New distribution records and variation of the two common lowland salamanders

With 20 currently accepted species, the genus Bolitoglossa is the second most species-rich amphibian genus in Panama, after Craugastor (Hertz et al. 2012). Most of these salamanders inhabit the high mountains of the Talamancan mountain range in the Panama-Costa Rica border region. Only four known species occur in the lowlands of western Panama. Among these are, besides B. biseriata, Tanner 1962 and B. schizodactyla, Wake and Brame 1966, the two common species B. colonnea (Dunn 1924) and B. lignicolor (Peters 1873). Bolitoglossa colonnea is distributed primarily along the Caribbean slopes of the central mountain ranges of Costa Rica and Panama and into the Caribbean lowlands, including offshore islands. In contrast, B. lignicolor inhabits the Pacific drainage of southwestern Costa Rica and western Panama including the Isla de Coiba and the Azuero eninsula. However, there is an overlap in the distribution areas of both species in the humid lowlands of the Osa peninsula and the Golfo Dulce region of Costa Rica (McDiarmid and Savage 2005) and possibly marginally into extreme western Panama, where both species may be found sympatrically. These two species probably also meet in the Fortuna depression area, where the continental divide drops to less than 1000 m asl, interrupting the effect of the central mountain range as a barrier between the Caribbean and Pacific side climates. However, both species have not been recorded at the same site. Although both salamanders are frequently collected, documented collection sites are widely separated, creating a mosaic-like distribution pattern. We present new collection sites that close the largest gaps in the known distribution of both species for Panama. We further present coloration, morphology, and molecular data of the corresponding voucher specimens and discuss their variation. All specimens were encountered during opportunistic searches at night, caught by hand, and preserved the day Abstract: We revise the geographic distribution of two common lowland salamanders in western Panama. We close the widest gap in the known distribution of Bolitoglossa colonnea with a first record for the province of Veraguas and extend its known vertical distribution to a third life zone. For B. lignicolor we present additional localities in the province of Chiriquí and the first record from Comarca Ngöbe-Buglé that close the gap between extreme western Panama and the Azuero Peninsula. We present morphological, molecular, and coloration data for both species. 1 Senckenberg Forschungsinstitut und Naturmuseum, Senckenberganlage 25, 60325 Frankfurt am Main, Germany. 2 Goethe-University, Institute for Ecology, Evolution & Diversity, Biologicum, Building C, Max-von-Laue-Straße 13, 60438 Frankfurt am Main, Germany. * Corresponding author. E-mail: ahertz@senckenberg.de Andreas Hertz 1,2,*, Sebastian Lotzkat 1,2 and Gunther Köhler 1 New distribution records and variation of the two common lowland salamanders Bolitoglossa colonnea (Dunn, 1924) and B. lignicolor (Peters, 1873) in Panama (Amphibia: Caudata: Plethodontidae)

Coordinates and elevation data were obtained using a Garmin GPS receiver with built-in barometric altimeter.All coordinates are in WGS 1984 datum, and elevations are rounded to the nearest 10 m.We obtained locality information for Bolitoglossa colonnea via HerpNet (http:// www.herpnet.org)from the herpetological collections AMNH, FMNH, KU, MCZ, MVZ, and USNM, as well as from UP (Tejera and Dupuy 2003) and the web portal of the Círculo Herpetológico de Panamá (accessed through GBIF data portal, Datos de los Especimenes de Anfibios y Reptiles del Círculo Herpetológico de Panamá, http://ara.inbio.ac.cr/SSTN-IABIN/datasets/resource/37).The locality information for B. lignicolor was taken from ANSP, CAS, MVZ, and USNM via HerpNet, as well as from Brame and Wake (1963), Bauer et al. (1993), De la Riva (1997), and Cedeño et al. (2006).The map was created using ArcGIS 10.We obtained map layers from the map server of the Smithsonian Tropical Research Institute (STRI) (accessed through http://mapserver.stri.si.edu/geonetwork/srv/ en/main.home).
The following morphological measurements of voucher specimens were made with a caliper under a dissecting microscope: snout-vent length (SVL), from tip of snout to posterior angle of vent; tail length (TL), from posterior angle of vent to tip of tail; head length (HL), from tip of snout to gular fold; head width (HW), between angles of jaw; hind limb length (HLL), from groin to tip of longest toe; hand width (HAW), at greatest width; hind foot width (HFW), at greatest width.Tooth counts were made directly under a dissecting microscope: premaxillary teeth (PMT), maxillary teeth (MT), and vomerine teeth (VT).
A fragment of the mitochondrial 16S rRNA gene of selected voucher specimens was sequenced for comparison with sequences available on GenBank.Sequences were aligned with ClustalW (Larkin et al. 2007) using the default settings in Geneious (Drummond et al. 2010).We determined the Tamura 3-parameter model (Tamura 1992) as the best-fitting substitution model.Using MEGA5 (Tamura et al. 2011) we calculated p-distances and conducted Maximum Likelihood analyses (with 10000 bootstrap replicates).Using TCSv1.21 (Clement et al. 2000), we conducted a statistical parsimony network analysis, with gaps considered as a fifth character state.Because some haplotypes were not connected to each other with the 95% limit of probability of parsimony, we decreased the connection probability in both networks to a minimum of 90% resulting in a connection limit of 14 steps in our analysis.
A list of specimens examined, including corresponding GenBank accession numbers, is in the appendix.(Dunn, 1924) Holotype: MCZ 9406, by original designation.Type locality: "La Loma, on trail from Chiriquicito to Boquete Diagnosis: Bolitoglossa colonnea is a moderate-sized species of salamander with fully webbed digits and only 0-11 maxillary teeth in adults, but up to 48 vomerine teeth.It is readily identified by a fleshy transverse ridge between the eyes.

Bolitoglossa colonnea
Geographic distribution: An overview map that combines literature records with our own records is provided in Figure 1.Previous collection sites on the Caribbean side of the central mountain range extend over the humid lowlands and premontane elevations from northern Costa Rica to western Panama.The easternmost Caribbean records come from La Loma, Comarca Ngöbe-Buglé on the mainland (Figure 1, locality 1), and from Isla Escudo de Veraguas, Bocas del Toro (Figure 1, locality 2).On the Pacific side it is known from several Costa Rican sites around Golfo Dulce, and in Panama from three sites in the La Fortuna area (Figure 1, localities 3-5), and from the southern slopes of Cerro La Campana in central Panama (Figure 1, locality 6), the last of which is also the easternmost collection site for B. colonnea.
Our records from Cerro Negro, Río Chilagre, and Cerro Narices (Figure 1, localities 7-9) in the vicinity of Santa Fé, Veraguas represent the first records for that province and bridge the gap between Quebrada Bijau, La Fortuna Forest Reserve, Province of Chiriquí (Figure 1, locality 5) approximately 130 km west, and Altos de Campana, Province of Panamá (Figure 1, locality 6) approximately 130 km east of Santa Fé.The nearest collection site is the island Escudo de Veraguas, Province of Bocas del Toro (Figure 1, locality 2) about 85 km from Santa Fé in the Caribbean Sea.Our record from Río Changena, Province of Bocas del Toro, 1650 m (Figure 1, locality 10) extends the known vertical distribution of Bolitoglossa colonnea from 1250 m (Köhler 2011).This location expands the known habitat of B. colonnea from the Lowland and Premontane Life Zones to the Lower Montane Life Zone, specifically the Lower Montane Wet Forest (Holdridge 1967).
Variation: Coloration: Coloration of Bolitoglossa colonnea includes mostly shades of drab brown and beige, often with indistinct stripes and stippling.A compilation of color morphs is shown in Figure 2. Coloration in life of two specimens was recorded in the field as follows: SMF 94460 (Figure 2B).Dorsal ground color Cinnamon (39).Two Vandyke Brown (121) furcating lines start at interorbital transverse ridge, and meet posterior to head, thence continuing as single vertebral stripe to base of tail.Laterally Prout´s Brown ( 121 Morphology: Two males and seven females of B. colonnea were included in the morphological analysis.Ratios of TL, HL, HW, HLL, HAW and HFW in relation to SVL, as well as tooth counts for each specimen, are in Table 1.We calculated mean and standard deviation for males and females separately.Most specimens examined agree well with the morphological descriptions of other authors, but there is a discrepancy in some tooth counts.Savage (2002) gave the number of maxillary teeth as 0-6, while in our sample many females have more than six and up to eleven maxillary teeth.Further, a male specimen (MHCH 2600) from Río Chilagre, Veraguas (Figure 1, locality 8) raises the known maximum of vomerine teeth of this species from 36 (Savage 2002) to 48. Figure 3 illustrates the VT/SVL ratio for males and females from eastern and western localities separately.The highest vomerine tooth counts were recorded in specimens from eastern localities.On average, males seem to have longer tails and more premaxillary teeth than females, although our sample of males is too small to confirm this (Table 1).
Molecular genetics: We compared 16S mtDNA sequences of two specimens (SMF 94460-61) collected at Río Changena and Río Clarito (Figure 1, localities 10 and 11) in highland Bocas del Toro, to two specimens on GenBank, one from Finca de Enrique Quintero, Río Changuinola (Figure 1, locality 12) in lowland Bocas del Toro and one from La Fortuna Forest Reserve (Figure 1, near Locality 4, exact locality unknown).We chose the sister species Bolitoglossa schizodactyla as an outgroup (Figure 4).Our two specimens (SMF 94460-61.)from higher elevations of Bocas del Toro share the same haplotype, they differ by 1.9% from one specimen on GenBank (CH 6526) that was collected at lower elevation at Río Changuinola (Figure 1, locality 12; Table 2).All three specimens have been connected in a haplotype network (Figure 4), with a calculation of 10 unsampled haplotypes between our two specimens from upland Bocas del Toro and the one from the lowlands.The fourth specimen (AY526119, no voucher) from Hornito, La Fortuna Forest Reserve, Chiriquí (Figure 1, locality 4), referred to as B. colonnea, has not been connected to the haplotype network and shows a p-distance of 4.2 to 5.4% to the specimens collected in Bocas del Toro, while its distance to the outgroup is only 5.2%.
Remarks: There is no other salamander species in lower Central America that is so easily recognized, owing to its conspicuous and unique fleshy interorbital ridge.Nevertheless, there is variation in morphology (Table 1; Figures 2 and 3) and genetics (Table 2; Figure 4).We found the highest count of vomerine teeth in two specimens from Río Chilagre, Veraguas.This difference is considerable between the two males (Figure 3), from widely separated localities, one (SMF 94460, Figure 2B) from Río Changena, Bocas del Toro (Figure 1, locality 10) in extreme western Panama on the Caribbean slopes of the Talamancan mountain range, the other (MHCH 2600, Figure 2F) from Río Chilagre, Veraguas (Figure 1, locality 8) on the eastern end of the central mountain range.Although both specimens are about the same size, the male from Río Chilagre (MHCH 2600) has 48 vomerine teeth, 31 more than the male from Río Changena (SMF 94460).Savage (2002) gave a maximum of 36 vomerine teeth for adults of B. colonnea.In females, the specimen (SMF 94461, Figure 2A) from Río Clarito, Bocas del Toro (Figure 1, locality 11), the largest in our sample, has 11 vomerine teeth fewer than the considerably smaller female (SMF 94463, Figure 2E) from Río Chilagre, Veraguas (Figure 1, locality 8), which has 34 vomerine teeth.Another subadult female (SMF 85066, Figure 2C) specimen from Isla Colón, Bocas del Toro (Figure 1, locality 13), the smallest in our sample, has one vomerine tooth fewer than the next largest female (SMF 94464, Figure 2D) from Cerro Negro, Veraguas (Figure 1, locality 7).Our data suggest that specimens from the eastern portion of the range tend to have more vomerine teeth than those from the west, even considering that the number of teeth increases with size and age (Figure 3).
We also observed genetic differences in 16S gene sequences.The distance between the specimen (AY526119, no voucher) from Hornito, La Fortuna Forest Reserve, Chiriquí (Figure 1, locality 4) and the others (SMF 94460-61; CH 6526) from Bocas del Toro (Figure 1, localities 10-12) is large enough to suggest that two different species may be involved.Unfortunately, there is no voucher specimen corresponding to the sequence of the specimen from Hornito, La Fortuna Forest Reserve (Parra-Olea et al. 2004).During our field work in the La Fortuna Forest Reserve we could not obtain specimens of B. colonnea or any other species that may resemble B. colonnea.This is a subject for future study.designated lectotype by Bauer et al. 1993.Type locality: "Chiriqui", Panama; corrected by Bauer et al. (1993) to "Camarón, Provinz Chiriqui", Panama (Figure 1, locality 17, see remarks).
Diagnosis: Bolitoglossa lignicolor is a relatively large and robust species with fully webbed hands and feet.It usually has a light brown dorsum, while the venter is dark brown.
Geographic distribution: An overview map that combines literature records with our own records is given in Figure 1  ) and the Azuero Peninsula (Brame and Wake 1963;Figure 1, localities 20-22 and Cedeño et al. 2006;Figure 1, locality 33), approximately 200 km to the east.Brame and Wake (1963) believed "Tiger Ridge Camp" and "Cerro Mangillo" to be located in the province of Los Santos.Actually, the collection data on the ANSP specimens read "Veraguas prov., Cerro Mangillo, 2800 ft." and "Veraguas prov.?, Tiger Ridge Camp 2600 ft." (Ned Gilmore, ANSP pers. comm. 2012).However, none of these place names is traceable on recent maps.There is only one mountain named Cerro Manguillo (or Cerro Manglillo) on Azuero Peninsula, located near the present day province triangle Veraguas, Herrera, and Los Santos.We assume that E.R. Dunn, who collected the specimens, started his expedition to Cerro Manguillo (Figure 1, locality 21) from the Veraguas side and was not sure if he was still in Veraguas when he reached Tiger Ridge Camp, which he expressed by the question mark.We suspect Tiger Ridge Camp (Figure 1, locality 22) has been on the crest above Quebrada El Tigre, about 4 km north of Cerro Manguillo in Herrera province.It is the only place that is name-wise linked to Tiger Ridge, as well as at the stated elevation.This view is supported by the fact that ANSP specimens with interjacent numbers were collected at Macaraquito Camp which is situated between these two places.The documented vertical distribution ranges from sea level to approximately 1200 m asl.
We present two more records from the Pacific slopes of the central mountain range that fill the gap in the distribution of Bolitoglossa lignicolor.The first new locality is Meseta de Chorcha, Chiriquí, about 25 km east of the city of Davíd (Figure 1, locality 23).The second is Alto Tólica, Comarca Ngöbe-Buglé (Figure 1, locality 24), about 80 km east of Meseta de Chorcha, also representing the first record for the Comarca Ngöbe-Buglé.Moreover, we present additional localities on the Azuero Peninsula (Figure 1, localities 25 and 26) and confirm a previous collection site at Montuoso Forest Reserve (Figure 1, localities 20 and 27).
Variation: Coloration: The usual coloration pattern is a broad, light-colored dorsal band, which may be light tan, beige or reddish, on darker, mostly chocolate brown ground coloration.However, the light dorsal coloration may be broken up into blotches and even smaller spots.In contrast, some individuals are predominantly light colored with darker mottling.Color photographs of selected specimens are in Figure 5.The coloration in life of three specimens has been recorded in the field as follows: SMF 91996 (not pictured).Dorsum Tawny (38) with Raw Umber (223) streaks.Iris Verona Brown (223 B).SMF 91994 (Figure 5F).Dorsal ground color Sepia (119) with dirty white stipples grading into Tawny Olive (223 D) on tail.Ventral surfaces the same as dorsal surfaces, but with finer and less dense stippling.Iris Drab Gray (119 D), peripherally Mars Brown (223 A).SMF 94458 (Figure 5B).Dorsal ground color Clay Color (26) with longitudinal, broken lines of Sepia ( 119) and Cream Color (54).Ventral ground color Dark Brownish Olive (129) powdered with dirty white spots.Limbs Dark Brownish Olive (129) dorsally suffused with Clay Color (26).
Morphology: Six males and 12 females of Bolitoglossa lignicolor were included in the morphological analysis.Ratios of TL, HL, HW, HLL, HAW and HFW in relation to SVL, as well as tooth counts, for each specimen are in Table 3.There is not much variation within sexes in our sample, while variation between sexes is evident.As in B. colonnea, males have longer tails in relation to their snout-vent length.We also observed slightly higher tooth counts for males.In contrast, Brame and Wake (1963) stated that females of B. lignicolor have more vomerine teeth, because the number of teeth increases with age and size and females grow bigger than males.However, in our sample males have higher vomerine tooth counts, and males in our sample are on average larger than females, [Atlantic side], altitude about 2000 feet [610 m], Province of Bocas del Toro [today: Comarca Ngöbe-Buglé], Panama".(Figure 1, locality 1) A) demarcated above by a fine Pale Pinkish Buff (121 D) line.The ventral surfaces mostly Pale Pinkish Buff (121 D) with fine Mikado Brown (121 C) longitudinal lines, except Vandyke Brown (221) gular region.SMF 94463 (Figure 2E).Dorsal ground color Chamois (123 D), irregularly mottled with Sepia (119).Distal 20% of tail grading into Pale Horn Color (92).Ventral surfaces Pale Horn Color (92) with fine mottling of Pratt´s Payne's Gray (88) and Sepia (119) spots and blotches, the larger ones grading into Dark Neutral Gray (83).

Figure 3 .
Figure 3. Vertical scatter plot of vomerine teeth/snout-vent-length ratio for Bolitoglossa colonnea.Males and females from eastern and western localities are shown separately.Horizontal bars indicate mean values.

Figure 4 .
Figure 4. Maximum Likelihood consensus tree of 16S mtDNA of selected specimens of Bolitoglossa colonnea, with B. schizodactyla as an outgroup, and the corresponding haplotype network.Specimen labels refer to collection number or, if there is no collection number available, to GenBank accession number.The upper two specimens have been sequenced for this study.Numbers in rectangles refer to the respective collection site in Figure 1.Other collection sites are those denoted on GenBank: Coclé = Parque Nacional G. D. Omar Torrijos H., El Copé, Distrito La Pintada, Cocle Province, 800 m elevation.
AMNH 11725 and KU 66164) that are in fact based on specimens of B. alvaradoi.Additional collection sites in Panama are Coiba Island (De la Riva 1997; Figure 1, locality 19

Figure 6 .
Figure 6.Vertical scatter plot of vomerine teeth/snout-vent-length ratio for Bolitoglossa lignicolor.Males and females kept separated.Centered horizontal bars indicate mean values; upper and lower horizontal bars standard deviation.

Table 2 .
Estimates of evolutionary divergence between sequences of specimens of Bolitoglossa colonnea.Bolitoglossa schizodactyla has been chosen as an outgroup.The number of base substitutions per site from between sequences are shown.Analyses were conducted using the Tamura 3-parameter model.

Table 1 .
Morphological characters and tooth counts for specimens of Bolitoglossa colonnea from Panama.

Table 3 .
Morphological characters and tooth counts for specimens of Bolitoglossa lignicolor from Panama.