagnostic characters for H.alienus and provide a few notes on the natural history of this species. Our results highlight skull similarities among Histiotus species and reinforce the usefulness of external morphology for their correct identification. Despite our new insights into the taxonomy of the genus, several taxonomic issues remain, and a comprehensive revision of the genus is needed.


Introduction
Histiotus Gervais, 1856 is endemic to South America and currently includes 11 species (Cláudio 2019;Velazco et al. 2021). The number of species recognized in the genus, however, has varied over the last decades (see Simmons 2005;Handley and Gardner 2008;Cláudio 2019;Rodríguez-Posada et al. 2021), and three new species were recently described (Feijó et al. 2015;Rodríguez-Posada et al. 2021;Velazco et al. 2021). The generic status of Histiotus has also been debated over the last decades since the genus is consistently recovered within Eptesicus in molecular phylogenies (= Eptesicus Rafinesque, 1820 + Cnephaeus Kaup, 1829 + Neoeptesicus Cláudio et al., 2023; see Cláudio et al. 2023). Eptesicus (sensu lato) occurs in both the New and Old World (Hoofer Roehrs et al. 2010Roehrs et al. , 2011Juste et al. 2013;Yi and Latch 2022). To solve the paraphyly of Eptesicus (sensu lato), two differing arrangements were proposed and have been used since: (1) to treat Histiotus as a subgenus of Eptesicus (sensu lato); or (2) to treat Histiotus as a full genus and allocate Old World forms of Eptesicus (sensu lato) in the available name Cnephaeus (Hoofer and Van Den Bussche 2003;Roehrs et al. 2010Roehrs et al. , 2011Juste et al. 2013;Yi and Latch 2022). More recently, Cláudio et al. (2023) reevaluated the taxonomy of New World Eptesicus (sensu lato) and Histiotus and proposed a new arrangement based on molecular and morphological data. In this arrangement, Histiotus is treated as a full genus, while Eptesicus (sensu lato) was split into three separate genera: Eptesicus, which includes only E. fuscus and E. guadeloupensis; Cnephaeus, which includes all Old World species of the former Eptesicus; and Neoeptesicus, a newly described genus which includes the Neotropical and smaller species of Eptesicus (sensu lato; Cláudio et al. 2023). Here we follow Cláudio et al. (2023) in treating Histiotus at the genus level.
Despite the continued efforts on the taxonomy of Histiotus, no broad revision has been hitherto made, and several problems are still persist in the systematics of the genus, such as the use of name combinations, subspecies and synonyms, specimens that do not fit the species description, and species that are poorly known (Handley and Gardner 2008;Cláudio 2019;Rodríguez-Posada et al. 2021;Velazco et al. 2021). Histiotus alienus was described by Oldfield Thomas in 1916, and only its holotype, captured in Joinvile, Santa Catarina state, southern Brazil, has been known ever since. Most authors consider this species valid, but it has also been treated either as a subspecies of H. montanus or as part of H. macrotus, and its taxonomic status is still uncertain (Handley and Gardner 2008;Cláudio 2019). Except for the brief morphological description and little more than a dozen measurements available in the original description of H. alienus, knowledge of this species is limited, and a fuller diagnosis and comparisons with its congeners is imperative.
Here, we present the second known record of H. alienus, a specimen captured by us in Paraná state in southern Brazil. In addition, we also provide an amended diagnosis of H. alienus based on our morphological analysis of the two known specimens, and we offer a detailed comparison with all congeners.

Methods
The second specimen of Histiotus alienus was captured during a field survey in November 2018 in the Refúgio de Vida Silvestre dos Campos de Palmas (Palmas Grasslands REVIS), which is a 16,600-ha protected area in southern Brazil. Palmas Grasslands REVIS encompasses mainly natural grasslands with small, isolated fragments of moist Araucaria forest and anthropized areas that include agriculture and silviculture patches (ICMBio 2014). The surrounding region of the reserve has wind farms for energy generation, which negatively impact the bat fauna. Bats were sampled using 10 mist nets (9 × 3 m, 20 mm mesh) placed at forest edges, on trails across forest patches, over water bodies, and near previously identified roosts. Mist nets remained open for 6 h each night from sunset and were inspected at average intervals of 30 min. All field activities followed biosafety and bioethics standards and have legal permission (SISBIO 19037-1; SISBIO 63846-1; CEUA-Fiocruz LM-6/18; SisGen A0E5902). For qualitative, quantitative, and morphometric analyses, we used external (N = 3) and craniodental (N = 16) measurements based on, but not limited to, Thomas (1916), Handley and Gardner (2008), Feijó et al. (2015), Rodríguez-Posada et al. (2021), and Velazco et al. (2021). Both external and skull measurements are presented and described in Table 1 and were taken from adults with closed epiphyses. All the measurements were taken with digital calipers, and craniodental measurements were determined under binocular microscopes with low magnification (usually 6×) to the nearest 0.01 mm. Color nomenclature used in the description of the species follows Ridgway (1912).
For morphometric analyses, we employed a canonical variate analysis (CVA) using all 16 craniodental measurements. The CVA was used to discriminate samples and compare skull morphology among Histiotus species. The analysis was performed in R (R Development Core Team 2020), using the MASS (Venables and Ripley 2002) and Lattice (Sarkar 2008) packages. To balance the number of samples within each species, we selected a subset of the total specimens analyzed, including a total of 58 samples, as follows: H. alienus (N = 2), H. colombiae (N = 6), H. humboldti (N = 4), H. laephotis (N = 10), H. macrotus (N = 8), H. magellanicus (N = 10), H. montanus (N = 8), and H. velatus (N = 10). Specimens with incomplete datasets were removed from the analysis, and eventual missing values (<5% of the total dataset) were estimated from the existing raw data using the Amelia II package (Honaker et al. 2011) implemented in R.

Results
On 21 November 2018, we captured an adult male of H. alienus (Fig. 1) in a mist net set at ground level 4h after sunset (ca 11 pm). It had been foraging on the edge of a forest fragment next to a grassland. The area is located in Cerro Chato Farm (26°30'10"S, 51°40'04"W, 1208 m a.s.l.; Figs 2, 3), within the Chopim River hydrographic basin. This river is one of the main tributaries of Iguaçu River in the Palmas Plateau, a subdivision of the Brazilian Southern Plateau. It was collected and preserved in spirit (alcohol 70 °GL) with the skull removed and deposited in Museu Nacional da Universidade Federal do Rio de Janeiro (MN 91624). At the same site, we also collected the following vespertilionid species: Myotis riparius Handley, 1960   The only known record of H. alienus until now was from the type locality in Joinville, Santa Catarina state, southern Brazil. With the new record from Palmas, Paraná state, we expand the geographic distribution of this species by about 280 km to the west at the same latitude (Fig. 3). The new record is from Araucaria forest, a different type of forest cover than at the type locality. Araucaria forests are even more threatened than the coastal Atlantic Forest. Thus, H. alienus, is now considered to occur from dense rainforests to Araucaria and riparian forests and grasslands, at altitudes from sea level to over 1200 m a.s.l.
Diagnosis. Histiotus alienus is distinguished from all other congeners by the following combination of characters: bicolored and dark dorsal fur; ventral fur bicolored and only slightly lighter than dorsal fur; ears intermediate in size when compared to congeners (EL ~ 27.5 mm) and slightly triangular; medial lobe of ear small (WMLE ~ 4.5 mm); transverse band of skin between pinnae low, 1-2 mm high at the edges and weakly fading toward the central portion, where it is practically absent.
Description. Histiotus alienus is a medium-sized species within the genus (FL 43.3-44.5 mm; Table 2). Dorsal fur long (LDF ~ 11.5 mm) and bicolored, with Bone Brown bases that extend to about half the length of hairs and Light Brownish Olive tips; contrast between bands not well delimited. Ventral fur long (LVF ~ 9.5 mm) and bicolored, slightly lighter than dorsal fur, with Brownish Olive bases that extend to about half the length of hairs, and Light Yellowish Olive tips; contrast between bands visible, but not well delimited. Wing membranes naked, dark brown. Plagiopatagium attached to the base of the toe. Dorsal surface of the uropatagium slightly paler than wing membranes, almost naked. Ventral surface of the uropatagium dark brown, with scarce hairs close to the base of the tail. Ears greatly enlarged, slightly triangular, connected by a low band of skin; tragus wider at the base, slightly curved outward, long (~ 13 mm), notched, and pointed. Muzzle broad and slightly inflated. Skull delicate; rostrum short and flattened dorsoventrally, straight in lateral profile; braincase slightly wider than the rostrum. Posterior region of the braincase rounded, regular. Nasal opening U-shaped in dorsal view. Frontals expanded laterally towards the orbit. Sagittal and lambdoidal crests weakly developed, not connected, occipital helmet absent. Triangular, flattened bony plate weakly developed where the sagittal and lambdoidal crests connect. Zygomatic arches thin and greatly widened medially. Basisphenoid pits absent. Palate extends well beyond molars, ending in a concave posterior edge, with a weakly developed medial spine (Fig. 4).
Dental formula I 2/3, C 1/1, P 1/2, M 3/3 (×2) = 32. I 1 separated, spatulate, and strongly bilobed; wide and short, with well-developed inner and outer cusps. I 1 about three times the size of I 2 . I 1 not aligned to I 2 on a transversal axis of the skull. I 2 and C 1 separated by a small gap, C 1 with two slightly concave faces on the lingual region, and one slightly concave face on the labial region. P 1 well developed, reaching half of C 1 in height; P 1 in contact with C 1 and molars. M 1 and M 2 about the same size, almost square shaped, with W-shaped cusps. M 3 reduced, triangular, with only 3 cusps. I 1 -I 3 reduced, trilobed, and occupying the whole space between canines. P 2 about three times P 1 in height. Molars have well-developed cusps and decrease in size from M 1 to M 3 .
Comparisons. Histiotus alienus most resembles H. colombiae Thomas, 1916, H. magellanicus (Philippi, 1866 Figure 4. Dorsal, ventral, and lateral views of the skull of the holotype of Histiotus alienus Thomas, 1916 (BM 9.11.19.1 , which has a greater influence of the shape of the skull, almost all species overlap in the morphospace; this highlights the resemblance of skull shapes among Histiotus species. Considering the correlations of CV2 (Fig. 6), there is an evident contrast between the POB, WFH, and BCB subset of measurements with the remaining measurements, indicating some degree of differentiation in the shape of the skull between these taxa despite the overall resemblance. Histiotus alienus is recovered as most similar to H. montanus in the morphometric analysis, which considers skull shape and size; however, these species are strikingly different in their external morphology and easily distinguished.

Discussion
The amended diagnosis of Histiotus alienus aims to facilitate the diagnosis of the species both in the field and in museum collections. The review of specimens, mainly from southern Brazil, already in scientific collections could reveal additional records of H. alienus. The general results of our morphometric analysis indicate some degree of similarity among species in the shapes of their skulls, which demonstrates the usefulness of external morphology in correctly identifying Histiotus species.
We reinforce the need for a broad taxonomic review of Histiotus and suggest that other species not yet described likely exist, as attested by recent studies (Rodríguez-Posada et al. 2021;Velazco et al. 2021). Most descriptions of Histiotus species are more than a century old and somewhat vague, and the taxonomic limits between species are not clear. Here, we hope to aid in the correct identification and delimitation of Histiotus species.
The lack of information on the natural history of H. alienus and its apparent rarity, with only two records in over more than 100 years, has led to its classification as Data Deficient by the International Union for the Conservation of Nature (González and Barquez 2016;Cláudio 2019). This species is associated with the Atlantic Forest, which is highly fragmented due to historical land occupation and is currently under pressure from agricultural activities (Ribeiro et al. 2009). In Palmas, threats to grasslands include the growth of the wind power sector and interests of the hydroelectric energy sector in the Chopim river basin. Despite that, the new record of H. alienus in Palmas is in a protected area, which indicates that at least one population of the species may be protected. We note the importance of protected areas for the maintenance of wildlife such as this species.

Funding
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brazil (CAPES, Brazil) -Finance Code 001. VCC has also received support from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil;E-26/205.820/2022 andE-26/205.821/2022); and research grants from the Field Museum of Natural History and American Museum of Natural History. RLMN has received support from FAPERJ (E-26/204.243/2021; E26/200.631/2022 and E26/200.395/2022). RM has received financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil;313963/2018-5) and FAPERJ (E-26/200.967/2021).The fieldwork in Palmas was funded for LMT by CNPq are arranged alphabetically by species and major political units. Specimens marked with asterisks were included in the canonical variate analysis.