A new species of Caecilia (Gymnophiona, Caeciliidae) from the Magdalena valley region of Colombia

Abstract A new species of the genus Caecilia (Caeciliidae) from the western foothills of the Serranía de los Yariguíes in Colombia is described. Caecilia pulchraserranasp. nov. is similar to C. degenerata and C. corpulenta but differs from these species in having fewer primary annular grooves and a shorter body length. With this new species, the currently recognized species in the genus are increased to 35. Mitochondrial DNA sequences, including newly sequenced terminals representing two additional, previously unanalyzed species, corroborate the phylogenetic position of the new species within Caecilia and the monophyly of the genus. This analysis also included newly sequenced terminals of Epicrionops aff. parkeri (Rhinatrematidae) and trans-Andean Microcaecilia nicefori (Siphonopidae). Evidence was found for the non-monophyly of the family Siphonopidae and the siphonopid genera Microcaecilia and Siphonops. The implications of these results for caecilian systematics are discussed and the status of the trans-Andean populations of Caecilia degenerata is commented upon.


Introduction
The Neotropical caecilian amphibian genus Caecilia Linnaeus, 1758 (Gymnophiona: Caeciliidae) currently comprises 34 nominal species (Wilkinson et al. 2011;Frost 2018;, 18 of which occur in Colombia, with eight being endemic to this country. Seven species occur in the Magdalena valley region of Colombia (Dunn 1942;Lynch 1999) and external morphology segregates them into two groups. A first group comprises four species that lack secondary annular grooves: C. caribea Dunn, 1942, endemic to Colombia, from the eastern slope of the Cordillera Central, Caldas Department, between 10-1700 m above sea level (a.s.l); C. corpulenta Taylor, 1968, from the type locality in Peru, with a Colombian record from the Andean forests on the 1750 m a.s.l., Santander Department; C. subdermalis Taylor, 1968, from northern Ecuador and eastern slopes of the Cordillera Central, Huila and Caldas Departments in Colombia, between 850-2320 m a.s.l.; and C. degenerata Dunn, 1942, endemic to Colombia, from both flanks of the Cordillera Oriental, between 800-2100 m a.s.l., Boyacá, Cundinamarca, and Santander Departments (Dunn 1942;Taylor 1968;Ruiz-Carranza et al. 1996;Lynch 1999;Acosta-Galvis 2000;Rivera-Correa 2006;Castro-Herrera et al. 2007;Frost 2018; Appendix 1).
A second group includes three species that have secondary annular grooves: C. guntheri Dunn, 1942, with a wide distribution from northern Ecuador to Colombia, where the records are discontinuous and include the sub-Andean forests of the Cordillera Occidental and the region of Muzo at Quípama Municipality, Boyacá Department, western slope of the Cordillera Oriental, 1000 m a.s.l.; C. subnigricans Dunn, 1942, from northern Venezuela and lowlands of the Caribbean and Magdalena Valley regions of Colombia, with a record from Mariquita Municipality, Tolima Department; and C. thompsoni Boulenger, 1902b, endemic to the middle Magdalena valley in Colombia, 240-1571 m a.s.l. (Dunn 1942;Taylor 1968;Ruiz-Carranza et al. 1996;Lynch 1999;Acosta-Galvis 2000;Bernal et al. 2005Acosta-Galvis et al. 2006Lynch and Romero 2012;Mueses-Cisneros and Moreno-Quintero 2012;Paternina-H et al. 2013;Acevedo-Rincón et al. 2014;Angarita-M et al. 2015;Restrepo et al. 2017;Frost 2018; Appendix 1).
During a recent herpetological survey in wet tropical forests of the Serranía de los Yariguíes, in the Department of Santander, Colombia (Fig. 1), we collected several specimens of a small Caecilia that lack secondary annular grooves and dermal scale pockets, suggesting that they correspond to either C. degenerata or C. corpulenta. However, a low number of primary annular grooves and a combination of morphometric characters indicate instead that these specimens belong to a new species, which we describe herein. To test the generic placement of the new species and to explore the relationships of other Neotropical caecilians, we perform a phylogenetic analysis of DNA sequences. We discuss the implications of our results for caecilian systematics and comment on the status of the trans-Andean populations of C. degenerata.

Fieldwork and reference collections
The new species was collected during fieldwork carried out in the Serranía de los Yariguíes, vereda La Belleza, municipality of El Carmen de Chucurí, Santander Department, Colombia (06°34'N, 73°34'W, 731-789 m a.s.l.; Fig. 1), from 17 February to 1 May 2018, during the dry season. Specimens were found in two separate humid spots near the Río Cascajales, which drains Tropical moist broadleaf forests, within the ecoregion of the Magdalena valley montane forests, in the foothills of the Cordillera Oriental, Colombia (Dinerstein et al. 1995;Olson and Dinerstein 2002).
Previous fieldwork conducted between 1998-1999 by John Lynch in collaboration with the first author, successfully allowed the detection of microhabitats and several specimens of Microcaecilia nicefori (Lynch 1999); subsequently, between 2000 to date, fieldwork with caecilians such as Oscaecilia polyzona (Lynch and Acosta 2004), Caecilia sp., C. thompsoni, and C. isthmica (unpublished data) allowed successful detection of microhabitats and multiple specimens.
The collecting technique, which was used to obtain specimens of the new species, consists of first asking local people about the locations where they have spotted caecilians using the common names of "blind snakes", or "captain worms" ("lombrices capitanas"), or "motolas" (this common name is specific for the Department of Santander). Subsequently, the reported sites are visited and inspected to select sites under the shade of vegetation, and where the soil is not compact and very humid (usually associated with water springs that form a mosaic of marshy and dry areas). Collecting efforts are focused in the selected damp microhabitats, digging with a hoe to a depth of approximately 20 cm (approximate sampling effort of 2-person-hour to collect five specimens). Coordinates and elevations were obtained with a Garmin GPSMAP 64SC (map datum WGS 84). Collected specimens were euthanized using 20% benzocaine (Chen and Combs 1999), fixed in 10% formalin, and preserved in 70% ethanol. Tissue samples from two individuals were obtained immediately after euthanasia and preserved in 96% ethanol. Specimens were deposited at the Biological Collections of the Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, Villa de Leyva, Boyacá, Colombia (IAvH-Am and IAvH-CT) and the Amphibian Collection of the Universidad Industrial de Santander, Bucaramanga, Santander, Colombia (UIS-MHN-A).

Phylogenetic analysis
To test the generic assignment of the new species and to explore the relationships of other endemic caecilians from Colombia, available mitochondrial DNA sequences of the genes 16S and CO1 from members of all Neotropical caecilian families (Caeciliidae, Typhlonectidae, Siphonopidae, Dermophiidae, and Rhinatrematidae) were analyzed ( Table 1). The analysis included a fragment of COI (ca. 651 bp) and a fragment of 16S (ca. 510 bp). Sequences for most terminals were obtained from GenBank (Table 1). We added new sequences for eight Colombian terminals representing the new species, Caecilia thompsoni, C. isthmica, Typhlonectes natans, Epicrionops aff. parkeri, and Microcaecilia nicefori (Appendix 1). The cryptobranchid Cryptobranchus alleganiensis was used to root the tree. Laboratory protocols and primers are those of Palumbi (1996), Ivanova et al. (2006), and Carr et al.(2011). Bidirectional PCR products were used for Sanger sequencing at the Instituto de Genética of the Universidad Nacional de Colombia. Resulting sequences were visualized, assembled, checked for stop codons (COI), and edited in Geneious Pro v 10.2.3 (Kearse et al. 2012). All sequences were deposited in the Barcode of Life Data System (BOLD; Ratnasingham and Hebert 2007) and GenBank (Table 1). Sequences of each gene were aligned independently using the MAFFT plugin v 7.388 within Geneious, considering the secondary structure of RNA in 16S and implementing the G-INS-I algorithm. Subsequently, sequences of both genes were concatenated in a single dataset using Geneious, which was used to construct a Maximum Likelihood phylogeny using IQ-TREE (Nguyen et al. 2015), performing a partitioned analysis based on four partitions (16S, COI first codon position, COI second codon position, COI third codon position) using the partition finder algorithm (-m option TESTMERGE; Lanfear et al. 2012) in IQ-TREE and best fitting models for each partition selected by the same program (Chernomor et al. 2016;Kalyaanamoorthy et al. 2017). Each partition was allowed to have its own set of branch lengths (-sp option). Branch support analysis was performed with 1000 ultrafast bootstrap replicates (Hoang and Chernomor 2017).

Morphology
Criteria and terminology for morphological descriptions, diagnostic characters, and data for other species of Caecilia follow Lynch (1999), Gower and Wilkinson (2002), Maciel et al. (2009), Maciel and Hoogmoed (2011), Kamei et al. (2009), Wilkin-  (2010), Donnelly and Wake (2013), and Wilkinson et al. (2009Wilkinson et al. ( , 2013Wilkinson et al. ( , 2015. For comparative purposes, specimens of C. guntheri, C. isthmica, C. thompsoni, and C. subdermalis were examined (Appendix 1). Morphological observations were made using a stereoscope Nikon optical device SMZ-1B, with High Intensity Illuminator NI-150 Nikon and App Scope 3xSRA41. Measurements were taken using a Mitutoyo precision digital caliper to ± 0.1 mm; and using the following abbreviations for anatomical features and ratios of measurements: Dermal scale pockets and subdermal scales were searched using the criteria proposed by Wilkinson et al. (2013) and sex and maturity were determined by examination of gonads. Live specimens were photographed with a digital camera model Canon EOS 70D and preserved specimens with a digital camera Canon EOS 5D Mark II.

Phylogenetic analysis
The final concatenated molecular dataset consisted of a matrix of 1273 bp, 567 sites were parsimony-informative, 111 were singletons, and 595 were constant sites. The best fitting substitution model for both CO1 and 16S was TIM2+F+I+G4 after testing the large selection of models in IQ-TREE. The ML tree is shown in Fig. 2 (LnL: -15725.921). Our phylogenetic analysis recovered the new species nested within a moderately well-supported (84%) monophyletic Caecilia, in a maximally supported monophyletic Caeciliidae. The new species appears most closely related, of the sampled species, to C. volcani but support for this relationship is not strong (58%). Rhinatrema nigrum and R. bivittatum were recovered as monophyletic with the sister group Epicrionops. Siphonops was inferred to be paraphyletic with respect to Luetkenotyphlus (Siphonopidae), and Microcaecilia nicefori was recovered as the sister group of Dermophidae + Siphonophidae, the latter including the remaining Microcaecilia (with Brasilotyphlus guarantanus nested within it) and the paraphyletic Siphonops.

Description of new species
Generic assignment. The new species is assignable to the genus Caecilia because its eyes are not covered by bone and it has tentacles below the nostrils (Type D sensu Lynch, 1999 Type Locality. (Fig. 1) Colombia, Santander Department, El Carmen de Chucurí Municipality, vereda La Belleza, Cascajales River, 06°34'8.9"N, 73°34'20.2"W, 789 m a.s.l.
Description of holotype. An adult female (Fig. 3). Head dorsoventrally flattened and slightly narrower than body; head width at CM 63% of width at midbody, head width at CM 72% of head length; head length 3.5% of total length; interorbital distance 40% of head width. Snout projects 1.6 mm beyond mouth; tip of snout rounded in dorsal and lateral view (Fig. 3); area between the eye and naris flattened. Eyes visible but small, eye diameter 4% of head length and 13.5% of eye-nostril distance; nares small, margins slightly protuberant, directed posterodorsally, visible from above. Tentacular openings circular and small, slightly raised above skin, laterally positioned near margin of mouth (Type D sensu Lynch 1999, Fig. 3D, E), slightly closer to corner of mouth than to nostrils. Tongue anteriorly attached, surface smooth with some longitudinally oriented grooves. Teeth pointed, recurved, with size decreasing posteriorly; premaxillary-maxillary and dentary teeth monocuspid and visible externally. Premaxillary-maxillary teeth 13, posterior maxillary teeth smaller. Premaxillary-maxillary series extending behind level of choanae. Vomeropalatine teeth 10, monocuspid, relatively uniform, moderately recurved, not visible externally, similar in size. Dentary teeth 12, moderately recurved, faintly larger than premaxillary-maxillary teeth. Choanae subovoid; narial plugs visible (Fig. 3F). Nuchal grooves indistinct dorsally and ventrally, incompletely encircling body with transverse grooves on the collars, in ventral surfaces. First collar shorter than second. Body subcylindrical, slightly deeper than wide (Fig. 3A, B); body width at midbody 4% of total length. Width along body varies slightly, narrower at terminal region. Primary annuli 104 incomplete dorsally and ventrally. Primary annular grooves completely encircling the body. Secondary grooves absent (Fig. 3G-I). Dermal scale pockets absent. Vent circular; disc around vent conspicuous enlarged (Fig. 3I) with seven denticulations anterior, seven nearly equal posterior denticulatios (Fig. 3I); anal papillae absent, and unsegmented terminal shield of 4.9 mm length.
Color in life (Fig. 4): Jaw margins, area between the eye and naris, and tentacular regions pink-orange (salmon); eyeballs completely violet blue (Fig 4b); periorbital region salmon; body dark brownish with thin salmon-colored chromatophores; ventral surface of body slightly paler than dorsum; annular grooves on sides of body slightly darker than general body color. Color in preservative (ethanol 70%; Fig. 3): Body dark slate gray dorsally with diffuse khaki chromatophores; jaw margins, rostral and periocular regions yellowish; ventral and lateral surfaces slightly paler than dorsum; vent disk jaw margins and area between the eye and naris yellowish.
Distribution and natural history. Caecilia pulchraserrana sp. nov. is currently known from two adjacent, relictual tropical wet forest localities on the western slope of the Cordillera Oriental of Colombia (Serranía de los Yariquíes; Fig. 1) at elevations between 731-789 m a.s.l. The Serrania of the Yariguies corresponds to an isolated mountain range that is part of the western slope of the Cordillera Oriental of Colombia (Fig. 1). Caecilia pulchraserrana sp. nov. is a fossorial species associated with marshy areas surrounded by secondary vegetation at the forest edge (Fig. 6). The specimens were collected during the dry season in very wet soils lacking rocks (i.e., bogs; Fig. 6), in a slightly inclined area (nearly 5°of slope) covered with vegetation of the family Heliconiaceae (Heliconia spp., Fig. 6).
Caecilia pulchraserrana sp. nov. was obtained during the initial 10 minutes of removal with a hoe.We extracted the first specimen in intermediate substrates between marshy and dry areas; after 40 minutes of excavation in these selected areas, we obtained four additional specimens. Using these same criteria, when moving two kilometers above the original point, an area with similar characteristics was located and within 20 minutes we collected two additional specimens. Caecilia pulchraserrana sp. nov. was collected on black sandy soils with high organic matter content. These caecilians move   quickly under the substrate, so once the first specimen is detected it is important to quickly create channels to surround and block them from escaping. Etymology. The specific epithet is formed from the Latin pulchra (nominative feminine singular of pulcher), meaning beauty, and the Spanish adjective serrana (feminine singular of serrano), from the sierra or serranía. This specific name refers to the type locality of the species: vereda La Belleza (beauty in English) in the western foothills of the Serranía de Los Yariguíes. The specific name was chosen using a citizen science approach. First, scientists and inhabitants of the El Carmen de Chucurí municipality gathered a list of possible names for the new species. Then, the list of potential names and their meanings was shared with the local people, who voted to choose their preferred name.

Phylogenetic relationships
Our description of Caecilia pulchraserrana sp. nov. brings the number of known species of Caecilia to 35 (Frost 2018). Molecular data are currently available for only six of these species (including the three newly sequenced species analyzed here), which precludes a thorough analysis of the relationships within the genus. Consequently, our phylogenetic analysis (Fig. 2) was designed mainly to test the generic placement of C. pulchraserrana sp. nov. in addition to exploring the relationships of C. isthmica and C. thompsoni (two other species that are endemic to Colombia). Our results recovered C. pulchraserrana sp. nov., C. isthmica, and C. thompsoni within Caecilia (Fig. 2), corroborating the generic placement of the new species and the monophyly of the genus, as previously hypothesized by Wilkinson et al. (2011). Our analysis recovered Oscaecilia as the sister group of Caecilia, which agrees with the results of San Mauro et al. (2014) but disagrees with those of Pyron and Wiens (2011), who instead recovered Caecilia as paraphyletic with respect to Oscaecilia.
Our phylogenetic analysis only included two mitochondrial loci and a small number of species and should not be considered as a robust resolution of caecilian relationships. Nevertheless, our results highlight several potential cases of non-monophyletic taxa and suggest that a taxonomic revision, including a major generic rearrangement, is warranted. Our study includes, for the first time, the Colombian endemics Epicrionops aff. parkeri (Rhinatrematidae) and Microcaecilia nicefori in molecular phylogenetic analyses. On one hand, recent contributions ) have allowed taxonomic rearrangements within Rhinatrematidae, with Rhinatrema nigrum and R. bivittatum being recovered as monophyletic, supporting previous claims (Wilkinson and Gower 2010;Wilkinson et al. 2011;Pyron and Wiens 2011;San Mauro et al. 2014) that Epicrionops could be transferred to Rhinatrema. Our analysis recovers Epicrionops aff. parkeri nested within a monophyletic Epicrionops (E. marmoratus+ E. aff. parkeri with 89%), which was sister to Rhinatrema (Fig. 2), corroborating the results obtained by .
On the other hand, Microcaecilia nicefori was recovered as the sister taxon to a clade formed by the dermophiids Gymnophis multiplicata + Dermophis mexicanus and the remaining siphonopids, including Microcaecilia, Brasilotyphlus guarantanus, Siphonops, and Luetkenotyphlus. In addition, Microcaecilia and Siphonops were recovered as paraphyletic with respect to Brasilotyphlus guarantanus and Luetkenotyphlus brasiliensis, respectively (Fig. 2). Recently, Correia et al. (2018) also presented evidence that Microcaecilia is paraphyletic with respect to Brasilotyphlus. The placement of Luetkenotyphlus brasiliensis within Siphonops contrasts with results of Pyron and Wiens' (2011) and Maciel et al.'s (2019) analyses that found Luetkenotyphlus and Siphonops to be sister taxa. Although analyses by San Mauro et al. (2006), San Mauro et al. (2014) and Correia et al. (2018) also recovered Luetkenotyphlus and Siphonops as sister groups, these studies only included one species of Siphonops (S. annulatus). Therefore, additional molecular data are needed to clarify the delimitation of these clades. Consistent with previous findings (i.e., Correia et al. 2018), our phylogenetic analysis recovers Microcaecilia as non-monophyletic. Previously, based on evidence from dentition (relationship between VPs and rows of PM) and orbit (open versus closed orbit), Wilkinson et al. (2013) suggested that some Microcaecilia, including the type species of the genus (Dermophis albiceps Boulenger, 1882; not included herein), are more closely related to M. nicefori (Gymnophis nicefori Barbour, 1925, the type species of Parvicaecilia, currently in the synonymy of Microcaecilia; analyzed here for the first time) than to other species of Microcaecilia. That is, the position of trans-Andean Microcaecilia nicefori compared to other cis-Andean members of the genus suggests the revalidation of the genus Parvicaecilia. However, our analysis does not represent solid evidence due to several aspects, such as the low number of genes used, the low support values (a bootstrap value of only 45%), and the absence of key terminals, such as the type species of the Amazonian Microcaecilia (M. albiceps (Boulenger, 1882). Thus, inclusion of relevant taxa, such as M. albiceps, in future phylogenetic analyses is key to guiding taxonomic changes. At the interfamilial level, our results provide evidence for the first time that Shiphonopidae is paraphyletic with respect to Dermophiidae due to the placement of M. nicefori (Fig. 2). Additional, large scale phylogenetic studies are required to rigorously test this finding. Lynch (1999) suggested that Caecilia degenerata is restricted to the Cordillera Oriental of Colombia (Departments of Boyacá, Cundinamarca and Santander). However, morphological and biogeographical evidence suggests that the cis-and trans-Andean populations are not conspecific. The type series was collected at two cis-Andean localities: Garagoa (Boyacá Department), the type locality, and Choachí (Cundinamarca Department), ca. 90 km southwest of the type locality (Dunn 1942). Later, Ruiz-Carranza et al. (1996) and Lynch (1999) examined a series of trans-Andean specimens collected at Muzo (Boyacá Department), Tena and Sasaima (Cundinamarca Department), and Charalá (Santander Department), and referred them to C. degenerata, based on morphological similarity and (presumably) relative geographical proximity. Although the absence of secondary annular grooves, the number of primary annular grooves (127-138 in the cis-Andean populations vs. 123-137 in the trans-Andean populations), and the ratio of length/diameter (32-60 in the cis-Andean populations vs 48-58 in the trans-Andean populations; Ruiz-Carranza et al. 1996, Lynch 1999 are consistent with the hypothesis of conspecific populations. The cis-and trans-Andean populations are isolated by biogeographic barriers that includes high and steep mountains, xerophytic areas, and rainy environments, factors that usually play a fundamental role in the speciation of Andean amphibians (Lynch et al. 1997). To test the conspecificity of the populations of C. degenerata, a more extensive sampling of specimens, populations, and additional molecular data are required. Finally, although Taylor (1968) recorded specimens of C. degenerata in Tomaque (probably in Colombia or Peru) and Río Pache (probably in Peru), we agree with Lynch (1999) that C. degenerata is restricted to the (eastern) Cordillera Oriental of Colombia.

Conclusions
Caecilia pulchraserrana sp. nov. is described as an endemic species from the Serranía de los Yariguies. The species is similar to C. degenerata, from which it can be distinguished using morphological characters. According to their morphology, we hypothesize there is a group of closely related species that comprises C. caribea, C. corpulenta, C. degenerata, C. orientalis, and C. subdermalis. The trans-Andean Microcaecilia nicefori is an endemic and poorly known species from Colombia. We provide here the first analysis of molecular data that tests its phylogenetic position. Our results address the need to evaluate with more evidence the status of the genus Parvicaecilia Taylor, 1968 (cur-rently under the synonymy of Microcaecilia), and the potential non-monophyly of the family Siphonopidae. Further analyses sampling additional taxa and molecular markers are required to establish a more robust classification for Gymnophiona.