Cryptic diversity and range extension in the big-eyed bat genus Chiroderma (Chiroptera, Phyllostomidae)

Abstract Since the last systematic review of Chiroderma (big-eyed bats) more than two decades ago, we report on biodiversity surveys that expand the distribution and species diversity of this Neotropical genus. The Caribbean endemic species Chiroderma improvisum is documented for the first time from Nevis in the northern Lesser Antilles. A broader geographic sampling for a molecular analysis identifies a paraphyletic relationship in Chiroderma trinitatum with respect to Chiroderma doriae. Cis-Andean populations of C. trinitatum are most closely related to the morphologically distinctive and allopatrically distributed C. doriae in the Cerrado and Atlantic Forest of Brazil and Paraguay. The sister taxon to this grouping includes trans-Andean populations of C. trinitatum, which we recommend to elevate to species status as C. gorgasi. This is an example of a cryptic species because C. gorgasi was previously considered morphologically similar to C. trinitatum, but more detailed examination revealed that it lacks a posterolabial accessory cusp on the lower second premolar and has a narrower breadth of the braincase. We provide an amended description of Chiroderma gorgasi.


Introduction
Cryptic species, phenotypically similar organisms that are classified as a single species but are genetically divergent lineages, are being discovered at a greater rate due to the increasing prevalence of molecular methods, such as DNA barcoding (e.g., Hebert et al. 2004). It has been estimated that Neotropical mammalian biodiversity is underestimated by one-third (Lim 2012). At typical lowland tropical forest sites, bats comprise the majority of mammal species diversity (Voss and Emmons 1996), so more species are expected to be recognized in this group as traditional taxonomic hypotheses are tested by genetic techniques. In addition, new surveying methods such as the use of triple-high netting systems to catch higher flying aerial insectivorous bats, and harp traps to target species that may be able to better detect mist nets, is decreasing the sampling bias associated with traditional mist nets set just above ground level.
The big-eyed bats in the genus Chiroderma Peters (Phyllostomidae) are characterized by greatly reduced nasal bones in the skull and a combination of external features including a white dorsal stripe that does not extend onto the head; legs and interfemoral membrane conspicuously hairy; and relatively large eyes (Straney 1984;Gardner 2008). The genus currently comprises six species (Simmons 2005, Taddei andLim 2010): C. doriae Thomas, 1891 occurs in central-eastern Brazil and Paraguay; C. improvisum Baker & Genoways, 1976 is endemic to the Lesser Antillean islands of Guadeloupe, Montserrat, and Saint Kitts (Beck et al. 2016); C. salvini Dobson, 1878 is found from Mexico to Bolivia (recent records from Brazil are misidentifications of C. villosum Peters, 1860 -see Brandão et al. 2019); C. trinitatum Goodwin, 1958 is distributed from Honduras (Turcios-Casco et al. 2020) and Costa Rica to Amazonian Brazil and Trinidad; C. villosum ranges from Mexico to southeastern Brazil and Trinidad; and C. vizottoi Taddei & Lim, 2010 is found only in northeastern Brazil.
The systematics of Chiroderma was last reviewed by Baker et al. (1994) based on a phylogenetic study of the mitochondrial DNA cytochrome b (Cytb) gene; however, each of the five species known at the time was represented by a single specimen. With broader geographic coverage, we re-assess the distributional range, genetic diversity, and morphological differences in the genus.

Fieldwork
We conducted a survey of bats on the Caribbean island of Nevis from 24-29 April 2016. Live traps used included a harp trap and 6 m or 12 m mist nets set singly in the forest understory or on a triple-high telescoping pole system. Traps were regularly monitored for the first 2-3 hours after sunset when bat activity is the highest after they leave their roosts to feed. Individuals not kept as part of the representa-tive collection documenting the species diversity were released at point of capture. A combined scientific research and export permit (F002) was issued through the authority of the Nevis Historical and Conservation Society. An Animal Use Protocol (2016-01) was obtained from the Royal Ontario Museum Animal Care Committee. An import permit (#2016-02101-4) was authorized by the Canadian Food Inspection Agency. Use of wild mammals followed the guidelines of the American Society of Mammalogists (Sikes et al. 2016).

Molecular analyses
The cytochrome c oxidase subunit 1 (CO1) gene is the best represented molecular marker for Chiroderma on the genetic sequence database GenBank (www.ncbi.nih.gov/ genbank). There are 117 samples from nine countries in Central and South America (Brazil, Ecuador, El Salvador, French Guiana, Guatemala, Guyana, Mexico, Panama, and Suriname). We add 26 new sequences to bring the sample size to 143 sequences representing 12 countries in the Neotropics, including Venezuela, Peru, and Nevis, and five species in the genus (Appendix 1). There are no tissue samples or nucleotide sequences on GenBank of any genes for the recently described Chiroderma vizottoi (Taddei and Lim 2010). Outgroup taxa were other genera in the subtribe Vampyressina Baker et al., 2016(Platyrrhinus incarum Thomas, 1912and Uroderma bilobatum Peters, 1866 of the New World leaf-nosed bats, for direct comparison to Baker et al. (1994) in their analysis of Cytb. Alternative phylogenetic relationships within the subtribe are given by Baker et al. (2016) and Rojas et al. (2016). We also analyzed Cytb, but there are only 11 sequences on GenBank, although we did add one new sequence of Chiroderma trinitatum gorgasi from Panama (Appendix 2).
Molecular methods for new sequences of CO1 follow the protocol for DNA extraction, PCR amplification, and automated nucleotide sequencing outlined in Lim (2017). For Cytb, extraction, amplification, and sequencing followed Lim et al. (2008). Base calls were confirmed with bidirectional sequences and aligned using Sequencher version 4.8 (Gene Code Corporation, Ann Arbor, Michigan). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 6 (Tamura et al. 2013). For a robust comparison of phylogeny, we used parsimony as a method that minimizes evolutionary change without an explicit model of evolution and maximum likelihood as a probabilistic method with an explicit model of evolution. Maximum parsimony used the subtree pruning regrafting inference method with 500 bootstrap replicates to test branch supports. Maximum likelihood used the Tamura 3-parameter substitution model and gamma distributed rates with invariant sites for COI as determined by the best fit test. For Cytb, the Tamura Nei model and gamma rates were the best fit. Tree inference used nearest neighbor interchange heuristic inference with 500 bootstrap replicates. Genetic distances were calculated with the Tamura 3-parameter model with gamma distributed rates among sites for the larger COI dataset.

Morphological analyses
Morphological and morphometric comparisons included 138 specimens from five species of Chiroderma, including two C. improvisum, four C. doriae, seven C. salvini, 58 C. trinitatum, and 66 C. villosum (Appendix 3). We also analyzed the holotypes of C. trinitatum gorgasi Handley, 1960 andC. trinitatum trinitatum Goodwin, 1958, but did not have specimens of the most recently described C. vizottoi. Only adults (defined as having closed cranial sutures and complete epiphyseal ossification of metacarpal and phalanx joints) of both sexes were examined. Specimens are deposited in the following institutions; Royal Ontario Museum (ROM, Toronto, Canada); National Museum of Natural History (USNM, Washington, DC, USA); American Museum of Natural History (New York, USA); Texas Tech University (Lubbock, USA); and Field Museum of Natural History (Chicago, USA).
Measurements defined below were taken with digital calipers accurate to 0.01 mm following the descriptions of Handley (1960): forearm length (FA); greatest length of skull (GSL); interorbital width (IOW); postorbital width (POW); braincase width (BCW); condyloincisive length (CIL); zygomatic breadth (ZB); width across upper molars (M-M); width across upper canines (C-C); and length of maxillary toothrow (C-M). An analysis of variance (ANOVA) for each measurement and a multivariate analysis of variance (MANO-VA) were performed to examine the significance of morphometric divergence among species of Chiroderma. The level of significance was p = 0.05 for all statistical tests. The homoscedasticity of each variable was tested using Bartlett's test with the R package mvoutliers. Statistical analyzes were performed using R 3.1.0 (R Core Team 2005) and PAST 2.17. Variables were log-transformed and a correlation matrix was used in a Principal Components Analysis (PCA) to assess phenetic differences in multivariate morphological space.

Results
We report the first occurrence of Chiroderma improvisum ( Fig. 1) from Nevis in the northern Leeward Islands of the Lesser Antilles in the Caribbean. An adult male was caught at Barnes Ghaut on April 28, 2016, in a harp trap set across a dry ravine in forest bisected by a road and surrounded by residential homes (Fig. 2). Other equipment deployed included 6 m mist nets set on a triple-high telescoping pole system, a single 6 m mist net, and a single 12 m mist net from 1900-2100 h. In addition to the new distributional record for the island, one Ardops nichollsi, one Noctilio leporinus, and 12 Artibeus jamaicensis were captured.

Molecular analyses
For COI, the 657 basepairs (bp) at the 5' end were available for most (82%) of the specimens analyzed. The complete 1140 bp of Cytb were available, including the newly generated sequence, for most (75%) of the specimens analyzed. The topology of the Chiroderma COI maximum likelihood tree identified six primary terminal clades with (1) C. salvini as   Interspecific genetic distances of the larger COI dataset ranged from 11.3% between C. doriae and C. salvini to 2.5% between C. doriae and C. t. trinitatum (Table 1). The sequence divergence between C. t. trinitatum and C. t. gorgasi was 3.9%. Intraspecific distances were 1% within C. villosum, 0.9% within C. t. trinitatum, and 0.2% within C. doriae, but three taxa were represented by only one sample.

Morphological analyses
Cranial and body measurements for the six taxa of Chiroderma identified in the molecular analyses are shown in Table 2. Chiroderma trinitatum gorgasi and C. trinitatum trinitatum are the smallest members of the genus, whereas C. improvisum is the largest for most measurements. In the PCA, there are three main groups of species (Fig. 4). The Table 1. Genetic divergence of cytochrome c oxidase subunit 1 for the big-eyed bat Chiroderma and outgroup taxa Uroderma and Platyrrhinus. Interspecific distances shown in the lower left matrix; intraspecific distances shown in bold in the diagonal.   5.3 (4.9-5.6) 5.3 (5.8-5.8) 5.9 (5.3-6.3) 6.2 (5.9-6.3) 6.4 (6.1-6.6) 6.6 (6.5-6.6) IOW 5.6 (5.2-5.9) 5.5 (5.0-6.2) 6.0 (5.5-6.8) 6.8 (6.1-7.3) 7.6 (7.1-7.8) 7.4 (7.4-7.4) BCW 9.4 (8.9-9.8) 9.6 (9.2-10.4) 10.7 (10.1-11.5) 11.21 (11.0-11.5) 11.9 (11.3-12.1) 12.0 (11.5-12.5) C-M 7.0 (6.5-7.3) 7.1 (6.7-7.8) 8.7 (8.1-9.4) 9.4 (9.1-9.  first group is formed by the smaller taxa C. t. gorgasi and C. t. trinitatum. The second group has species with medium size, C. villosum and C. salvini, and the third group is formed by the largest species of the genus, C. doriae and C. improvisum. The first and second principal components (PC1 and PC2) explained 94.5% of the total variation. PC1 shows a pattern in general size variation and is explained mostly by C-M, C-C, and FA. PC2 has positive loadings for most measurements, especially IOW, with the exception of C-M, C-C, M-M, and ZB that have negative loadings (Table 3). All the species seem to occupy the entire range of PC2, indicating that the contrast among measurements is negligible and it is not responsible for the separation of groups. Although similar in size, C. t. trinitatum has a more robust breadth of the braincase than C. t. gorgasi. Chiroderma t. trinitatum also has an accessory cusp on the second lower premolar, which is absent in C. t. gorgasi (Fig. 5). In the genetic analyses, C. t. trinitatum is well supported as the sister species to C. doriae and does not share a most recent common ancestor with C. t. gorgasi. We consider this as a previous example of a cryptic species and therefore now recognize C. gorgasi as a distinct species from C. trinitatum. Sáez and Lozano (2005: 111) considered cryptic species to be "groups of organisms that are morphologically indistinguishable from each other, yet found to belong to different evolutionary lineages". They also stated that "after detailed comparisons of morphological and non-morphological features, we can often establish key morphological characters for their identification. In those cases, we can then refer to pseudo-cryptic or pseudo-sibling species". Because Handley's original description was qualitative and univariate, we offer an amended description of this taxon.
Description. Chiroderma gorgasi is a small species of Chiroderma ) that is similar in size to C. trinitatum (sensu stricto) (Table 2). Overall, the dorsal pelage is tricolor varying from light to dark brown (Fig. 7). The dorsal hairs have a dark brown band at the base, a buff coloration in the middle, and brown tips. A white medial stripe extends from the interscapular region to the base of the rump. Proximal two-thirds of forearm hairy. Basal third of uropatagium hairy. Conspicuous white facial stripes extend from the noseleaf to the inner base of the ears, and from the posterior part of the upper lip to the base of the ears. The uropatagium is medium brown. The skull has an elongated braincase with an undeveloped sagittal and lambdoidal crest. The nasal aperture is short, not extending beyond the second premolar. The occipital is rounded in posterior view. The upper incisors are thin and elongated with parallel or convergent tips, which may or may not touch apically. The second lower premolar lacks a third cusp (Fig 5). The postorbital processes are undeveloped and rounded (Fig. 8).
Comparisons. Chiroderma gorgasi is morphologically very similar to C. trinitatum. Both species have a small cranial and body size for the genus (Table 2, Fig. 4), an  undeveloped sagittal and lambdoidal crest, a rounded occipital complex, a short nasal aperture, and undeveloped supraorbital region. However, C. trinitatum has a third posterior cusp on the second lower premolar, which is absent in C. gorgasi (Fig. 5). This cusp in C. trinitatum may vary from very pointed and developed to rounded and less marked, but is always present. In addition, C. gorgasi tends to have a broader braincase (Table 4) and a flatter supraorbital region, which tends to be deeper in C. trinitatum.
Chiroderma gorgasi is easily distinguished from other species of the genus by its smaller cranial and body size ( Table 2). C. villosum shares with C. gorgasi an elongated braincase, rounded occipital region in dorsal view, and absence of a third cusp on the second lower premolar. However, C. gorgasi has an undeveloped postorbital processes, a short nasal aperture, and conspicuous white stripes on the face and back, whereas C. villosum has a very developed and pointed postorbital processes, a long nasal ap- erture, which extends beyond the first molar, a conspicuous posterior palatine spine, and usually incipient white stripes on the face and back. Chiroderma salvini resembles C. gorgasi in the undeveloped sagittal and lambdoidal crest and by the rounded postorbital processes, but a set of other cranial characters distinguish both species, such as a triangular occipital complex and a long nasal aperture. In the dentition, C. gorgasi can be readily distinguished from C. salvini and C. villosum by having a tall first lower premolar, with a crown height approximately 2/3 the height of the crown of the second lower premolar, and placed approximately in the middle of the distance between the canine and the second lower premolar. In C. salvini and C. villosum, this tooth is much smaller, usually with a low crown, shorter than the mesiodistal length of the tooth, and is nearer to the canine than to the second lower premolar.
Chiroderma doriae and C. improvisum are the largest species of the genus, and unlike C. gorgasi have a triangular occipital complex in dorsal view, a pointed and developed supraorbital region, a relatively more developed sagittal and lambdoidal crest, and a long nasal aperture. In addition, C. doriae also tends to have a relatively broader braincase than C. gorgasi and the presence of an undeveloped third cusp in the second lower premolar. We were not able to examine specimens of the more recently described C. vizottoi, but it is larger than C. gorgasi and most similar to C. doriae in qualitative craniodental traits.

Discussion
The only big-eyed bat species occurring in the Caribbean is Chiroderma improvisum, which until recently was known from Guadeloupe (Baker and Genoways 1976) and Montserrat (Jones and Baker 1979;Pierson et al. 1986) by six individuals (Larsen et al. 2007). Subsequently, it was caught on Saint Kitts by Beck et al. (2016) and we are the first to report its occurrence on Nevis. Although this species has been sporadically documented since its discovery, the distribution has broadened in the northern Lesser Antilles but this may be ephemeral depending on weather systems such as hurricanes (Larsen et al. 2007).
Chiroderma gorgasi was originally described by Handley (1960) using five specimens from the type locality in Panama. The author distinguished the new species from C. trinitatum by its smaller size, deeper brain case, shorter rostrum, shaper lacrimal ridge, bulging forehead, larger upper incisors, and thicker white band in the dorsal hairs. But at that time, C. trinitatum was only known by the holotype from Trinidad (Goodwin 1958) so the extent of variation within each species was poorly understood. Based on a specimen from Mitu in Amazonian Colombia, Barriga-Bonilla (1965) recognized the taxon as two subspecies and assigned his Colombian specimen to C. t. gorgasi. The subspecies were considered to be distributed from eastern Panama to western Venezuela for C. t. gorgasi and Trinidad to the Amazon basin for C. t. trinitatum (Jones and Carter 1976). However, with more geographic sampling the initial distinctions between the two taxa were less obvious due to individual and geographic variation (Simmons and Voss 1998), as also demonstrated by our morphometric analysis. But the taxonomy and distributional limits were still contentious with Gardner (2008) recognizing the Andes as the delineation of the subspecies and reassigning the specimen of Barriga-Bonilla (1965) from Mitu, Colombia, to C. t. trinitatum.
Our morphological review identified the presence of three cusps on the second lower premolar in cis-Andean populations referable to C. trinitatum and two cusps in trans-Andean populations referable to C. gorgasi that also match the taxonomic boundaries of Gardner (2008). Morphometrically, C. trinitatum averages smaller than C. gorgasi in all cranial measurements except for a proportionately broader braincase. Furthermore, our genetic analyses recovered C. trinitatum as the well-supported sister species to the larger and morphologically distinctive C. doriae, and not to the superficially similar C. gorgasi. Based on this morphological and molecular evidence, we recognize C. gorgasi as a distinct species and divergent lineage that does not share the most recent common ancestor with C. trinitatum (sensu stricto).
The overall topology of the Cytb tree proposed by Baker et al. (1994) is identical to our tree except for the recognition of C. gorgasi, which they did not have a sample of, as the sister species to C. trinitatum and C. doriae. The evolution of Chiroderma was suggested as occurring primarily by allopatric speciation (Baker et al. 1994). More specifically, C. improvisum arose by peripatric speciation in the Lesser Antilles after dispersing from its most recent common ancestor with C. villosum in South America. The Andes is an obvious geographic barrier separating C. gorgasi from the most recent common ancestor of C. trinitatum and C. doriae. A dated phylogeny is needed to test whether this is an older sundering event associated with the uplift of the northern Andes in the Late Miocene or a more recent dispersal event followed by isolation and the cessation of gene flow. Rojas et al. (2016) date the divergence of Chiroderma species to the Pliocene-Holocene, but C. gorgasi was not included in their dataset. The allopatric distribution of C. trinitatum and C. doriae suggests that perhaps the Cerrado Savanna in Brazil acted as a barrier after colonization of the Atlantic Forest from the Amazon, but the records of C. doriae for the Cerrado and the discovery of a species of Chiroderma in the dry deciduous forests of the Brazilian Caatinga, C. vizottoi, indicates that species of the genus can adapt to more harsh habitats. The speciation event that gave rise to C. salvini and the most recent common ancestor of the other species of Chiroderma is speculative without a thorough biogeographic analysis with a dated phylogeny.
Although not an overly species-rich genus, biodiversity surveys and molecular analyses are finding new distributional and taxonomic discoveries in Chiroderma. However, there are still large geographic gaps in sampling throughout the Neotropics, such as the Amazon basin in Brazil and northern South America in Colombia and Venezuela. In addition, this has hindered detailed study of the biogeography of the genus and more broadly the evolution of bats in the Neotropics.