Molecular and morphological identification of Phylloderma stenops Peters , 1865 ( Chiroptera , Phyllostomidae ) and new records for Colombia

Based on revisionary work of recently collected material in Colombian museums we confirm the presence of Phylloderma stenops Peters, 1865 in 6 new localities for the country, including the first record of the species in the dry lowlands of the northern Caribbean coast, and the increase by more than 800 m of the elevational range of the species in Colombia. DNA-barcoding confirmed our morphological identification, and supported a paraphyletic composition of the cis-Andean populations. Our records exemplify the little knowledge on the ecogeographic distribution of this species and provide further evidence to consider this as a widespread but rare species.


Introduction
The pale-faced Bat, Phylloderma stenops Peters, 1865, is the only representative of its genus and includes 3 recognized subspecies primarily based on morphometric and distribution data: P. s. boliviensis Barquez & Ojeda, 1979, from southeastern Bolivia, P. s. septentrionalis Goodwin, 1940, distributed in Central America, and the nominal subspecies P. s. stenops Peters, 1865, distributed throughout most of South America from the Guianas to southeastern Brazil (Goodwin 1940, Husson 1962, Handley 1966, Barquez and Ojeda 1979, Simmons and Voss 1998, Emmons and Feer 1999, Simmons 2005, Williams and Genoways 2008).Phylloderma stenops is considered a large and robust bat that externally may be confused with taxa of the genus Phyllostomus (Emmons and Feer 1999, Wetterer et al. 2000, Williams and Genoways 2008), an event that has facilitated its misidentification with species of the latter genus not only in the field but also in biological collections.
In Colombia, the species is known from a few records primarily from cis-Andean localities which are mostly categorized as terrestrial ecoregions (sensu Dinerstein et al. 2017) of moist forests (Fig. 1: localities 2, 5, 8 and 9) (Marinkelle and Cadena 1972, Lemke et al. 1982, Alberico 1994, Montenegro and Romero-Ruiz 1999), a single locality at the ecotone between montane and dry   2.
records are distributed in an elevational range between 0 and 900 m a.s.l.Herein, we review the distribution of the genus in Colombia, providing new localities based on museum specimens as well as recently collected material, and we additionally corroborate our morphology-based identification using a DNA-barcoding approach.
To further corroborate the identification of the species we DNA-barcoded specimen CSJ-m 995-an adult individual that exhibits all the diagnostic characters of the species (see below)-and performed a phylogenetic analysis.Following a total DNA extraction using the UltraClean Tissue & Cells DNA Isolation Kit (MO BIO Laboratories, Inc.), part of the mitochondrial marker Cytochrome Oxidase Subunit I (COI) was PCR-amplified using the primer cocktail and thermal cycling conditions described in Ivanova et al. (2012).Amplicons were sequenced using amplification primers and dye-terminator chemistry on an ABI-3730xl automated sequencer.Sequences were edited and assembled in GENEIOUS ® 9.1.8(http://www.geneious.com,Kearse et al. 2012).The obtained sequence was deposited in GenBank with accession number MH298327.We downloaded all the available COI sequences of Phylloderma stenops from GenBank (Table 1) and to corroborate the genus-level identification we also downloaded sequences from at least one species of all the genera within the phyllostomid subfamily Phyllostominae (access numbers JF435806, JF447419, JF447421, JF447429, JF448940, JF449253, JF455242, JF455365, JF455386).All sequences were aligned with MUSCLE (Edgar 2004) and the resulting alignment was analyzed using maximum likelihood as implemented in GARLI 2.0 (Zwickl 2006), specifying 5 independent search replicates, and HKY+G+I as the best fitted nucleotide substitution model-based on the Bayesian Information Criterion implement in JMODELTEST We confirm the presence of P. stenops in 14 localities (19 specimens) for Colombia, 8 of which were previously published and 6 are new (Fig. 1, Table 2).Specimen CSJ-m 995 collected in northwestern Andean montane forests (Fig. 1: locality 4) represents an increase of 830 m in the elevational distribution of the species for the country, and specimen CTUA 1692 corresponds to the first record of the species in the xeric scrubs of northern Caribbean coast (Fig. 1: locality 13), extending the known distribution of the species in Colombia northwared by 534 km.All examined material conforms to the description of the species and the most diagnosticrelevant characters are illustrated in Figures 2 to 5.This is a large species with reported forearm length between 65-81.7 mm and the maximum length of the skull between 29-34.5 mm (Husson 1962, Barquez and Ojeda 1979, Williams and Genoways 2008).It has a face with prominent chin and lip facial projections (excrescences not warts like those found in Trachops), noseleaf wide at the base with a pointed tip, short dorsal hairs with reddish-brown color as reported by Salas et al. (2014) (Fig. 2), white wingtips, calcar nearly equal to or shorter than the length of the foot, and the tail reaches only half of the inter-femoral membrane (Fig. 3) (Husson 1962, Barquez and Ojeda 1979, Emmons and Feer 1999,Williams and Genoways 2008, Reid 2009, Díaz et al. 2011, Díaz et al. 2016).Although the species seems to be easily identified, specimen ICN 4465 identified by Ramírez-Fráncel et al. ( 2015) as P. stenops, was re-identified by us as Phyllostomus elongatus (É.Geoffroy St.-Hilaire, 1810).
As previously mentioned, the species is not uncommonly misidentified with members of the genus Phyllostomus.Nonetheless, our examined material of P. stenops can be easily differentiated from the former genus using dental characters: while Phylloderma has 3 lower premolars (the second of which is small and labially oriented) the genus Phyllostomus has only 2 premolars similar in size (Figs 4, 5) (Husson 1962, Emmons and Feer 1999, Williams and Genoways 2008).
Our phylogenetic analysis of COI sequences shows 3 salient features (Fig. 6).First, haplotype of specimen CSJ-m 995, identified as P. stenops using morphological characters, is nested with other sequences of the species with strong support-and not with any other Phyllostomine genera-implying that the morphological characters are reliable for an accurate identification of this species and delimitation from phyllostomine genera.Second, the Colombian haplotype is sister to the only other available sequence from a trans-Andean locality (Panama), suggesting a trans-Andean clade for the species.Third, cis-Andean haplotypes appear to be paraphyletic and as previously found by other authors (Clare et al. 2011), the material from the Guiana Shield does not form a monophyletic group (Fig. 6).

Discussion
One of our new records, represented by the specimen CSJ-m995, increases the elevational distribution of the species in Colombia by 830 m, as the species had been recorded in the country only up to the 900 m a.s.l.(Alberico 1994); nonetheless, across its distribution (e.g., in Ecuador) this species was known to occur up to 1,750 m a.s.l.(Brito and Arguero 2012), and according to Emmons and Feer (1999) it can reach elevations of 2,600 m a.s.l.Additionally, before our identification of CTUA 1692 from the xeric scrubs in the Caribbean of Colombia (Fig. 1: locality 13), the northernmost known locality of Phylloderma stenops for Colombia was from northwestern Andean montane forests, (Fig. 1: locality 6) (Alberico 1994), and consequently our record increases the latitudinal distribution of the species in Colombia by 534 km.Despite our efforts for examining all the available material at the 2 visited collections, we were unable to find several specimens reported by Sánchez-Palomino et al. (1993) and Ramírez-Fráncel et al. (2015).In particular, the single available specimen from the Llanos ecoregion (Fig. 1: locality 10) was not found, and only 2 (out of 5) specimens from the ecotone between montane and dry forests (Fig. 1: locality 11) were found and positively identified as P. stenops (Table 2).
Although our phylogenetic analysis using mitochondrial data had only identification purposes, it does show that despite its wide distribution, this species has a reduced mitochondrial variation-only 2.1% within species (p-) distance-across haplotypes that cover a wide longitudinal gradient from French Guiana to Panama, and only 0.2% of pairwise (p-) distance between the Colombian haplotype and its sister clade of Guyanese sequences (Fig. 6).Although relevant, the issue of the likely paraphyly of Guyanese mitochondrial haplotypes is out of the scope of this paper and should be addressed elsewhere.
Many studies have catalogued P. stenops as locally rare but widespread (Handley 1976, Trajano1984, Arita 1993, McDade 1994, Simmons and Voss 1998, Emmons and Feer 1999, Nadkarni and Wheelwright 2000, Reid 2009) and our study seems to provide evidence to reinforcing this concept.As an example, as part of a mammal inventory at Parque Nacional Natural Las Orquídeas, from more than 300 netted bats only a single individual of P. stenops was captured (Díaz-Nieto unpublished).Although for Colombia the species is only known for 14 localities, these encompass almost the entire latitudinal and longitudinal gradient of the country as well as a great  New field-based studies with increased sampling effort should be developed to gain knowledge on the ecogeographic limits of the species, in particular in areas with likely suitable habitat where the species has yet to be found (e.g., biogeographic Choco, Andean montane forests of Cordillera Central and Oriental).Additionally, a denser genetic (multiple loci) and geographic sampling should be useful in testing both the ecogeographic and evolutionary limits of the proposed subspecies, in particular, relevant samples such as those from dry ecoregions and those from the northern and southern limits of its distribution would be welcome.1, 2).Bolded terminal corresponds to the sequence obtained in this report.

Figure 1 .
Figure 1.Collecting localities and ecoregions of Phylloderma stenops for Colombia.Red circles represent new records; blue circles correspond to previously published records (see text).Numbers correspond to localities in Table2.
Such wide geographical distribution includes mostly humid ecoregions (see above) but also at least 2 dry ecoregions, such as dry and xeric shrublands (Fig.1: localities 11, 13 and 14).

Figure 6 .
Figure 6.A maximum-likelihood topology of COI sequences of Phylloderma stenops.Numbers on branches correspond to bootstrap support values.Each terminal is identified by country of origin and an alphanumeric specimen identifier (see Tables1, 2).Bolded terminal corresponds to the sequence obtained in this report.

Table 1 .
Sequences of Phylloderma included in this report.*Correspondtounpublisheddata, ** corresponds to a sequence without voucher.As part of a mammal inventory at the Parque Nacional Natural Las Orquídeas on 9 October 2014, an adult female of Phylloderma stenops (Fig.2) was captured with mist net in secondary forest (northwestern Andean Montane Forest ecoregion) at El Macho, municipality of Urrao, Antioquia Department, Colombia (Fig.1: locality 4).The specimen CSJ-m 995 is an adult female with an open pubic symphysis, preserved as skin and skeleton, with liver tissue preserved in ethanol (96%), and deposited in the mammal collection of the Museo de Ciencias Naturales de la Salle of Instituto Tecnológico Metropolitano (CSJ-m).It has the following measurements: total length 122 mm; tail length 18 mm; hind foot length 22 mm; ear length 26 mm; forearm length 73 mm; and maximum length of the skull 33 mm.

Table 2 .
List of records of Phylloderma stenops in Colombia.Locality numbers and ecoregions are mapped in Figure1; asterisk (*) indicates material that was not examined by us (see Methods).