Systematic revision of the Eyelash Palm-Pitviper Bothriechis schlegelii (Serpentes, Viperidae), with the description of five new species and revalidation of three

We present a taxonomic review and systematic revision of the Eyelash Palm-Pitviper Bothriechis schlegelii (Berthold, 1846) based on examination of 400 museum specimens, a phylogeographic analysis of 818 locality records, and 80 individuals sampled for molecular characters. We find morphological and phylogenetic support for five new species of Bothriechis Peters, 1859, which we describe here based on their unique combination of molecular, meristic, hemipenial, and color pattern characteristics . They are: B. klebbai sp. nov. , B. rasikusumorum sp. nov. , B. khwargi sp. nov. , B. rahimi sp. nov. , and B. hussaini sp. nov. Finally, we revalidate the names B. nigroadspersus (Steindachner, 1870), B. nitidus (Günther, 1859), and B. torvus (Posada Arango, 1889a), and provide a redescription of B. schlegelii .


Introduction
The Eyelash Palm-Pitviper Trigonocephalus schlegelii Berthold, 1846 was described by Arnold Berthold in 1846 based on a juvenile specimen (ZFMK 32554) collected in Popayán, Colombia (Böhme 2010).The species was diagnosed primarily based on its enlarged supraciliary scales that resemble "eyelashes" and is now placed in the genus Bothriechis Peters, 1859.We do not review the history of generic reassignments in depth but refer to the synonyms in their original genus on first mention below.
Other eyelash viper species distinct from Berthold's Trigonocephalus schlegelii were described for Latin America in the period between 1859 and 1954.We refer to this group as the Bothriechis schlegelii species complex or the "eyelash clade" of the genus Bothriechis.The first of these was Lachesis nitidus Günther, 1859, described by Albert Günther based on a specimen (BMNH 1946.1.17.73) from the "western Andes of Ecuador," and differing from B. schlegelii primarily based on its green coloration with reddish dorsal stripes.The second was Bothrops nigroadspersus Steindachner, 1870, described by Franz Steindachner based on a specimen (NMW 18811) from "Central America," and differing from B. schlegelii primarily based on its bright yellow coloration and higher number of ventral scales and dorsal scale rows.The third was Thanatophis torvus Posada-Arango, 1889a, described by Andrés Posada Arango based on individuals (types not designated but reported as lost by Campbell and Lamar 2004) from the warm regions of Antioquia, and differing from B. schlegelii primarily based on the immaculate yellowish white ventral coloration.The fourth was Thanatophis colgadora García, 1896, described by Evaristo García based on individuals (specimens not designated) from the mountains of Cauca department, Colombia, and differing from B. schlegelii primarily based on its bluish-green dorsal coloration and larger body size.The fifth was named Teleuraspis birri, Posada Arango, 1909, a different name given to the reprinted description of Thanatophis torvus.The latter is therefore an objective junior synonym.
A sixth taxon, Bothrops schlegelii supraciliaris Taylor, 1954, previously considered conspecific or a subspecies of Bothriechis schlegelii, has since been revalidated and given full species status (Solórzano et al. 1998).Individuals of this species have been shown to be readily separated from Central American B. schlegelii based on scale counts and dorsal coloration (Solórzano et al. 1998).Using a sample of B. supraciliaris and one of B. schlegelii from Costa Rica, Castoe et al. (2009) presented evidence that both species were also genetically distinct.However, the inclusion of additional DNA sequences of B. schlegelii from Ecuador, Honduras, and Nicaragua by Daza et al. (2010) and Townsend et al. (2013) rendered B. schlegelii paraphyletic with respect to B. supraciliaris.
Bothriechis schlegelii is not monophyletic (Townsend et al. 2013) and is probably a species complex (Hurtado-Gómez 2009).In the words of Jay M Savage (cited in Campbell and Lamar 2004), "the great individual variation in coloration has precluded any attempt to fragment the species."To address this issue, we present a comprehensive taxonomic review and systematic revision of B. schlegelii based on 400 museum specimens, 818 locality records, and 80 individuals sampled for DNA-sequence data.We resolve the paraphyly of B. schlegelii, describe five new species, and revalidate the names B. nigroadspersus, B. nitidus, and B. torvus.Although additional diversity and potential taxonomy complexity exists in the group, to be examined by future researchers, the current proposal offers a robust and stable platform for additional study.

Preparation of hemipenial morphology
The hemipenes were removed and prepared from museum specimens using the procedures of Pesantes (1994) and Zaher (1999).Hemipenial terminology is based on Dowling and Savage (1960), Zaher (1999), and Myers and MacDowell (2014).

Sampling and laboratory techniques
Genomic DNA was extracted from 96% ethanol-preserved tissue samples (liver, muscle tissue, or scales) using either a guanidinium isothiocyanate extraction protocol (Peñafiel et al. 2020) or a modified salt precipitation method based on the Puregene DNA purification kit (Gentra Systems).The nucleotide sequences of the primers and the PCR conditions applied to each primer pair are detailed in Appendix 1. PCR products were cleaned with either ExoSAP-IT (Affymetrix, Cleveland, OH) or Exonuclease I and Alkaline Phosphatase (Illustra ExoProStar by GE Healthcare) before they were sent to Macrogen Inc (Seoul, South Korea) for Sanger sequencing.All PCR products were sequenced in both forward and reverse directions with the same primers that were used for amplification.The sequences for the holotypes NMW 18811 and ZMFK 32554 were obtained using ancient DNA methods as described in Agne et al. (2022).Raw sequence data underwent removal of adapters (1 bp overlap) and short reads (> 30 bp) using Cutadapt v2.8 (Martin 2011), and of PCR duplicates (-q 30) using Samtools v1.10 (Li et al. 2009).Mitochondrial genes were assembled using the iterative mapper MITObim v. 1.9.1 (Hahn et al. 2013) and default values except for a mismatch parameter of six.For the assembly, the following sequences were downloaded from GenBank as refe rences: 12S gene (MK313305.1),16S gene (AF057260.1),COI gene (MH140083.1),CYTB (AF039270.1),and ND4 gene (MK313435.1).We generated sequence data for samples marked with an asterisk in Suppl.material 2. The sequences were deposited in GenBank and the accession numbers are listed in Suppl.material 2. Interrictals are counted across the head between the last supralabials.We consider interoculolabials to be only the scales directly below the subocular and above the supralabials.We do not include perisupraoculars (scales bordering the supraoculars) in the count of canthals.Method for counting ventrals follows Dowling (1951).Illustrations by Valentina Nieto.

DNA phylogenetic analyses
A total of 279 DNA sequences were used to build a phylogenetic tree of the genus Bothriechis, of which 206 were generated during this work and 73 were downloaded from GenBank.Of these, 39 sequences are 398-408 bp long fragments of the 12S gene, 52 are 376-495 bp long fragments of the 16S gene, 20 are 493 bp long fragments of the COI gene, 78 are 143-712 bp long fragments of the CYTB gene, 71 are 378-675 bp long fragments of the ND4 gene, 9 are 185-522 bp long fragments of the NT3 gene, and 10 are 185-522 bp long fragments of the RAG1 gene.New sequences were edited and assembled using the program Geneious Pro TM 2021.1.1 (Drummond et al. 2021) and aligned with those downloaded from GenBank (Suppl.material 2) using MAFFT v.7 (Katoh and Standley 2013) under the default parameters.Gene fragments were concatenated into a single matrix with 20 partitions, one per non-coding gene and three per protein coding gene corresponding to each codon position.The best partition strategies along with the best-fit models of evolution were obtained in PartitionFinder 2.1.1 (Lanfear et al. 2016) under the Bayesian Information Criterion.
Phylogenetic relationships were assessed under a Bayesian inference (BI) approach in MrBayes 3.2.0(Ronquist and Huelsenbeck 2013).Four independent analyses were performed to reduce the chance of converging on a local optimum.Each analysis consisted of 20,000,000 generations and four Markov chains with default heating settings.Trees were sampled every 1,000 generations and 25% of them were arbitrarily discarded as ''burn-in."The resulting 15,000 trees saved per analysis were used to calculate the posterior probabilities (PP) for each bipartition in a 50% majority-rule consensus tree.We used Tracer 1.6 (Rambaut et al. 2022) to assess convergence and effective sample sizes (ESS) for all parameters.Additionally, we verified that the average standard deviation of split frequencies between chains and the potential scale reduction factor (PSRF) of all the estimated parameters approached values of ≤ 0.01 and 1, respectively.

Distribution maps and ecological niche models
We present ranges of occurrence for the ten species of Bothriechis of the "eyelash clade."Presence localities are derived from museum vouchers (Suppl.material 1), photographic records (iNaturalist), and the literature (all summarized in Suppl.material 3).For each species, a binary environmental niche model (ENM) is presented.These ENMs estimate potential areas of distribution based on observed presences and a set of environmental predictors (Elith and Leathwick 2009).To delimit the occupancy areas and the potential species distribution, we used the BAM diagram proposal (Soberón and Peterson 2005;Peterson et al. 2011).To create the models, we used presence localities listed in Suppl.material 3, 19 bioclimatic variables from Worldclim 1.4 (Hijmans et al. 2005), and Maxent 3.4.1k,an algorithm based on the principle of maximum entropy (Phillips et al. 2006;Elith et al. 2011;Renner and Warton 2013).
For the first explorative exercise, we used the 19 climate layers from the WorldClim project and assessed which variables were the most important for the model, according to the Jackknife test calculated in MaxEnt (Royle et al. 2012).Correlated environmental variables (r < 0.8) were identified using the PEARSON correlation test of PAST 3. In a second modelling exercise, we used the locality records for each species and the variables identified in the first approach to generate the species distribution.5,000 iterations were specified to the program with clamping and no extrapolation.All other parameters in MaxEnt were maintained at default settings.To create the binary environmental niche models, suitable areas were distinguished from unsuitable areas by setting a "minimum training presence" threshold value.The logistic format was used to obtain the values for habitat suitability (continuous probability from 0 to 1), which were subsequently converted to binary presence-absence values based on the established threshold value, defined herein as "the minimum training presence."The convergence threshold was set to 10 -5 , maximum iterations to 500, and the regularization parameter to "auto."

Rationale for definition of species-level candidate taxa
We here recognize species limits following an integration by congruence approach (Padial et al. 2010) based on the intersection of evidence from three or more independent taxonomic characters.We follow the general species concept of de Queiroz (2007) in defining species as independent evolutionary if two or more independent lines of evidence support their distinctness.We start the species delimitation procedure by seeking mitochondrial clades divergent from other mitochondrial clades by sequence divergences > 4% in a 700 bp fragment of the CYTB gene (Table 1), given that this degree of divergence has been found to correspond to species-level units in other Bothriechis, such as the species pairs B. lateralis-B.guifarroi (Townsend et al. 2013), with some exceptions explained below.We then tested if the individuals belonging to these mitochondrial lineages can be unambiguously diagnosed based on at least 4 of the 21 morphological characters summarized under Table 2.We considered in decreasing order of importance: (1) lepidosis; (2) hemipenial morphology; (3) coloration; and (4) size.We subsequently tested if males belonging to each species-level candidate taxa can easily be diagnosed on hemipenial morphology.We also evaluated if these mitochondrial lineages are congruent geographically and supported by the two nuclear genes studied.Thus, in our species delimitation approach, we consider groups of individuals as species if they (1) form a monophyletic group based on mtDNA; (2) differ from other such groups by > 4% sequence divergence (mean p-distance); (3) are diagnosable based on external characters of coloration and lepidosis; (4) are diagnosable based on hemipenial morphology; and (5) are congruent ecologically and biogeographically.

Molecular phylogeny
Selected partitions and models of evolution are presented in Table 3.We consider strong support for a clade when Bayesian analyses yield posterior probability values > 95%, following Felsenstein (2004).The topology and support (Fig. 2) of our phylogenetic tree is identical to that of Doan et al. (2016) and Mason et al. (2019) for all species of Bothriechis other than those within the B. schlegelii species complex.The most notable similarity is the relationship of the "eyelash vipers" as sister to the remaining members of the genus.Below, we comment on the topology of the "eyelash" clade.
At a continental scale, we found two strongly supported reciprocally monophyletic clades within the Bothriechis schlegelii species complex.The first is a Central American (CA; red in Fig. 3) clade and the second is a South American (SA) clade (Fig. 3).The CA clade includes B. supraciliaris and another species for which the name B. nigroadspersus is available (see account below).We sequenced the holotype of B. nigroadspersus (NMW 18811; Fig. 4).In our analysis, this specimen is deeply nested within this clade and is closely related to specimens from Costa Rica (Fig. 5).We also found two strongly supported subclades within B. nigroadspersus: one represents an almost entirely Panamanian population with one record in Cerro Tacarcuna, Colombia (COLZOOCH 581), and the other represents all other populations of B. nigroadspersus throughout Central America.
The SA clade includes eight species-level candidate taxa.The most early divergent of these is also the most geographically isolated: a new species endemic to Huila department, Colombia, at the junction between Cordillera Central and Cordillera Oriental (Dark blue in Fig. 3).This is the only species in the Bothriechis schlegelii complex known to occur along the Amazonian slopes of the Andes and is sister to all other SA Bothriechis species.
A second species occurs along the Chocó-Río Magdalena valley biome in Colombia (turquoise clade in Figs 2, 3).This species marginally enters Darién province in Panama, where it occurs in sympatry (see section on the distribution of Bothriechis nigroadspersus) with B. nigroadspersus along the Serranía de Pirré.Samples of this species, for which the name B. torvus is available, are sister to the remaining SA Bothriechis.We restrict the name B. torvus to this clade based on this species' type locality ("warm regions of Antioquia") and description of its coloration: belly yellowish white without spots.This description (Posada Arango 1889a) unambiguously rules out the other Antioquian species, B. schlegelii sensu stricto (see below for our updated definition), which occurs only in the highlands (Fig. 3) of the department and has a dark ventral coloration.
We restrict the name Bothriechis schlegelii to the magenta clade in Figs 2, 3, which includes samples from throughout Colombia's Cordillera Occidental and Cordillera Central, based on the inclusion of DNA sequences from the holotype (ZFMK 32554; Fig. 6) in this group.Consequently, B. schlegelii sensu stricto is primarily a highland species, endemic to Colombia, and sister to the remaining species in the SA clade exclusive of the species from Huila, Colombia and B. torvus.Its distribution only overlaps slightly with that of the previous species (turquoise clade), although we did not find areas of sympatry.The holotype of B. schlegelii is listed (Böhme 2010) as having been collected at Popayán, Cauca province, and our results agree with this view (see Discussion for additional detail on this locality).
A second new species occurs on the lowlands and mid-elevations of Colombia's Cordillera Oriental towards the Río Magdalena valley, in the northeast of the country (cyan clade in Fig. 3).The included samples are sister to the remaining SA species.This taxon occurs nearby, but is not sister to, a third new species that is distributed on the highlands of the western slopes of the Cordillera Oriental.The predicted distribution of both species overlaps (21-29%; darker shade of cyan in Fig. 3) and we found them to occur in sympatry at elevations above 1,700 m on the western slopes of the Serranía de los Yariguíes.This third new species (rose red clade) is sister to the remaining three SA Bothriechis, all of which occur south of the aforementioned ones, primarily in Ecuador.A fourth new species (orange clade; Figs 2, 3) is sister to the remaining southernmost members of the SA group.This species is known only from the Chocoan lowlands of extreme southwestern Colombia and adjacent northwestern Ecuador.We apply the name Bothriechis nitidus to a taxon endemic to west-central Ecuador (purple clade) based on three lines of evidence (see Discussion): the number of ventrals in the holotype, the travel logs of the collector Louis Fraser, and the illustration of the holotype by George Henry Ford (Fig. 7), which depicts a specimen that agrees in coloration with members of the purple clade, not those of the yellow clade.Finally, the yellow clade corresponds to the southernmost species, a fifth new species that inhabits dryer ecosystems in the transition area between the Chocó and the Tumbes biogeographic regions, in southwestern Ecuador and extreme northwestern Peru.

Systematic accounts
Species delimitation and the distinction between species-level and intraspecific variation is a complex topic (Carstens et al. 2013;Burbrink et al. 2022).In this instance, we name and provide descriptions only for species that are monophyletic in our molecular phylogeny and share diagnostic features of their coloration pattern, lepidosis, hemipenial morphology, and biogeography.Future studies might elucidate species boundaries based on explicit species-delimitation analyses.Based on these delimitation criteria, which follow the general lineage species concept of de Queiroz (2007), we describe five new species of Bothriechis and revalidate B. nigroadspersus, B. nitidus, and B. torvus.While additional genetic variation and potentially undescribed species may exist in the group and in the region, we are confident that these species represent ecologically, geographically, morphologically, and phylogenetically differentiated lineages that are equivalent in their evolutionary distinctiveness as compared to other recognized snake taxa.Consequently, we revise the B. schlegelii species complex to include: (Steindachner, 1870)   Figs 4, 5, 8 Bothrops nigroadspersus Steindachner, 1870: 348. Holotype NMW 18811 (Fig. 4), an adult female from Central America.Garman, 1884:  Comparisons.Bothriechis nigroadspersus is compared to other species of the genus previously subsumed under B. schlegelii sensu lato (differences summarized in Table 2).It differs from all of them by having a higher number of ventral scales in both males and females (although there is overlap with some species), two raised and spinelike supraciliary scales (vs low and granular or broad and triangular in the other species), and by lacking dorsal bands.Instead, most individuals of B. nigroadspersus have either opposing kidney shaped reddish dorsal marks o are of the golden morph (=oropel) (Fig. 5).Bothriechis nigroadspersus differs from B. supraciliaris by having a higher number of ventral scales, no broad blotches or dorsal bands, and a different pattern on the dorsal aspect of the snout.Bothriechis nigroadspersus differs from B. torvus by having opposing kidney-shape dorsal marks, a higher number of ventrals, loreal not in contact with preocular, and comparatively smaller spines on the hemipenial body (Fig. 8).
Hemipenial morphology.(n = 2, Fig. 8) Everted and inflated, the organ is deeply bilobed, unicalyculate and slightly capitate; hemipenial lobes thick and cylindrical; in sulcate and asulcate views, lobe crotch ornamented with scattered spinules; sulcus spermaticus centrolineal, bifurcate and with walls weakly defined, bifurcation occurs below bilobation point and proximal to the base of the hemipenial body; sulcus spermaticus branch runs to lobe tips; distal one third of each hemipenal lobe ornamented with calyces with spinulate edges.In sulcate view, hemipenial body surface nude, but with a pair of enlarged and strongly calcified lateral spines (basal hooks), one on each side; each hemipenial lobe ornamented with 1-3 mesial spines and 5-7 lateral spines, all about a third of the size of the basal hooks; the spines in each lobe are replaced distally by calyces with spinulate edges.In lateral view, hemipenial body nude with the exception of two basal spines; lobes also largely nude but with 5-6 smaller spines that are replaced distally by calyces.In asulcate view, the hemipenial body is nude with the exception of the pair of large lateral spines and also a pair of smaller mesial spines; hemipenial lobes largely nude but ornamented with smaller spines that decrease in size towards the lobe crotch.
Natural history.Bothriechis nigroadspersus is an arboreal snake that inhabits evergreen lowland/foothill forests, plantations, and rural gardens.In Panama (Sorrell 2007(Sorrell , 2009) ) and Costa Rica (Solórzano 2004) these vipers were found to be mostly active at night or at dusk and on the base of tree or on low shrubby vegetation, and Leenders (2019) and Witold Lapinski (pers.comm.to AA) reports that they have been found in the canopy at heights of 32-35 m in Costa Rica.Sorrell (2007Sorrell ( , 2009) ) observed that some snakes reside in the same perch for up to 14 days, 70.2% of individuals relocated each night, and only 6.4% remained at the same daytime perch site for more than two days.According to Rand and Myers (1990), Seifert (1983), Savage (2002), and our own observations, B. nigroadspersus is primarily nocturnal.During the day, most individuals of B. nigroadspersus remain in hunting posture on or close to their night perches, but others hide inside bromeliads, or occasionally remain active and move at ground level or on vegetation.Sorrell (2009) showed that members of this species are primarily ambush predators, but they also forage actively in search for food.In a study by Antonio (1980), captive juveniles fed mostly on frogs and attracted them by means of moving their bright yellow tails as a lure.Campbell and Lamar (2004), Sorrell (2009), Barrio-Amorós (2015), Morgan and Barrio-Amorós (2015), and Entiauspe-Neto et al. ( 2021) provide details on the dietary preference of adults encountered in the wild.These authors found that this age category also feeds on frogs (primarily treefrogs and rainfrogs), but also on lizards (anoles, whiptails, and geckos), birds (including hummingbirds), and mammals (bats, mice, and mouse opossums).We report on additional specimens found feeding on bats: CH 5651 from Coclé, Panama is preserved with an Artibeus jamaicensis  in its mouth.An uncollected specimen photographed by Philipp Hoenle in Chiapas, Mexico was feeding on an unidentified bat, as well as it was another uncollected individual found by Barrio-Amorós near Arenal, Costa Rica, on the ground on a dirt road feeding on a bat, with only one wing out of its mouth.Gerhardt et al. (1993), Laurencio (2005), and Chavarría and Barrio-Amorós (2014) provide accounts of predation on this viper by hawks (Buteogallus urubitinga and Herpetotheres cachinnans) and snakes (Clelia clelia).Solórzano (2004) suggests that in Costa Rica, breeding in Bothriechis nigroadspersus coincides with the rainy season.Blody (1983) observed that females become sexually mature at an age of less than three years and can produce more than one litter per year.Antonio (1980) described the courtship and copulatory behavior of captive B. nigroadspersus from Honduras and Gómez et al. (2015) recorded a case of a female from Costa Rica that produced a litter after presumably storing sperm for no less than ~35 months (slightly under three years).A specimen from Lago Yojoa, Honduras, kept at Centro El Ocotal, produced a litter of four eggs and one live young after being kept in a terrarium without a male for 18 years (Alejandro Velasquez pers.comm.to AB).Antonio (1980), Blody (1983), Gómez et al. (2015), and Murphy and Mitchell (1984) report a gestation period of 150-166 days (~5 months) and litters of 6-23 neonates that measure 16-22.5 cm in total length at birth.Campbell and Lamar (2004) report that captive B. nigroadspersus have lived up to 20 years.
Venom.In a series of 477 snakebite cases in Costa Rica in 1979, 18.9% were caused by Bothriechis nigroadspersus (Bolaños 1984).Mekbel and Céspedes (1963) report that four of a series of 27 autopsied snakebite cases in San José were caused by this species.Savage (2002) observed that between 90 and 100 bites by B. nigroadspersus are reported in Costa Rica in a typical year, with 3-6 resulting in deaths according to Seifert (1983).An average bite results in the injection of ~0.5 cc of venom.The venom is hemotoxic and strongly myonecrotic when compared to other Central American vipers (Gutiérrez and Chaves 1980).In humans, it causes intense localized pain, progressive hemorrhagic edema, and, in some cases, hemorrhagic blisters or hives, ecchymoses, and necrosis (Lomonte et al. 2008;Pla et al. 2017).Prezotto-Neto et al. ( 2016) studied the composition of the venom of specimens of B. nigroadspersus from Costa Rica and found that its properties differ drastically from specimens of Bothriechis torvus from Vegachi, Antioquia.When compared to the latter, the venom of B. nigroadspersus was found to be more edematous, hemorrhagic, and lethal.LD50 estimated as 1.7-5.6 mg/kg in B. nigroadspersus vs 9.24 mg/kg in B. torvus (Bolaños 1972;Gutiérrez and Chaves 1980;Lomonte et al. 2008Lomonte et al. , 2012;;Prezotto-Neto et al. 2016).
Distribution.Bothriechis nigroadspersus is known from at least 335 localities (listed in Suppl.material 3) throughout much of the Mesoamerican biome, from the isthmus of Tehuantepec in Mexico to extreme Etymology.The specific epithet nigroadspersus comes from the Latin words nigrum (meaning "black") and adspersus (meaning "sprinkled").It refers to the minute black specks scattered throughout the dorsum of the holotype (Fig. 4).
Conservation status.We consider Bothriechis nigroadspersus to be included in the Least Concern category following IUCN Red List criteria (IUCN 2012) because the species is widely distributed (but see Discussion), present in dozens of protected areas, tolerates moderate habitat degradation, and is presumably not declining fast enough to qualify in a threatened category.In a rainforest locality in Panama, the occurrence rates of B. nigroadspersus have increased by a factor of ten in the period from 2006 to 2012 (Zipkin et al. 2020).In another locality in Panama, the species was found to be extremely common in forest islands within a matrix of pastures (Sorrell 2007).However, it is unsure whether such "forest islands" will sustain the species without the presence of a dense population nearby that may act as a source of individuals that can immigrate to the fragmented habitat (Sorrell 2007).
Diagnosis.Bothriechis supraciliaris is diagnosed based on the following combination of characters: (1) two or three raised and spinelike supraciliary scales; (2) anterior dorsal head scales smooth or barely keeled; (3) gular scales much smaller than chinshields; (4) 7-24 interoculolabials; (5) 4-5 canthals, most of which have raised triangular projections; (6) loreal in contact with preocular; (7) oropel morph extremely rare and with noticeably dark dorsal blotches on a yellowish background, rather than uniform golden-yellow; (8) reddish-brown irregular oval blotches or bands on dorsum; (9) opposing kidney shaped dorsal marks absent; (10) black speckles on dorsal scales absent; (11) black speckling on ventral surfaces usually absent or faint; (12) ventral surfaces not entirely white but yellowish anteriorly and light bluish-green posteriorly; Comparisons.Bothriechis supraciliaris occurs near and is most similar to B. nigroadspersus.It can be identified from this other much more variable and widely distributed viper species primarily by having fewer ventral scales (Table 2), loreal in contact with preocular, anterior head scales smooth or barely keeled (vs keeled in B. nigroadspersus), and by having reddish dorsal blotches or bands (Fig. 10).Lotzkat (2014) accurately pointed out that the two species differ in the dorsal pattern of the snout in contrastingly marked specimens.In B. supraciliaris there is a single anteriormost dorsal blotch centrally on the snout (Figs 9a, 10b-e); in B. nigroadspersus, there are two paramedian blotches that may touch each other or be fused to form a short transverse band, but nevertheless remain discernible.Lotzkat (2014)  Hemipenial morphology.(n = 2, Fig. 11) Everted and inflated, the organ is deeply bilobed, calyculate and semicapitate; hemipenial lobes cylindrical and with minimal separation between them; in sulcate and asulcate views, lobe crotch ornamented with densely packed spinules; sulcus spermaticus centrolineal, bifurcate and with walls weakly defined, bifurcation occurs below bilobation point and proximal to the base of the hemipenial body; sulcus spermaticus branch runs to lobe tips; distal one third of each hemipenal lobe densely ornamented by calyces with strongly defined spinulate edges.In sulcate view, hemipenial body surface nude medially, but with enlarged and strongly calcified lateral spines followed by 1-2 rows of smaller obliquely-arranged mesial spines that become gradually smaller towards each lobe's capitulum; each hemipenial lobe ornamented with 1-2 mesial spines and 3-4 lateral spines, all about half the size of the larger lateral spines at the base of the hemipenial body; the spines in each lobe are replaced distally by calyces with strongly spinulate edges.In lateral view, hemipenial body and lobes with rows of spines, but otherwise smooth; distal 1/4 strongly calyculate.In asulcate view, the center of hemipenial body is nude except for a pair of large lateral spines; hemipenial lobes ornamented with 3-4 rows of smaller spines that decrease in size towards the lobe crotch.
Natural history.Bothriechis supraciliaris is an arboreal snake that inhabits evergreen lower-montane forests, cloud forests, clearings with coffee and banana plantations, edges of farm fields, and rural gardens (Campbell and Lamar 2004;Lotzkat 2014;Leenders 2019;Solórzano et al. 2021).Solórzano et al. (1998) and   Solórzano (2004) report that individuals of B. supraciliaris are crepuscular, nocturnal, and tend to spend more time on the ground than other Bothriechis: they found 22 out of 25 individuals on the forest floor.However, all individuals seen by CLBA (n=10) were on bushes from 30 cm to 2 m above the ground.Another naturalist, Norberto Solano, with more than 80 sighting of the species, only found one on the ground (Norberto Solano, pers.comm.to CLBA).During the day, individuals have been found resting on the ground or at the base of trees and shrubs (Solórzano 2004).Lotzkat (2014) and Batista et al. (2020) found individuals on low understory vegetation 0.5-2 m above the ground, either in ambush posture, crawling, or hanging from the tail.These authors also report that SMF 89764 was in the process of swallowing a comparatively large Craugastor fitzingeri.Solórzano et al. (1998) found mostly small forest-floor rodents in the stomach contents of four dissected specimens.They also report that specimens found in resting positions were curled in a loose circle and mostly on horizontal, wide surfaces.
Venom.Lomonte et al. (2012) studied the venom of this species in Costa Rica and found that it has a potent hemorrhagic action, moderate myotoxicity, and very weak procoagulant activity.These authors estimated its lethal activity at LD50 6.04 mg/kg and found that the equine polyvalent (Viperidae) antivenom from Instituto Clodomiro Picado was able to neutralize the lethal effect of B. supraciliaris venom.The antivenom tested was produced from the plasma of equines immunized with a mixture of the venoms of Bothrops asper, Crotalus simus, and Lachesis stenophrys (Lomonte et al. 2012).Distribution.Bothriechis supraciliaris is known from at least 24 localities (listed in Suppl.material 3) along the Pacific slopes of the Cordillera de Talamanca in southwestern Costa Rica and western Panama.The species occurs over an area of approximately 3,568 km 2 and has been recorded at elevations 734-1,506 m above sea level (Fig. 3).We found that 100% of the predicted area of distribution of B. supraciliaris is included in the predicted area of distribution of B. nigroadspersus.
Etymology.The specific epithet supraciliaris, which comes from the Latin words supra (meaning "above") and cilium (meaning "eyelash"), refers to the prominent spinelike scales above the eye.
Conservation status.We consider Bothriechis supraciliaris to be included in the Endangered category (instead of Least Concern; see Solórzano et al. 2021) following the IUCN criteria B1a, b (i, iii, iv) (IUCN 2012), because the species' extent of occurrence is estimated to be less than 5,000 km 2 (Fig. 3) and its habitat is severely fragmented and declining in extent and quality due to deforestation.Although B. supraciliaris occurs in three protected areas (Estación Biológica Las Cruces, Las Nubes Biological Reserve, and Parque Binacional La Amistad), the remaining localities where the species has been recorded (Suppl.material 3) are in forest patches surrounded by a matrix of pastures and small settlements.Based on the species distribution model presented in Fig. 3 in combination with maps of vegetation cover of Central America (CATHALAC 2011), we estimate that nearly 37% of the forest cover throughout the species' potential distribution area has been destroyed, mostly due to the expansion of the agricultural frontier.
Diagnosis.Bothriechis schlegelii is diagnosed based on the following combination of characters: (1) two or three triangular and moderately raised supraciliary scales; (2) anterior dorsal head scales smooth; (3) gular scales much smaller than or similar in size to chinshields; (4) 3-11 interoculolabials; (5) 1-3 canthals which may be flat or slightly raised forming a ridge along the canthus; (6) loreal in contact with preocular in ~1/3 of specimens; (7)  Comparisons.Bothriechis schlegelii sensu stricto is compared to other species of the genus previously subsumed under B. schlegelii sensu lato (differences summarized in Table 2).It differs from the other species except B. klebbai sp.nov.and B. khwargi sp.nov. in having two triangular and moderately raised supraciliary scales (supraciliaries either low and granular or enlarged and spinelike in the other species).Bothriechis schlegelii occurs near the distribution of B. torvus (Fig. 3), from which it differs by having anterior dorsal head scales smooth, 1-3 canthals (rather than 3-8), bands black or dark brown (rather than pink or red), ventral surfaces never entirely white, a lower number of ventrals in both males and females, and larger basal hooks on the hemipenial body (Fig. 14).Bothriechis schlegelii is most similar to B. klebbai sp.nov.and B. rasikusumorum sp.nov., but these other species occur parapatrically and are characterized by having small supraciliary scales, dorsal and ventral surfaces heavily speckled with black, gular scales similar in size or larger than chinshields, and no enlarged basal hooks on the hemipenial body.Bothriechis schlegelii differs from B. khwargi sp.nov.by having smooth anterior dorsal head scales (instead of keeled), a lower number of canthals, no reddish bands, and an entirely pale white belly.
Hemipenial morphology.(n = 7; three depicted in Fig. 14) Everted and inflated, the organ is deeply bilobed, calyculate and noncapitate; hemipenial lobes uniformly cylindrical or tapering towards the tip; in sulcate and asulcate views, lobe crotch ornamented with densely packed spines that become larger distally; sulcus spermaticus centrolineal, bifurcate and with walls strongly defined (weakly in IAVH-R 5465 and ICN PR 13225), bifurcation occurs below bilobation point and proximal to the base of the hemipenial body; sulcus spermaticus branch runs to lobe tips; distal one third to one half of each hemipenal lobe densely ornamented with small calyces with strongly defined spinulate edges.In sulcate view, hemipenial body surface with small spinules medially, but with enlarged and strongly calcified lateral spines (basal hooks) followed by 2-4 rows of much smaller obliquely-arranged mesial spines that become gradually smaller towards each lobe's capitulum; each hemipenial lobe ornamented with mesial and lateral spines about 1/3-1/10 the size of the large basal hooks; the spines in each lobe are replaced distally by calyces with strongly spinulate edges.In lateral view, hemipenial body and lobes with rows of spines, but otherwise non-spiculate; distal 1/3-1/2 of lobes strongly calyculate.In asulcate view, the center of hemipenial body is nude to barely spiculate; hemipenial lobes ornamented with 3 rows of smaller spines that decrease in size towards the lobe crotch.
Natural history.Bothriechis schlegelii is an arboreal snake that inhabits evergreen montane forests, planted forests, coffee plantations, and urban/rural gardens.Based on our own field notes, iNaturalist records, and the literature (Rojas-Morales 2012; Galofre-Ruiz 2016), vipers of this species are found at night perched on low (less than 3 m above the ground) shrubby vegetation or at ground level during the day, either in ambush posture or moving.Most observations are of snakes active on rocks, leaf-litter, grass, and even crossing roads during daylight hours.Santiago Ayerbe (pers.comm.to AA) reports that in Cauca, individuals of this species prey upon lizards (anolines and Pholidobolus), rodents, and hummingbirds.Rojas-Morales (2012) reports that "though generally docile, individuals of B. schlegelii defend themselves with frontal displays and striking." Venom.Galofre-Ruiz (2016) reports two cases of human envenomation by this snake species in departments Caldas and Tolima.The victims developed pain at the site of the wound, paresthesia, and edema, but recovered with the use of polyvalent antivenom.Sevilla-Sánchez et al. ( 2021) report that most accidents caused by this snake involve coffee plantation workers bitten on the hands or face during the harvesting work.In a series of 1,653 cases of envenomation in Cauca province between 2009 and 2018, 8.4% were caused by B. schlegelii (Sevilla-Sánchez et al. 2021), whereas 5.5% of 218 cases in departments Antioquia and Chocó between 1989 and 1990 were caused by a combination of B. schlegelii and B. torvus (Otero et al. 1992a).Of these, one envenomation was moderate, 11 were mild, and none involved systemic complications.The venom in this Antioquian-Chocoan mixed Bothriechis sample was found to be the least edematous, hemorrhagic, and lethal (LD50 estimated as 10.3 mg/kg) among a sample of eight Colombian venomous snakes (Otero et al. 1992b).Ayerbe et al. (1979) provided a report of a coffee plantation worker that died after being bitten on the tongue.
Distribution.Bothriechis schlegelii is endemic to Colombia.It is known from at least 161 localities (listed in Suppl.material 3) on Colombia's Cordillera Central and Occidental, from Cauca department in the south to Antioquia department in the north.The species occurs over an area of approximately 40,432 km 2 and has been recorded at elevations 1,034-2,597 m above sea level (Fig. 3).Approximately 8.8% of the predicted area of distribution of B. schlegelii overlaps with that of B. torvus, but we did not find evidence of sympatry.In Valle del Cauca department, we found the two species separated by an airline distance of about 10 km.
Conservation status.We consider Bothriechis schlegelii to be included in the Near Threatened category following IUCN Red List criteria (IUCN 2012) primarily because the species' extent of occurrence is estimated to be much larger than the 20,000 km 2 needed to meet B1 criteria for the Vulnerable category.However, although the species occurs in numerous protected areas (no less than 13; see Suppl.material 2), the majority of the species' montane forest habitat has been destroyed.Based on the distribution model presented in Fig. 3 in combination with maps of vegetation cover of Colombia (IDEAM 2014), we estimate that only ~23% of the species' forest habitat is still standing.Comparisons.Bothriechis torvus is compared to other species of the genus previously subsumed under B. schlegelii sensu lato (differences summarized in Table 2).It differs from all of them by having three raised and triangular supraciliary scales, anterior dorsal head scales keeled, a high number of canthals (3-8) and interoculolabials (6-18), thin pink dorsal bands, and entirely pale white belly.Bothriechis torvus differs from B. schlegelii sensu stricto by having anterior dorsal head scales keeled, 3-8 canthals (rather than 1-3), bands pink or red (rather than black or dark brown; Fig. 15), ventral surfaces entirely white, a higher number of ventrals in both males and females, smaller body size, and smaller basal hooks on the hemipenial body (Fig. 16).Bothriechis torvus is sympatric with B. nigroadspersus along the Serranía de Pirre, Darién province, Panama (lime green shade in Fig. 3).The two species differ in coloration (Figs 5,15).Bothriechis torvus has dorsal bands (vs opposing kidney shaped marks), three broad and triangular enlarged supraciliary scales (vs two thin and spinelike), a lower number of ventral scales, and lateral surface of the hemipenial body and lobes more heavily packed with larger spines (Fig. 16).Bothriechis torvus resembles B. rahimi sp.nov., but this other species occurs south of the known distribution of B. torvus and is characterized by having a lower number of ventrals, enlarged spinelike supraciliary scales, faint dorsal banding, and presence of the yellow morph.Bothriechis torvus differs from B. khwargi sp.
Hemipenial morphology.(n = 1; Fig. 16) Everted and inflated, the organ is deeply bilobed, calyculate and noncapitate; hemipenial lobes cylindrical, but tapering towards the capitulum; in sulcate and asulcate views, lobe crotch ornamented with densely packed spines that become larger distally; sulcus spermaticus centrolineal, bifurcate and with walls strongly defined, bifurcation occurs below bilobation point and proximal to the base of the hemipenial body; sulcus spermaticus branch runs to lobe tips; distal half of each hemipenal lobe densely ornamented with small calyces with defined spinulate edges.In sulcate view, hemipenial body surface spinulate medially with enlarged and strongly calcified lateral basal hooks followed by rows of smaller obliquely arranged mesial spines that become gradually smaller towards each lobe's capitulum; each hemipenial lobe ornamented with mesial and lateral spines about 1/5 the size of the large basal hooks; the spines in each lobe are replaced distally by calyces with strongly spinulate edges.In lateral view, hemipenial body and lobes non-spiculate but with rows of spines; distal half of lobes strongly calyculate.In asulcate view, the center of hemipenial body is nude to barely spiculate; hemipenial lobes ornamented with 2 rows of smaller spines (about 1/3 the size of the lateral basal hooks) that decrease in size towards the lobe crotch.
Natural history.Bothriechis torvus is an arboreal snake that inhabits evergreen lowland/foothill forests.We have found vipers of this species active at night crawling on ferns, tree branches, and trunks from 1.8 to 5 m above the ground or during the day perched on arboreal vegetation up to 3.5 m above the ground.Specimens from Cerro Sapo and Pirre Mountain ranges, eastern Panama, were found in a cloud forest consisting predominantly of trees covered with moss and a large variety of understory and midstory bromeliads.In captivity, TH 145 (Fig. 15c) and another specimen from Morromico, Chocó department, Colombia, consumed lizards (Anolis and Lepidodactylus) and frogs (Craugastor and Pristimantis).
Venom.Prezotto-Neto et al. ( 2016) studied the composition of the venom of specimens of Bothriechis torvus from Vegachi, Antioquia, and found that its properties differ drastically from specimens of B. nigroadspersus from Costa Rica.When compared to the latter, the venom of B. torvus was found to be less edematous, hemorrhagic, and lethal (LD50 estimated as 9.24 mg/kg vs 5.60 mg/kg).
Distribution.Bothriechis torvus is known from at least 37 localities (listed in Suppl.material 3) along the Chocó and Río Magdalena valley regions of northern Colombia and extreme southeastern Panama.The species occurs over an area of approximately 61,466 km 2 and has been recorded at elevations 61-1,413 m above sea level (Fig. 3).The ENM suggests the distribution is not continuous across the valley of the Río Atrato (see Discussion).
Etymology.The specific epithet torvus is a Latin word meaning "fierce."It refers to the protruding supraciliary scales, which according to Andrés Posada Arango, give this species an "air féroce." Conservation status.We consider Bothriechis torvus to be included in the Least Concern category following IUCN Red List criteria (IUCN 2012) because the species is distributed over a region that holds large areas of continuous unspoiled forest.Based on the species distribution model presented in Fig. 3 in combination with maps of vegetation cover of Colombia (IDEAM 2014) and Panama (CATH-ALAC 2011), we estimate that more than half (~59%) of the species' forest habitat is still standing.Unfortunately, vast areas of the Chocó rainforest in northern Colombia and towards central Panama have already been converted to pastures (Myers et al. 2000).However, B. torvus occurs over an area greater than 50,000 km 2 and is presumably not declining fast enough to qualify for a threatened category.Paratypes: All labeled Bothriechis khwargi sp.nov. in Suppl.material 1.
Diagnosis.Bothriechis khwargi sp.nov. is diagnosed based on the following combination of characters: (1) two triangular and moderately raised supraciliary scales; (2) anterior dorsal head scales keeled; (3) gular scales much smaller than chinshields; (4) 7-14 interoculolabials; (5) 3-4 canthals, some of which with raised triangular projections; (6) loreal in contact with preocular in ~1/3 of specimens; (7) yellow morph absent; (8) dorsal bands absent, faint, or restricted to top of dorsum in  Comparisons.Bothriechis khwargi sp.nov. is compared to other species of the genus previously subsumed under B. schlegelii sensu lato (differences summarized in Table 2).It differs from all of them by having two triangular and moderately raised supraciliary scales, anterior head scales keeled, gular scales much smaller than chinshields, entirely pale white belly in most individuals, and dorsal bands faint or absent.Bothriechis khwargi sp.nov.resembles B. torvus, but this other species does not occur along the slopes of the Cordillera Oriental and is characterized by having thin pink bands on the dorsum (Fig. 15).Bothriechis khwargi sp.nov.occurs in sympatry with B. klebbai sp.nov.along the upper slopes of the Serranía De Los Yariguíes, but it differs from this other species by being smaller in body size, having an entirely white belly, pale greenish iris, keeled anterior dorsal head scales, gulars much smaller than chinshields, and by lacking black speckling on dorsal and ventral surfaces (Fig. 18).
Natural history.Bothriechis khwargi sp.nov. is an arboreal snake that inhabits evergreen foothill forests.Vipers of this species have been seen at night or during the day, either at ground level or on low understory vegetation.EMP and CLBA found one individual at 1 m above the ground in a fern foraging at night.
Venom.We know of no snakebites caused by this species in Colombia.
Distribution.Bothriechis khwargi sp.nov. is known from at least 19 localities (listed in Suppl.material 3) along the western foothills of Colombia's Cordillera Oriental.The species occurs over an area of approximately 14,697 km 2 and has been recorded at elevations 167-1,800 m above sea level (Fig. 3).Approximately 29% of the predicted area of distribution of B. khwargi sp.nov.overlaps with that of B. klebbai sp.nov., and we found evidence of sympatry between the two species at elevations above 1,700 m on the western slopes of the Serranía de los Yariguíes.UIS-R 4447 is a B. klebbai sp.nov.and was found just 3.7 km airline distance from a photographic record of an B. khwargi sp.nov.
Etymology.This species is named in honor of Dr. Juewon Khwarg, in recognition of his support of the discovery and protection of new species of vipers in the Andes of Colombia.Khwarg is a conservationist with a special love for the lesser-known reptiles of the unexplored regions of the tropics.
Conservation status.We consider Bothriechis khwargi sp.nov. to be included in the Vulnerable category following the IUCN criteria B1a, b (i, iii, iv) (IUCN 2012), because the species' extent of occurrence is estimated to be less than 20,000 km 2 (Fig. 3) and its habitat is severely fragmented and declining in extent and quality due to deforestation.Although B. khwargi sp.nov.occurs in one national park (Serranía de los Yariguíes) and a privately protected area (Reserva Natural Reinita Cielo Azul), most localities where the species has been recorded (Suppl.material 3) are in heavily human-modified areas.Based on the species distribution model presented in Fig. 3 in combination with maps of vegetation cover of Colombia (IDEAM 2014), we estimate that nearly 79% of the forest cover throughout the species' potential distribution area has been destroyed, mostly due to the expansion of the agricultural frontier.Type material.Holotype: ICN 2786 (Fig. 19), adult male collected by Pedro M. Ruiz-Carranza on September 21, 1979 at Vereda Virolín, municipio de Charalá, Santander department, Colombia (6.10720, -73.19781;1819 m).
Diagnosis.Bothriechis klebbai sp.nov. is diagnosed based on the following combination of characters: (1) supraciliary scales low and granular or two raised, but not sharp; (2) anterior dorsal head scales smooth; Comparisons.Bothriechis klebbai sp.nov. is compared to other species of the genus previously subsumed under B. schlegelii sensu lato (differences summarized in Table 2).It differs from most of them by having supraciliaries low and granular or two raised but not sharp, a low number of canthals (2-4), gular scales similar in size to chinshields, and dorsal and ventral surfaces heavily speckled with black pigment.Bothriechis klebbai sp.nov.resembles B. rasikusumorum sp.nov., but this other species occurs on the southern portion of Colombia's Cordillera Oriental and is characterized by having more than 151 ventrals in females, loreal not in contact with preocular, and by lacking paired oblong blotches on each side of the dorsum (a characteristic variably present in B. klebbai sp.nov.).Furthermore, Bothriechis klebbai sp.nov.lacks enlarged basal hooks on the hemipenial body (present in B. rasikusumorum sp.nov.).Bothriechis klebbai sp.nov.also resembles B. schlegelii, but this other species does not occur on the Cordillera Oriental and is characterized by having two triangular and moderately raised supraciliary scales, a different iris color (pale orange, light green, or golden yellow usually without speckles vs rich dark reddish brown), and by lacking black speckles on the dorsal and ventral scales in most populations (Figs 19,20).Bothriechis klebbai sp.nov.occurs in sympatry with B. khwargi sp.nov.along the upper slopes of the Serranía De Los Yariguíes, but it differs from this other species by being larger in body size, having a dark green belly, smooth anterior dorsal head scales, gulars similar in size to or large than chinshields, and by having prominent black speckling on dorsal and ventral surfaces.
Hemipenial morphology.(n = 3; Fig. 21) Everted and inflated, the organ is deeply bilobed, calyculate and noncapitate; hemipenial lobes cylindrical or spindle shaped; in sulcate and asulcate views, lobe crotch ornamented with densely packed small spines that become larger distally; sulcus spermaticus centrolineal, bifurcate and with walls weakly defined (strongly in ICN 2786), bifurcation occurs below bilobation point and proximal to the base of the hemipenial body; sulcus spermaticus branch runs to lobe tips; distal half of each hemipenal lobe densely ornamented with small calyces with strongly defined spinulate edges.In sulcate view, hemipenial body surface spinulate medially; calcified spines at the base of the hemipenial body only slightly larger than adjacent rows of smaller obliquely arranged mesial spines that become gradually smaller towards each lobe's capitulum   (=no obvious basal hooks); each hemipenial lobe ornamented with mesial and lateral spines homogenous in size; the spines in each lobe are replaced distally by calyces with strongly spinulate edges.In lateral view, hemipenial body and lobes with rows of spines, but otherwise non-spiculate; distal half strongly calyculate.In asulcate view, the center of hemipenial body is nude to barely spiculate; hemipenial lobes ornamented with 4 rows of spines that decrease in size towards the lobe crotch.
Natural history.Bothriechis klebbai sp.nov. is an arboreal snake that inhabits montane cloud forests.Vipers of this species have been seen at night or during the day, either at ground level or on low understory vegetation.In captivity, this species is known to hybridize with B. khwargi sp.nov.INSV-SR-1015 is believed to be a hybrid between the two species.We (JV and EMP) have seen this species preying upon Anolis heterodermus.
Venom.We know of no snakebites caused by this species in Colombia nor any studies on the venom that could be assigned to this species.
Distribution.Bothriechis klebbai sp.nov. is known from at least 23 localities (listed in Suppl.material 3) along the western slopes of Colombia's Cordillera Oriental.The species occurs over an estimated 20,061 km 2 and has been recorded at elevations 1,418-2,380 m above sea level (Fig. 3).Approximately 21% of the predicted area of distribution of B. klebbai sp.nov.overlaps with that of B. khwargi sp.nov.
Etymology.Named after Casey Klebba, whose dedication to the preservation of tropical biodiversity is exemplary.As a co-founder of Minifund.org,alongside Carly Jones, he has been one of the most invaluable supporters of AA's field expeditions to remote areas of Ecuador and Colombia, resulting in the discovery of many new species to science.
Conservation status.We consider Bothriechis klebbai sp.nov. to be included in the Near Threatened category following IUCN Red List criteria (IUCN 2012) primarily because the species' extent of occurrence is estimated to be greater than the 20,000 km 2 needed to meet B1 criteria for the Vulnerable category.However, although the species occurs in at least two national parks (Serranía de los Yariguíes and Guanentá-alto Río Fonce), the majority of the species' montane forest habitat has been destroyed.Based on the distribution model presented in Fig. 3 in combination with maps of vegetation cover of Colombia (IDEAM 2014), we estimate that only ~14% of the species' forest habitat is still standing.
Proposed standard Spanish name.Víbora de pestañas de los Shah.
Local Spanish name.Culebra de tiro.
Diagnosis.Bothriechis rasikusumorum sp.nov. is diagnosed based on the following combination of characters: (1) supraciliary scales low and granular, or two pointy but not raised; (2) anterior dorsal head scales smooth; (3) gular scales similar in size to chinshields; (4) 5-10 interoculolabials; (5) 2-3 canthals which may be flat or slightly raised forming a ridge along the canthus; (6) loreal not in contact with preocular; (7)  Comparisons.Bothriechis rasikusumorum sp.nov. is compared to other species of the genus previously subsumed under B. schlegelii sensu lato (differences summarized in Table 2).It differs from most of them by having supraciliaries not raised, a low number of canthals (2-3), a low number of ventral scales, and dorsal and ventral surfaces heavily speckled with black pigment (Figs 22,23a).Bothriechis rasikusumorum sp.nov.resembles B. klebbai sp.nov., but this other species occurs on the northern portion of Colombia's Cordillera Oriental and is characterized by having less than 152 ventrals in females, a rich reddish-brown iris, a series of paired oblong blotches on each side of the dorsum, and no basal hooks on the hemipenial body (Fig. 21).Bothriechis rasikusumorum sp.nov. is also similar to B. schlegelii, but this other species occurs northwest of the known distribution of B. rasikusumorum sp.nov.and is characterized by lacking black speckles on the dorsal and ventral scales in most populations (Figs 12,13), having two triangular and moderately raised supraciliary scales, and basal hooks that are 3-10X the size of adjacent spines at the base of the hemipenial body (Fig. 14).
Hemipenial morphology.(n = 1; Fig. 24) Everted and inflated, the organ is deeply bilobed, calyculate and noncapitate; hemipenial lobes cylindrical, narrower towards the capitulum; in sulcate and asulcate views, lobe crotch ornamented with densely packed small spines that become larger distally; sulcus spermaticus centrolineal, bifurcate and with walls strongly defined, bifurcation occurs below bilobation point and proximal to the base of the hemipenial body; sulcus spermaticus branch runs to lobe tips; distal half of each hemipenal lobe densely ornamented with small calyces with strongly defined spinulate edges.In sulcate view, hemipenial body surface with small spinules medially, but with a pair of enlarged and strongly calcified lateral spines (basal hooks) followed by 4-5 rows of smaller obliquely-arranged mesial spines that become gradually smaller towards each lobe's capitulum; each hemipenial lobe ornamented with mesial and lateral spines about half the size of the basal hook; the spines in each lobe are replaced distally by calyces with strongly spinulate edges.In lateral view, hemipenial body and lobes with rows of spines, but otherwise non-spiculate; distal half of lobes strongly calyculate.In asulcate view, the center of hemipenial body covered with scattered spinules; hemipenial lobes ornamented with 3 rows of spines.
Venom.We know of no snakebites caused by this species in Colombia nor any studies on the venom that could be assigned to this species.
Distribution.Bothriechis rasikusumorum sp.nov. is known from at least 26 localities (listed in Suppl.material 3) along the upper watershed of the Río Magdalena in Huila department, Colombia.The species occurs over an area of approximately 6,578 km 2 along the eastern slope of Cordillera Central and on both slopes of Cordillera Oriental at elevations 1,298-2,180 m above sea level (Fig. 3).
Etymology.The specific epithet rasikusumorum is a patronym honoring Rasik Shah (1939Shah ( -2022)), Kusum Shah (1942-present), and their grandson Oscar Shah.The Shah family helped fund the expedition that led to the discovery of this new species.
Conservation status.We consider Bothriechis rasikusumorum sp.nov. to be included in the Vulnerable category following the IUCN criteria B1a, b (i, iii, iv) (IUCN 2012), because the species' extent of occurrence is estimated to be much less than 20,000 km 2 (Fig. 3) and its habitat is severely fragmented and declining in extent and quality due to deforestation.Although B. rasikusumorum sp.nov.occurs in three protected areas (Parque Nacional Cueva de los Guácharos, Bosque Protector Pompeya, and Reverva Chiyurco), most localities where the species has been recorded (Suppl.material 1) are in forest patches surrounded by a matrix of pastures and plantations.Based on the species distribution model presented in Fig. 3 in combination with maps of vegetation cover of Colombia (IDEAM 2014), we estimate that nearly 71% of the forest cover throughout the species' potential distribution area has been destroyed, mostly due to the expansion of the agricultural frontier.One of us (JV) interviewed coffee plantation workers in Huila, who report that they routinely kill these vipers when found.During a 10-night expedition to Huila department, a group of three field biologists led by JV failed to find a single specimen in four of the localities where the species has been reported nearby the city Pitalito.Type material.Holotype: ZSFQ 5055 (18), adult female collected on January 1, 2017 at Tundaloma Lodge, Esmeraldas province, Ecuador (1.18236, -78.7525;74 m).
Proposed standard Spanish name.Víbora de pestañas de Rahim.
Diagnosis.Bothriechis rahimi sp.nov. is diagnosed based on the following combination of characters: (1) two or three raised triangular or spinelike supraciliary scales; (2) anterior dorsal head scales keeled; (3) gular scales much smaller than chinshields; (4) 8-13 interoculolabials; (5) 4-5 canthals, some raised slightly forming a ridge along the canthus; ( 6 Comparisons.Bothriechis rahimi sp.nov. is compared to other species of the genus previously subsumed under B. schlegelii sensu lato (differences summarized in Table 2).It differs from all of them by having supraciliaries raised, anterior dorsal head scales keeled, 8-13 interoculolabials, faint pink dorsal bands, and low number of ventrals in both males and females.It is one of the two members of the SA Bothriechis group where the golden morph has been recorded (Fig. 26c), albeit it is not identical to the B. nigroadspersus golden morph (Fig. 5a-c), as it has faint pink bands.Bothriechis rahimi sp.nov.resembles B. torvus and B. nitidus.From the former, the new species differs by being smaller in body size, having less than 148 ventrals in males, supraciliaries spinelike (vs broad and triangular), and presence of yellow morph and red morph.From B. nitidus, the new species differs by being smaller in body size, having raised supraciliaries (vs low), a higher number of canthals (4-5 vs 2-3) and interoculolabials (8-13 vs 3-8), scales on the anterior dorsal surface of the head keeled (vs smooth), presence of a yellow morph, and lack of a green morph (all B. nitidus examined are green; Suppl.material 1).
Natural history.Bothriechis rahimi sp.nov. is an arboreal snake that inhabits evergreen lowland forests usually within 25 km from the coastline.We have found vipers of this species active at night perched on stems, branches, and tangled vegetation 0.4-8 m above the ground.One individual was perched in hunting posture on a heliconia stem facing the flowers of the plant, presumably on the wait for hummingbirds.One individual was found feeding on a treefrog (Smilisca phaeota) and another one regurgitated an unidentified Pristimantis species, probably P. esmeraldas (field observations by Javier Aznar and JHV).
Venom.We know of only one snakebite cause by this species in Ecuador.A photographer was bitten on the right index finger by MZUTI 3332.The victim experienced intense local pain and swelling but recovered shortly after receiving three doses of polyvalent antivenom (elaborated by Instituto Clodomiro Picado).
Distribution.Bothriechis rahimi sp.nov. is known from at least 12 localities (listed in Suppl.material 3) along the mouths of the rivers Esmeraldas, Santiago, Cayapas, and Mira in extreme northwestern Ecuador and southwestern Colombia.The species occurs over an area of approximately 6,003 km 2 of the Chocó biome and has been recorded at elevations of 11-200 m above sea level (Fig. 3).Approximately 7.6% of the predicted area of distribution of B. rahimi sp.nov.overlaps with that of B. nitidus, but we did not find evidence of sympatry between the two species.
Etymology.The specific epithet rahimi is a patronym honoring Prince Rahim Aga Khan, a firm environmentalist who has inspired many with his work focused on tackling climate change, primarily in countries where the Aga Khan Development Network is active, alongside his brother Prince Hussain Aga Khan.
Conservation status.We consider Bothriechis rahimi sp.nov. to be included in the Vulnerable category following the IUCN criteria B1a, b (i, iii, iv) (IUCN 2012), because the species' extent of occurrence is estimated to be much less than 20,000 km 2 (Fig. 3) and its habitat is severely fragmented and declining in extent and quality due to deforestation.Although B. rahimi sp.nov.occurs in two protected areas (Reserva Awá and Refugio de Vida Silvestre La Chiquita), the remaining ten localities where the species has been recorded (Suppl.material 3) are in heavily human-modified areas.Based on the species distribution model presented in Fig. 3 in combination with maps of vegetation cover of Colombia (IDEAM 2014) and Ecuador (MAE 2012), we estimate that nearly 49% of the forest cover throughout the species' potential distribution area has been destroyed, mostly due to the expansion of the agricultural frontier.Lachesis nitidus Günther, 1859: 414. Holotype BMNH 1946.1.17.73 (Fig. 7), an adult of undetermined sex from "Western Andes of Ecuador".Referred specimens.All labeled Bothriechis nitidus in Suppl.material 1.
Diagnosis.Bothriechis nitidus is diagnosed based on the following combination of characters: (1) supraciliary scales low and granular or two pointed but not raised; (2) anterior dorsal head scales smooth or barely keeled; (3) gular scales usually much smaller than chinshields or similar in size to chienshields in some cloud forest populations; (4) 3-8 interoculolabials; (5) 2-3 canthals, raised slightly and forming a ridge along the canthus; ( 6 Comparisons.Bothriechis nitidus is compared to other species of the genus previously subsumed under B. schlegelii sensu lato (differences summarized in Table 2).It differs from all of them by having supraciliaries not raised, a low number of canthals (2-3) and interoculolabials (3-8), a low number of ventral scales, and a green dorsum with dark reddish brown, red, or pink bands (Fig. 27).Bothriechis nitidus resembles B. hussaini sp.nov., but this other species occurs south of the known distribution of B. nitidus and is characterized by having the loreal scale in contact with the preocular (not in contact in B. nitidus), supralabials spotted, dorsal scales densely stippled with black pigment, black-spotted ventral surfaces, and basal hooks of hemipenial body 4X (instead of 2X) the size of adjacent spines.Also, unlike B. nitidus, the golden morph is present in B. hussaini sp.nov.Bothriechis nitidus also resembles B. rahimi sp.nov., but this other species occurs north of the known distribution of B. nitidus and is characterized by having two or three raised and spinelike supraciliary scales, keeled anterior dorsal head scales, a higher number of interoculolabials (8-13 vs 3-8), canthals (4-5 vs 2-3), and a completely different array of color morphs, including red and pink.
Hemipenial morphology.(n = 3; Fig. 28) Everted and inflated, the organ is deeply bilobed, calyculate and noncapitate; hemipenial lobes cylindrical, but tapering towards the capitulum; in sulcate and asulcate views, lobe crotch ornamented with densely packed spinules and spines that become larger distally; sulcus spermaticus centrolineal, bifurcate and with walls strongly defined, bifurcation occurs below bilobation point and proximal to the base of the hemipenial body; sulcus spermaticus branch runs to lobe tips; distal one third to one half of each hemipenal lobe densely ornamented with small calyces with strongly defined spinulate edges.In sulcate view, hemipenial body surface with small spinules medially, basal hooks present, and 2-3 rows of smaller obliquely-arranged mesial spines that become gradually smaller towards each lobe's capitulum; each hemipenial lobe ornamented with mesial and lateral spines about half the size of the basal hooks; the spines in each lobe are replaced distally by calyces with strongly spinulate edges.In lateral view, hemipenial body and lobes with rows of spines, but otherwise non-spiculate; distal half strongly calyculate.In asulcate view, the center of hemipenial body is nude to sparsely spiculate; hemipenial lobes ornamented with 5 rows of smaller spines (about 1/3 the size of the lateral basal hooks) that decrease in size towards the lobe crotch.
Natural history.Bothriechis nitidus is an arboreal snake that inhabits evergreen lowland and montane forests, cloud forests, planted forests, plantations (cacao, coffee, and banana), and rural gardens.We have found vipers of this species at night perched on arboreal vegetation up to 10 m above the ground or, rarely, moving at ground level during the daytime.Individuals of B. nitidus are known to prey on frogs (Craugastor longirostris, Pristimantis achatinus, P. walkeri, and Trachycephalus jordani; Valencia et al. 2016;Meza-Ramos et al. 2019), anoles (personal observation by JV), hummingbirds, and mice (Meza-Ramos et al. 2019).Valencia et al. (2016) reports that females of this species give birth to litters of 5-9 young.One specimen (FHGO 7795) lived for 12 years in captivity.
Venom.In coastal Ecuador, 0.2-10.3% of snakebites are attributed to this species (Betancourt 2012;Valencia et al. 2016).Kuch et al. (1996) found the venom of this species to be not hemorrhagic, weakly coagulant, and only moderately lethal (LD50 estimated as 6.5 mg/kg).
Distribution.Bothriechis nitidus is endemic to Ecuador.It is known from at least 121 localities (listed in Suppl.material 3) along the Chocoan lowlands and adjacent foothills of the Andes in Ecuador.The species occurs over an area of approximately 37,400 km 2 and has been recorded at elevations of 0-2,230 m above sea level (Fig. 3).An estimated 4.2% of the predicted area of distribution of B. nitidus overlaps with that of B. hussaini sp.nov.and we found evidence (MZUA.Re.288 and JM 75;Fig. 3) of sympatry between the two species where the model predicted it.Both species co-occur along the foothills of the Andes in the sector between Naranjal, Guayas province, and Cumandá, Chimborazo province.An estimated 3.3% of the predicted area of distribution of B. nitidus overlaps with that of B. rahimi sp.nov., but we did not find evidence of sympatry between the two species.Etymology.The specific epithet nitidus is a Latin word meaning "shining" or "elegant."It refers to the eye-catching dorsal pattern of the holotype, which is shared by most individuals of lowland populations of this species (Fig. 27).
Conservation status.We consider Bothriechis nitidus to be included in the Near Threatened category following IUCN Red List criteria (IUCN 2012) primarily because the species' extent of occurrence is estimated to be larger than the 20,000 km 2 needed to meet B1 criteria for the Vulnerable category.However, although the species occurs in numerous protected areas (no less than 30; see Suppl.material 3), the majority of the species' forest habitat has been destroyed.Based on the distribution model presented in Fig. 3 30g), subadult male collected on June 11, 2019 at Buenaventura Biological Reserve, El Oro province, Ecuador (-3.65467, -79.76794; 524 m).
Proposed standard Spanish name.Víbora de pestañas de Hussain.
Diagnosis.Bothriechis hussaini sp.nov. is diagnosed based on the following combination of characters: (1) supraciliary scales low and granular to triangular; (2) anterior dorsal head scales smooth or barely keeled; (3) gular scales 1/2 size of chinshields; (4) 5-8 interoculolabials; (5) 2-3 canthals, which may be flat or slightly raised forming a ridge along the canthus; (6) loreal in contact with preocular; (7)  Comparisons.Bothriechis hussaini sp.nov. is compared to other species of the genus previously subsumed under B. schlegelii sensu lato (differences summarized in Table 2).It differs from all of them by having supraciliaries not raised, a low number of canthals (2-3) and interoculolabials (5-8), a low number of ventral scales, and a green dorsum heavily speckled with black pigment (Fig. 30).Bothriechis hussaini sp.nov. is similar to B. nitidus, but differs from this species by having the loreal scale in contact with the preocular (not in contact in B. nitidus), supralabials heavily marked by black speckles, dorsal and ventral scales densely stippled with black pigment, basal hooks of hemipenial body 4X (instead of 2X) the size of adjacent spines (Fig. 31), and by the presence of a yellow morph (=víbora sol; Fig. 30b).Bothriechis nitidus also resembles B. rahimi sp.nov., but this other species occurs north of the known distribution of B. nitidus and is characterized by having two or three raised and spinelike supraciliary scales, keeled anterior dorsal head scales, a higher number of interoculolabials (8-13 vs 3-8), canthals (4-5 vs 2-3), and a completely different array of color morphs, including pale gray and pink (Fig. 26).
Hemipenial morphology.(n=2; Fig. 31) Everted and inflated, the organ is deeply bilobed, calyculate and noncapitate; hemipenial lobes cylindrical to spindle-shaped; in sulcate and asulcate views, lobe crotch ornamented with scattered spinules proximally and larger spines distally; sulcus spermaticus centrolineal, bifurcate and with walls strongly defined, bifurcation occurs below bilobation point and proximal to the base of the hemipenial body; sulcus spermaticus branch runs to lobe tips; distal half of each hemipenal lobe densely ornamented with small calyces with strongly defined spinulate edges.In sulcate view, hemipenial body surface with small mesial spinules, a pair of enlarged calcified spines (basal hooks), and 3-4 rows of smaller obliquely-arranged mesial spines that become gradually smaller towards each lobe's capitulum; each hemipenial lobe ornamented with mesial and lateral spines about 1/4 the size of the basal hooks; the spines in each lobe are replaced distally by calyces with strongly spinulate edges.In lateral view, hemipenial body and lobes with rows of spines, but otherwise non-spiculate; distal half strongly calyculate.In asulcate view, the center of hemipenial body is nude to sparsely spiculate; hemipenial lobes ornamented with 4 rows of smaller spines (about 1/4 the size of the lateral basal hooks) that decrease in size towards the lobe crotch.
Natural history.Bothriechis hussaini sp.nov. is an arboreal snake that inhabits evergreen lowland/foothill forests and plantations (coffee and banana).We have found vipers of this species at night or during the day perched on vegetation up to 1.2-3 m above the ground.Meza-Ramos et al. ( 2010) found a mouse of the genus Oryzomys in the stomach of DHMECN 2580 from El Oro province, Ecuador.
Venom.One biologist who was bitten in the shoulder by a Bothriechis hussaini sp.nov.from Azuay province reports intense localized pain, swelling, and tachycardia with two hours from the incident (personal comments to AA, 2022).The patient received antihistamines as treatment and recovered fully within 24 hours of the bite.
Distribution.Bothriechis hussaini sp.nov. is known from at least 55 localities (listed in Suppl.material 3) along the Pacific lowlands and adjacent Andean foothills in southwestern Ecuador and extreme northwestern Peru.The species occurs over an area of approximately 13,591 km 2 in the transition area between the humid Chocó rainforests and the Tumbesian dry forests and has been recorded at elevations 1-1,680 m above sea level (Fig. 3).Approximately 11.7% of the predicted area of distribution of B. hussaini sp.nov.overlaps with that of B. nitidus and we found evidence (MZUA.Re.288 and JM 75) of sympatry between the two species where the model predicted it.Both species co-occur along the foothills of the Andes in the sector between Naranjal, Guayas province, and Cumandá, Chimborazo province.
Etymology.The specific epithet hussaini is a patronym honoring Prince Hussain Aga Khan, who has devoted his life, influence, and wealth to environmental conservation since he was eleven years old.In 2014, he created a Swiss-based non-profit called Focused On Nature (FON), through which he protects endangered global biodiversity through local organizations worldwide.
Conservation status.We consider Bothriechis hussaini sp.nov. to be included in the Vulnerable category following the IUCN criteria B1a, b (i, iii, iv) (IUCN 2012), because the species' extent of occurrence is estimated to be much less than 20,000 km 2 (Fig. 3) and its habitat is severely fragmented and declining in extent and quality due to deforestation.Although B. hussaini sp.nov.occurs in four protected areas (Reserva Ecológica Manglares Churute, Reserva Biológica Buenaventura, Reserva Forestal Cerro Samama, and Reserva Nacional Tumbes), the remaining localities where the species has been recorded (Suppl.material 3) are historical and probably do not represent extant populations.Based on the species distribution model presented in Fig. 3 in combination with maps of Ecuador (MAE 2012), we estimate that approximately 78% of the forest cover throughout the species' potential distribution area has been destroyed, mostly due to the expansion of the agricultural frontier.

Discussion
This work marks the first comprehensive attempt elucidating the systematics of Bothriechis schlegelii sensu lato from an integrated taxonomic perspective.It solves the paraphyly of the species with respect to the previously recognized B. supraciliaris, finds support for the recognition of ten species within this complex, and provides a framework for future studies on this group.Hurtado- Gómez (2009) pointed out that the paraphyly of B. schlegelii sensu lato was probably the result of this nomen (or name) being a species complex.Our results confirm this view.We uncovered as much variation at the molecular level within B. schlegelii sensu lato as among all other currently recognized members of this genus (Fig. 2).We found that this genetic variation is geographically structured (Fig. 3) and corresponds to the existence of ten species that are easily diagnosed based on characters of coloration, lepidosis, hemipenial morphology, natural history, and venom properties (Table 2).
The name Bothriechis schlegelii (Berthold, 1846) is restricted to the species distributed on the central highlands of Colombia (magenta clade in Figs 2, 3) based on the inclusion of the holotype ZFMK 32554 (Fig. 6) in the phylogeny.The holotype was labeled as coming from Popayán.At the time of description, the city Popayán existed but the same name was applied to a colonial province that included nearly all of what is now western Colombia (Myers and Böhme 1996).Although broad, this region is inhabited by only two Bothriechis clades that correspond to species (magenta and turquoise clades in Figs 2, 3).The holotype (Fig. 6) belongs to the highland population and is genetically most similar to another sample from Colombia's Cordillera Occidental, rather than to samples from Cordillera Central.Although this is not enough to restrict the type locality to the city Popayán rather than the broader "Provinz Popoyán," it narrows down the provenance of the holotype to the highlands of the Cordillera Occidental of Colombia.
The name Bothriechis torvus (Posada Arango, 1889a) is restricted to the species distributed on the Chocó-Río Magdalena valley biome in Colombia (turquoise clade in Figs 2, 3) based on the description of the species as well as its type locality.In our phylogenetic analyses, we included samples of specimens assignable to this species from Darién province, Panama, and Chocó department, Colombia.However, all of these come from a western population, which according to our species distribution models, is separated from the one bordering the foothills of Colombia's Cordillera Occidental and Cordillera Central.In agreement with the model, we did not find records of Bothriechis torvus between the two populations, which now, appear to be isolated by the valley of the Río Atrato.We examined specimens from both populations and found minor differences among them, most notably in the condition of the loreal-preocular contact and in the color of the ventral surfaces.The inclusion of genetic samples from the eastern population will help shed light into the relationship between the two populations.
The name Bothriechis nitidus (Günther, 1859) is restricted to the species endemic to west-central Ecuador (purple clade in Figs 2, 3) based on the illustration and description of the holotype as well as on the travel logs of its collector.The illustration of the holotype (Fig. 7) resembles some individuals of B. nitidus in having immaculate supralabials and a bright green dorsum with red bands and minimal speckling on the dorsum (see Fig. 27f).The number of ventral scales in this specimen is 154, which is outside the range reported for B. hussaini sp.nov.but falls within the range reported for B. nitidus (Table 2).The holotype was collected by British zoologist and naturalist Louis Fraser in "western Ecuador" and almost surely in late 1858 or early 1859 based on the illustration of the holotype by George Henry Ford, which is based on a freshly preserved snake.This specimen was sent to the British Museum of Natural History in a shipment that contained hundreds of birds, fish, and reptiles, including the holotypes of Anadia rhombifera, Anolis fraseri, Leptophis occidentalis, Stenocercus iridescens, and Gonatodes caudiscutatus, all described by Günther in 1859.The birds were labeled as having been collected at Pallatanga, Chimborazo province, Ecuador where Mr. Fraser spent the months of August, September, and October 1858 (see Sclater 1859).Although during his trip to Ecuador, which lasted from 1857 to 1859, Mr. Fraser collected in other localities (see Gardner 1983 and references therein), none of these were in the range where B. hussaini sp.nov. is known to occur.
We bring attention to the comparatively low topological divergence between Bothriechis nitidus and B. hussaini sp.nov. in our phylogeny (Fig. 2).The relationship between these two species as reciprocally monophyletic is strongly supported but the genetic distances (1.5% in a 700 bp of the mitochondrial CYTB; Table 1) gene among them are smaller than intraspecific distances in another species of the group: B. nigroadspersus.We do not think this is an artifact of sampling because our molecular analysis includes samples from throughout each species' areas of distribution, including regions where the two species co-occur (Fig. 3).The fact that we found evidence of sympatry between the sister species where the model predicted it suggests a scenario of secondary contact after isolation.However, a comprehensive study of the genetics of this population is needed to evaluate whether introgressive hybridization is taking place in this contact zone.Although there is overlap in scale counts between B. nitidus and B. hussaini sp.nov.(Table 2), the two species can be diagnosed based on hemipenial morphology (Figs 28,31) and coloration (Figs 27,30) and the overlap between their predicted areas of distribution is small (4.2-11.7%;Fig. 3).The existence of B. hussaini sp.nov.mirrors the distribution of other recently described squamates that are restricted to the transition area between the humid Chocó rainforests and the Tumbesian dry forests in southwestern Ecuador and extreme northwestern Peru, including Enyalioides touzeti Torres-Carvajal et al. 2008, Anadia buenaventura Betancourt et al., 2018, Dipsas bobridgelyi Arteaga et al., 2018, Sibon bevridgelyi Arteaga et al., 2018, and Anolis nemonteae Ayala-Varela et al., 2021.All these reptiles are like B. hussaini sp.nov.not only in their distribution but in that their respective sister species are endemic to west-central Ecuador much like B. nitidus.We believe this shared pattern suggests the existence of a unique biogeographical province between the humid Chocó rainforests and the Tumbesian dry forests.
One of the new species described here, Bothriechis rahimi sp.nov., occurs along the mouths of the rivers Esmeraldas, Santiago, Cayapas, and Mira in extreme northwestern Ecuador and southwestern Colombia (orange clade in Fig. 2, 3).We find the existence and distribution of this species puzzling given that we know of no other snakes having a similar range and we found overlap between the predicted areas of distribution of these two species but no evidence of sympatry.Along the northern coast of Esmeraldas province, we found B. nitidus only west of the Río Esmeraldas and B. rahimi sp.nov.only east of the river, a pattern that could point to the role of this river as a potential biogeographical barrier between the two species.However, B. nitidus does occur east of the river towards the Andean foothills in Ecuador.The Río Esmeraldas has already been identified as a potential biogeographical barrier (Arteaga et al. 2016;Guayasamin et al. 2022) for other taxa and could likely help explain the distribution of B. rahimi sp.nov.
The name Bothriechis nigroadspersus (Steindachner, 1870) is restricted to the widespread Central American (red clade in Figs 2, 3) species based on the inclusion of the holotype NMW 18811 (Fig. 4) in the phylogeny.We found two major clades within this species: one distributed throughout Panama and another distributed from Mexico to extreme western Panama.Although we did not identify major biogeographical barriers separating the two populations, we did find a gap in Comarca Ngäbe Buglé, western Panama, where the species has not been reported.The oropel (=golden morph; Fig. 5a-c) has so far only been recorded in the western population, mainly from central and eastern Panama northwards to Honduras.Despite the genetic distances and differential occurrence of the oropel morph, we did not find additional morphological differences that could allow the separation of the two clades into distinct species.Additionally, we only examined the hemipenial architecture of specimens of the western population (Fig. 8).Finally, when locality records of this Panamanian clade were excluded from the species distribution model of B. nigroadspersus, the resulting model still predicted the species occurrence throughout Panama, including the gap in Comarca Ngäbe Buglé (Fig. 3).A more comprehensive sampling of this region in Panama is needed to ascertain whether the model is correct and B. nigroadspersus has a continuous distribution throughout Mesoamerica.We found that an estimated 1.0% of the predicted area of distribution of B. nigroadspersus overlaps with that of B. torvus and B. supraciliaris, and we found records of B. nigroadspersus 3.9-4.4km airline distance away from those of the other two species, respectively.It remains to be seen whether B. nigroadspersus occurs syntopically with one of the other two species and if they occupy different microhabitats when this is the case.
The status of Bothriechis supraciliaris as a valid species is maintained.We found that this species can easily be diagnosed from B. nigroadspersus based on characters of dorsal coloration (Figs 5,9) and number of ventrals (Solórzano et al. 1998), pattern on dorsal aspect of the head (Lotzkat 2014), natural history, venom properties (Lomonte et al. 2012), and hemipenial morphology (all summarized under Table 2).Lomonte et al. (2012) estimated the lethal activity of their sampled B. supraciliaris venom at LD50 6.04 mg/kg, less potent than what has been recorded for B. nigroadspersus, which has LD50 values in the range of 1.7-5.6 mg/kg (Bolaños 1972;Gutiérrez and Chaves 1980;Lomonte et al. 2008Lomonte et al. , 2012;;Prezotto-Neto et al. 2016).The fact that we did not find evidence of syntopy between B. nigroadspersus and B. supraciliaris is intriguing given that the distribution area of the latter is the only region where the presence of B. nigroadspersus is predicted but not confirmed (Fig. 3).The absence of B. nigroadspersus probably points out to a scenario of ecological exclusion.Solórzano et al. (1998) and Solórzano (2004) pointed out that the two species have different habits: primarily arboreal in B. nigroadspersus and mainly terrestrial in B. supraciliaris.However, all B. supraciliaris found by us were on bushes (see account for the species above).Natera-Mumaw et al. (2015) discussed the presence of Bothriechis in Venezuela and suggested that this species be removed from the herpetofauna of this country given the lack of confirmed museum vouchers.Our results agree with this view.We did not find any museum voucher or photographic assignable to this genus in Venezue-la, and neither the distribution models of B. klebbai sp.nov.or B. khwargi sp.nov.(the easternmost species in the group) predict their presence in Venezuela (Fig. 3).
Although this work marks the first comprehensive attempt at elucidating the systematics of Bothriechis schlegelii, it is still far from complete.First, the relationships between the Colombian members of the genus are not strongly supported in our phylogeny and they will likely benefit from an improved sampling that includes more nuclear loci.Adult males of B. khwargi sp.nov.and their corresponding hemipenial morphology remain unknown.Likewise, we were unable to produce information on the hemipenial architecture of B. rahimi sp.nov.The low genetic differentiation but notable morphological difference between cloud forest and lowland population of B. nitidus is intriguing.Conversely, the lack of major morphological differences but comparatively high genetic variation between Panamanian B. nigroadspersus and the population distributed elsewhere throughout Central America deserves further study.Finally, it remains to be seen whether populations of B. torvus from Darién and northwestern Colombia are conspecific with the Central Colombian population.We suggest that any future work focused on the systematics of the B. schlegelii species group include a more comprehensive sampling of molecular characters.Such work would gain much clarity by sampling species of Bothriechis occurring on Colombia's Cordillera Oriental.Until then, we hope that our work helps guide future studies into the biogeography and venom composition in this charismatic group of vipers. is of Colombia and Panama as well as for his suggestions to improve earlier versions of this manuscript.For granting access to the protected forests under their care, we are grateful to Daniel Arias and Raúl Arias of the Canopy Family lodges, to Martin Schaefer and David Agro of Fundación Jocotoco, to Guido Berguido of Fundación Adopta Bosque, and to Jason Crespo Whitney and Veronica Buenaño Bonilla (Bosque Protector la Perla).For providing DNA sequence data of Bothriechis schlegelii, we are grateful to Juan Manuel Daza (MHUA), Christopher Parkinson (Clemson University), and the whole team

Figure 1 .
Figure 1.Terminology of Bothriechis cephalic lepidosis used in this work.Interrictals are counted across the head between the last supralabials.We consider interoculolabials to be only the scales directly below the subocular and above the supralabials.We do not include perisupraoculars (scales bordering the supraoculars) in the count of canthals.Method for counting ventrals followsDowling (1951).Illustrations by Valentina Nieto.

Figure 3 .
Figure 3. Distribution of species of Bothriechis previously subsumed under B. schlegelii sensu lato.Each colored area is a geographic representation of the suitable environmental conditions for one of the clades recovered in the phylogeny of Fig. 2. Lime green shade in southeastern Panama represents overlap between between B. nigroadspersus and B. torvus.

Figure 4 .
Figure 4. Adult female holotype of Bothriechis nigroadspersus NMW 18811 in a. Dorsal and b.Ventral view.Photos by Alice Schumacher.

Figure 5 .
Figure 5. Photographs of some specimens of Bothriechis nigroadspersus in life.a. Adult female from a private collection in Costa Rica; b.MHCH 3269 adult from Guabito, Bocas del Toro province, Panama; c. Subadult from Parque Nacional Gandoca Manzanillo, Limón province, Costa Rica; d. Adult from a private collection in Costa Rica; e, f.Juveniles from Parque Nacional Gandoca Manzanillo, Limón province, Costa Rica; g.MHCH 3268 adult female from Portón, Chiriquí province, Panama; h.MHCH 3266 adult female from Chucantí Reserve, Darién province, Panama; i. Subadult from Parque Nacional Gandoca Manzanillo, Limón province, Costa Rica; j. Adult female from a private collection in Costa Rica; k.FP 001 from Cerro Gaital, Coclé province, Panama; l.MHCH 3267 juvenile male from Chucantí Reserve, Darién province, Panama.Photos by Jose Vieira.

Figure 7 .
Figure 7. Illustration of the holotype of Bothriechis nitidus by George Henry Ford taken from the original description (Günther 1859) at the Biodiversity Heritage Library.
also brings attention to the fact that specimens of B. supraciliaris have very little or no dark pigmentation on the ventral surfaces at least anteriorly in adult specimens (vs.extensive dark pigmentation throughout the venter which are not of the oropel morph in B. nigroadspersus).

Figure 9 .
Figure 9. Adult female holotype of Bothriechis supraciliaris KU 31997 in a. Dorsal and b.Ventral view.Photos by Hannah Som.

Figure 10 .
Figure 10.Photographs of some specimens of Bothriechis supraciliaris in life.a, d, e.From Reptilandia, Costa Rica; and b, c.From a private collection in Costa Rica.Photos by Jose Vieira.

Figure 12 .
Figure 12.Photographs of some specimens of Bothriechis schlegelii from Antioquia department, Colombia, in life.a, c, f.From the live collection at INSV-SR, Serpentario Nacional de Colombia; b, d, e, g. from the live collection at SUA, Serpentario de la Universidad de Antioquia.Photos by Jose Vieira.

Figure 13 .
Figure 13.Photographs of some specimens of Bothriechis schlegelii in life.a. Adult from Planes de San Rafael, Risaralda department, Colombia; b-f.Adults from Popayán, Cauca department, Colombia, photographed at the Serpentario de la Universidad del Cauca.Photos by Jose Vieira and Amanda Quezada.

Figure 15 .
Figure 15.Photographs of some specimens of Bothriechis torvus in life.a. Adult and b.Juvenile (same individual) from Morromico Reserve, Chocó department, Colombia; c. TH 145 adult female from Morromico Reserve, Chocó department, Colombia; d. Adult and e. Juvenile from Urabá, Antioquia department, Colombia.Photos by Jose Vieira.

Figure 25 .
Figure 25.Adult female holotype of Bothriechis rahimi sp.nov.ZSFQ 5055 in a. Dorsal and b.Ventral view.Photos by Alejandro Arteaga.

Figure 27 .
Figure 27.Photographs of living specimens of Bothriechis nitidus from Ecuador.a. AMARU SN adult female from Santa Rosa, Pichincha province; b. Adult from Reserva FCAT, Esmeraldas province; c.Juvenile from Verde Bambú, Pichincha province; d.Juvenile from Canandé Biological Reserve, Esmeraldas province; e. Adult from Gualpi, Esmeraldas province; f. Adult from Bosque Protector La Perla, Esmeraldas province; g. Adult from Santa Lucía Reserve, Pichincha province; h.Adult from Reserva FCAT, Esmeraldas province; i. Juvenile from Santa Lucía Reserve, Pichincha province; j.Juvenile from Canandé Biological Reserve, Esmeraldas province.Photos by Jose Vieira, Alejandro Arteaga, and Sebastián Di Doménico.

Figure 29 .
Figure 29.Adult male holotype of Bothriechis hussaini sp.nov.ZSFQ 5056 in a. Dorsal and b.Ventral view.Photos by Alejandro Arteaga.

Figure 30 .
Figure 30.Photographs of living specimens of Bothriechis hussaini sp.nov.from Ecuador.a. CZ-003 adult female from El Colorado, El Oro province; b.CZ-001 adult male from El Colorado, El Oro province; c.CZ-004 adult male from San Carlos, Guayas province; d.ZSFQ 5058 juvenile female from Buenaventura Biological Reserve, El Oro province; e. AMARU SN adult female Buenaventura Biological Reserve, El Oro province; f. subadult of undetermined sex from Buenaventura Biological Reserve, El Oro province; g.ZSFQ 5056 adult male holotype from Buenaventura Biological Reserve, El Oro province.Photos by Alejandro Arteaga and Jose Vieira.

Table 1 .
Between-group mean distance in percent (number of base differences per site from averaging over all sequence pairs between groups) for species of Bothriechis reviewed in this work, based on 700 bp of the mitochondrial CYTB gene.

Table 3 .
Partition scheme and models of evolution used in phylogenetic analyses.Numbers in parentheses indicate codon position. 108.
in combination with maps of vegetation cover of Ecuador (MAE 2012), we estimate that only ~32% of the species' forest habitat is still standing.