A new species of blunt-headed vine snake (Colubridae, Imantodes) from the Chocó region of Ecuador.

We describe a new species of Imantodes from the Chocó region of northwestern Ecuador. The new species differs most significantly from all other congeners in lacking a loreal scale. We analyze the phylogenetic relationships among species of Imantodes based on two mitochondrial genes, and postulate that the new species and Imantodes lentiferus are sister taxa. A key to the species of Imantodes from Ecuador is presented.

Introduction

The New World colubrid snake clade Dipsadinae Bonaparte 1838 includes more than 400 extant species assigned to approximately 25 taxa traditionally ranked as genera (Daza et al. 2009; Zaher et al. 2009). Most members of the Dipsadinae have unilobed (or nearly unilobed), unicapitate hemipenes, with the sulcus spermaticus dividing distally (Zaher et al. 2009). One of the most remarkable dipsadine genera is . Its long, thin body, disproportionately slender neck, and blunt head, makes easy to distinguish from all other New World snakes. This genus includes six currently recognized species (, , , , , and ) commonly known as blunt-headed vine snakes, occurring from Mexico to Argentina (Myers 1982).

Studies on phylogenetic relationships and species limits among dipsadines are scarce. However, recent work provides strong evidence from DNA sequence data for a clade containing and , although monophyly of remains controversial (Daza et al. 2009; Mulcahy 2007). Futhermore, as currently circumscribed appears to be paraphyletic (Daza et al. 2009; Mulcahy 2007). Future studies with increased taxon and character sampling will probably clarify the phylogenetic relationships and species limits within .

Three species of blunt-headed vine snakes are known from Ecuador; and occur west and east of the Andes, respectively, whereas is known from both versants (Torres-Carvajal and Salazar-Valenzuela 2012). In this paper we describe a new species of from northwestern Ecuador and infer its phylogenetic affinities to other species in the genus as currently circumscribed.

Materials and methods

Morphological data

All type specimens of the new species described in this paper are listed in the type series below, and were deposited at the Museo de Zoología, Pontificia Universidad Católica del Ecuador, Quito (QCAZ), the Museo Ecuatoriano de Ciencias Naturales, Quito (DHMECN), and the Amphibian & Reptile Diversity Research Center at The University of Texas at Arlington, USA (UTA). Specimens of other species of examined in this study are listed in the appendix. Snout-vent length (SVL) and tail length (tL) measurements were recorded to the nearest millimeter. All other measurements were made with digital calipers and recorded to the nearest 0.01 mm. Sex was determined by noting the presence of hemipenes, everted or by tail dissection. Partially everted hemipenes were prepared following standard techniques (Pesantes 1994; Zaher 1999). Differences in scale counts between the new species and other species of were evaluated with t-tests for normally distributed variables (i.e., Shapiro-Wilk test, P > 0.05), all of which had equal variances (i.e., F-test, P > 0.001). We used the program PAST 2.15 (Hammer et al. 2001) for all statistical tests.PageBreak

DNA Sequence Data

Total genomic DNA was digested and extracted from liver or muscle tissue using a guanidinium isothiocyanate extraction protocol. Tissue samples were first mixed with Proteinase K and lysis buffer and digested overnight prior to extraction. DNA samples were quantified using a Nanodrop® ND-1000 (NanoDrop Technologies, Inc), re-suspended and diluted to 25 ng/ul in ddH2O prior to amplification.

We amplified 1674 nucleotides (nt) encompassing two mitochondrial loci, NADH dehydrogenase subunit 4 (ND4, 651 nt) and cytochrome b (cyt-b, 1023 nt) from five individuals of , three of , three of the new species described herein and one of . Cyt-b was amplified using the primers Gludg, L14910, and H16064 (Burbrink et al. 2000; Parkinson et al. 2002), whereas ND4 was amplified using the primers ND4, LEU and ND412931L (Arévalo et al. 1994; Blair et al. 2009). Additionally, we used sequences of , , , and from GenBank. Although monophyly of has not been rigorously tested yet (see Discussion), for the purposes of this study we assume that forms a clade and root our tree with . Gene regions of taxa included in phylogenetic analyses along with their GenBank accession numbers and locality data are shown in Table 1. Amplification of genomic DNA consisted of an initial cycle at 94 C for 3.5 min, 42 C for 1 min, and 68 C for 1.5 min, followed by 40 cycles of a denaturation at 94 C for 30 s, annealing at 52 C for 30 s, and extension at 72 C for 60 s, as well as a final extension at 72 C for 15 min.

Table 1.

Vouchers, locality data, and GenBank accession numbers of taxa and gene regions included in this study. Asterisks indicate new sequences obtained for this study.

Taxon	Voucher	Locality	Genbank accession number
			Cyt-b	ND4
Imantodes cenchoa	MPEGLJV 5763	Brasil: Para	EF078556	EF078508
Imantodes cenchoa	JMD 1616	Colombia: Chocó	GQ334486	GQ334587
Imantodes cenchoa	MHUA R-14290	Colombia: Antioquia	GQ334484	GQ334585
Imantodes cenchoa	MHUA R-14500	Colombia: Antioquia	GQ334485	GQ334586
Imantodes cenchoa	MVZ 149878	CostaRica: Limón	EF078553	EF078505
Imantodes cenchoa	QCAZ 11115	Ecuador: Santo Domingo de los Tsáchilas	*KC176244	*KC176256
Imantodes cenchoa	QCAZ 6300	Ecuador: Esmeraldas	*KC176248	*KC176260
Imantodes cenchoa	QCAZ 4207	Ecuador: Orellana	*KC176247	*KC176259
Imantodes cenchoa	UTA R-42360	Guatemala: Izabal	EF078554	EF078506
Imantodes cenchoa	SIUCR 03724	Panama: Cocle	EF078555	EF078507
Imantodes cenchoa	CORBIDI 3794	Peru: Tumbes	*KC176245	*KC176257
Imantodes cenchoa	CORBIDI 8823	Peru: San Martín	*KC176246	*KC176258
Imantodes chocoensis sp. n.	QCAZ 7978	Ecuador: Esmeraldas	*KC176249	*KC176261
Imantodes chocoensis sp. n.	QCAZ 7984	Ecuador: Esmeraldas	*KC176250	*KC176262
Imantodes chocoensis sp. n.	UTA R-60205	Ecuador: Esmeraldas	*KC176254	*KC176266
Imantodes gemmistratus	UTA R-45922	Guatemala: San Marcos	GQ334487	GQ334588
Imantodes gemmistratus	LSUMZ 39541	Mexico: Sonora	EF078558	EF078510
Imantodes gemmistratus	UTA R-51979	Mexico: Sinaloa	EF078557	EF078509
Imantodes inornatus	MHUA R-14540	Colombia: Antioquia	GQ334488	GQ334589
Imantodes inornatus	ASL 307	CostaRica	GQ334489	GQ334590
Imantodes inornatus	MVZ 204109	CostaRica: Cartago	EF078559	EF078511
Imantodes inornatus	MVZ 204110	CostaRica: Heredia	EF078560	EF078512
Imantodes lentiferus	MPEGLJV 5581	Brazil: Para	EF078562	EF078514
Imantodes lentiferus	MPEGLJV 6880	Brazil: Amazonas	EF078561	EF078513
Imantodes lentiferus	QCAZ 8377	Ecuador: Pastaza	*KC176251	*KC176263
Imantodes lentiferus	QCAZ 8488	Ecuador: Zamora Chinchipe	*KC176252	*KC176264
Imantodes lentiferus	QCAZ 9187	Ecuador: Morona Santiago	*KC176253	*KC176265
Leptodeira septentrionalis	MHUA R-14403	Colombia: Antioquia	GQ334528	GQ334632
Imantodes septentrionalis	QCAZ 10550	Ecuador: Esmeraldas	*KC176243	*KC176255

Phylogenetic analyses

Editing, assembly, and alignment of sequences were performed with Geneious ProTM 5.3 (Drummond et al., 2010). Phylogenetic relationships were assessed under a Bayesian approach in MrBayes 3.2.0 (Ronquist and Huelsenbeck, 2003). The model of character evolution for each gene was obtained in JModeltest (Posada, 2008) under the Akaike information criterion. Genes were combined into a single dataset with two partitions, one per gene. Four independent analyses were performed to reduce the chance of converging on a local optimum. Each analysis consisted of five million generations and four Markov chains with default heating values. Trees were sampled every 1000 generations resulting in 5000 saved trees per analysis. Stationarity was confirmed by plotting the log-likelihood scores per generation in the program Tracer 1.2 (Rambaut and Drummond, 2003). Additionally, the standard deviation of the partition frequencies and the potential scale reduction factor (Gelman and Rubin, 1992) were used as convergence diagnostics for the posterior probabilities of bipartitions and branch lengths, respectively. Adequacy of mixing was assessed by examining the acceptance rates for the parameters in MrBayes and the effective sample sizes (ESS) in Tracer. After analyzing convergence and mixing, 500 trees were discarded as “burn-in” from each run. We then confirmed that the four analyses reached stationarity at a similar likelihood score and that the topologies were similar, and used the resultant 18,000 trees to calculate posterior probabilities (PP) for each bipartition on a 50% majority rule consensus tree.

Results

The taxonomic conclusions of this study are based on the observation of morphological features and color patterns, as well as inferred phylogenetic relationships. We consider this information as species delimitation criteria following the general species concept of de Queiroz (1998, 2007).PageBreak

sp. n.

Proposed standard English name: Chocoan blunt-headed vine snakes

Proposed standard Spanish name: Cordoncillos del Chocó

urn:lsid:zoobank.org:act:D47B3B06-B8B2–4FDC-A54E-3A1F99091044

http://species-id.net/wiki/Imantodes_chocoensis

Holotype.

– QCAZ 7984 (Figs. 1, 2), an adult male from 4 km N Durango, 1.0283°N, 78.5950°W (DD), 253 m, Provincia Esmeraldas, Ecuador, collected on 24 April 2007 by E. Carrillo-Ponce, I. G. Tapia, and E. E.Tapia.

Figure 1.

Holotype of sp. n. in dorsal (left) and ventral (right) views. Photographs by OTC.

Figure 2

. Head of holotype of sp. n. in dorsal (top), lateral (middle) and ventral (bottom) views. Photographs by OTC.

Paratypes (6).

– ECUADOR: Provincia Carchi: DHMECN 6753, Río San Juan, 1.1858°N, 78.5006°W (DD), 243 m, collected on 12 September 2009 by M. Yánez-Muñoz, L. Oyagata, and M. Altamirano; DHMECN 6757, Sendero Awa, 1.1643°N, 78.5071°W (DD), 257 m, collected on 16 September 2009 by M. Yánez-Muñoz, L. Oyagata, and M. Altamirano. Provincia Esmeraldas: UTA R-60205, San Lorenzo-Santa Rita, 1.0321°N, 78.7138°W (DD), 115 m, collected on 21 March 2008 by M. Alcoser, R. Betancourt, P. Loaiza L., L. Oyagata, S. Ramírez J., J. W. Streicher, C. Tobar, and E. N. Smith; QCAZ 7978, same collection data as holotype; QCAZ 10185, 4 km W Alto Tambo, 0.91241°N, 78.5809°W (DD), collected on 18 December 2009 by S. Poe, L. Gray, and I. Latella; QCAZ 10710, Playa de Oro, Estero Pote and Estero Angostura, lower part of Cotacachi Cayapas Ecological Reserve, 0.8285°N, 78.7220°W (DD), collected on 27 November 1994 by E. Toral-Contreras, V. Ortiz, and F. Nogales.

Diagnosis.

differs from all other known congeners in lacking a loreal scale. It can be further distinguished from its sister species (see Phylogenetic relationships) by having 17 longitudinal rows of dorsal scales at midbody and at nearly one head length anterior to the cloaca (15 in ), more ventrals (t = 7.27, P < 0.001), more subcaudals (t = -4.31, P < 0.001), more postoculars (2–3, mean = 2.43 ± 0.51; 1–2, mean = 1.81 ± 0.39 in ), more infralabials (12–15, mean = 13.21 ± 0.80; 9–12, mean = 10.68 ± 0.60 in ), and smaller dark blotches on dorsum (Fig. 3). Among other species of known from Ecuador, the new species differs further from (N = 2–3) in having more ventrals (t = 6.74, P < 0.001), more subcaudals (t = -5.05, P = 0.002), more infralabials (9–11, mean 10.00 ± 0.89 in ), a longer head (head length/width 1.54–1.71, mean = 1.63 ± 0.07 in sp. n.; 1.29–1.61, mean = 1.45 ± 0.16 in ), and dark blotches on dorsum (dark spots and flecks in ; Fig. 3). The new species can also be distinguished from by having a single anal scale (vrs. two), fewer ventrals (t = 7.73, P < 0.001), fewer subcaudals (t = -4.04, P < 0.001), more infralabials (7–12, mean 9.92 ± 0.85 in ), and dorsal dark blotches that include two or fewer vertebral scales and do not extend laterally onto ventrals (blotches are larger in and extend onto lateral tips of ventrals; Fig. 3). Scale counts and measurements of species of from Ecuador are presented in Table 2.

Figure 3.

Body segments of species of from Ecuador in dorsal (left) and lateral (right) views. A sp. n. (DHMECN 6753, paratype) B (DHMECN 8345) C (DHMECN 7826) D (DHMECN 5661). Photographs by MYM.

Table 2

. Scale counts and measurements of species of from Ecuador. Range (first line) and mean ± SD (second line) are presented when appropriate. Sample size is presented in parentheses if different from that in heading.

Character	Imantodes cenchoa N = 42	Imantodes inornatus N = 6	Imantodes lentiferus N = 30	Imantodes chocoensis sp. n. N = 7
Longitudinal scale rows on neck	17	17	15–17 15.07 ± 0.37	17
Longitudinal scale rows at midbody	17	17	15	17
Longitudinal scale rows anterior to cloaca	17	13–15 13.67 ± 1.03	15	17
Ventrals	249–280 262.62 ± 6.22	203–219 210.67 ± 5.99	216–237 226.80 ± 5.03	232–251 243.14 ± 5.84
Subcaudals	155–189 (37) 165.95 ± 8.01	109–126 (5) 117.80 ± 6.18	130–151 (27) 139.85 ± 5.89	140–161 (6) 151.83 ± 7.41
Anals	2	1–2 1.17 ± 0.41	1	1
Anterior temporals	1–3 2.13 ± 0.51	1–2 1.08 ± 0.29	1	1–2 1.43 ± 0.51
Posterior temporals	2–5 2.80 ± 0.53	1–2 1.92 ± 0.29	1–3 2.07 ± 0.36	2
Loreals	1	1	1	0
Preoculars	1–3 1.36 ± 0.53	1–2 1.25 ± 0.45	1–2 1.03 ± 0.18	1
Postoculars	1–4 2.11 ± 0.38	2–3 2.08 ± 0.29	1–2 1.81 ± 0.39	2–3 2.43 ± 0.51
Supralabials	7–9 7.99 ± 0.33	8	7–9 8.05 ± 0.34	9
Infralabials	7–12 9.92 ± 0.85	9–11 10.00 ± 0.60	9–12 10.68 ± 0.60	12–15 13.21 ± 0.80
Genials	2	2	2 (28)	2
Head length/width	1.35–1.80 1.56 ± 0.11	1.29–1.62 1.51 ± 0.13	1.37–1.91 1.62 ± 0.14	1.54–1.71 1.63 ± 0.07
Tail length/Total length	0.28–0.33 (37) 0.30 ± 0.01	0.27–0.30 (5) 0.28 ± 0.01	0.28–0.34 (27) 0.31 ± 0.01	0.29–0.32 (6) 0.31 ± 0.01
Maximum SVL (cm)	107.90	64.00	70.30	74.40
Maximum Total length (cm)	152.10	91.50	101.40	107.50

Description of holotype.

Male (Figs. 1, 2); SVL = 66.30 mm; tail length = 30.40 mm; head width = 7.98 mm; head length = 13.26 mm; head height = 5.37 mm.

PageBreakShort, blunt head 1.7 times longer than broad and 2.5 times longer than deep; head abruptly distinct from neck, three times wider than thinnest part of neck and also slightly wider than greatest width of body; eye large and protuberant, occupying 27% of length of head, with elliptical pupil visible from anterior, lateral, dorsal, and ventral aspects; rostral 1.6 times wider than high, concave in anterior view, and narrowly visible from above; paired prefrontals extending anteroventrally to level of center of eye, each in contact with its mate and with frontal, supraocular, preocular, nasal, and internasal; frontal pentagonal, 1.6 times longer than wide (greatest width), and about 1.2 times longer than distance from its anterior edge to tip of snout; supraocular anteriorly narrow and posteriorly nearly as wide as greatest frontal width; broad parietals, about 1.3 times longer than wide; interparietal suture 1.2 times longer than length of frontal, and 1.4 times longer than distance from frontal to tip of snout; nasal plate single, centrally pierced by large naris (0.79 mm in diameter), in contact with rostral anteriorly, internasal dorsally, prefrontal posterodorsally, preocular posteriorly, and first and second supralabials ventrally; loreal absent; one large and high preocular; two postoculars (an extra tiny scale on left side ventrally), the lower somewhat less than half the size of the upper; temporals 2+2+3; supralabials 9, first and second in contact with nasal, fourth in contact with preocular, and fourth to seventh bordering the orbit; infralabials 13, with first six in contact with anterior genial, and sixth to eighth touching posterior genial; first pair of infralabials in contact medially behind mental; anterior and posterior genials nearly equal in length; gular scales with posterolateral apical pit.

Body higher than wide, rounded ventrolaterally; dorsal scales smooth, juxtaposed or subimbricate; dorsal body scales in 17 rows throughout; scales of vertebral rowPageBreakPageBreak conspicuously enlarged, 2.5 times wider than adjacent dorsals, with concave posterior margins; ventrals 242; anal plate single; subcaudals 161.

Color in preservative of holotype (Figs 1, 2). Dorsal background light brown, with a longitudinal series of 63 dark brown middorsal blotches from head to cloaca; dark middorsal blotches longer anteriorly, 2–3 vertebral scales long, than posteriorly, 1–2 vertebral scales long, and extending laterally 1–3 (anteriorly) or more (posteriorly) dorsal scale rows, but never reaching ventral scales; each dark middorsal blotch irregularly bordered anteriorly and posteriorly by thin cream line; ventral aspect of body yellowish cream with dark brown spots and flecks; ventral aspect of tail yellowish cream with spots concentrating midventrally; dorsal surface of head light brown with several dark brown spots and two short dark stripes extending from posterior aspect of parietals to a point just posterior to head; ventral surface of head whitish cream.

Hemipenes (Fig. 4). The right hemipenis of the paratype DHMECN 6753 of was removed, fully everted and expanded (Fig. 4). The organ is bulbous and relatively long, 11.2 mm in length, and when adpressed to the outside of the tail it extends from the cloaca to the sixth subcaudal scale. The organ is longer than wide (width 46% of length), unilobed, symmetrical, calyculate, capitate, and arched towards the sulcal side. The sulcus spermaticus is simple, linear, semicentripetal, and thin, demarcated by thick bordering tissue at the base, particularly at the anterior border, and ending on the surface of the capitulum facing medially. The capitulum is ornamented with papillated calyces, spinulated proximally. The capitulum, approximately 45% the length of the hemipenis, is slightly demarcated by a groove, more prominent on the sulcal side and joining the sulcus spematicus. In the asulcate side the base of the capitulum has more prominent spines. Truncus covered by large spines, on the sulcate PageBreakand asulcate and surfaces, 23 in total, 13 to the right of the sulcus spermaticus and 10 to the left, and has a few rows of small spines, two at the base on the asulcate side and two to three rows just right of the sulcus spermaticus.

Figure 4.

Right hemipenis of sp. n. (DHMECN 6753, paratype) in sulcal (A), asulcal (B), and lateral (C) views D close-up of distal end showing spines interrupted by sulci. Photographs by MYM.

Variation.

Intraspecific variation in scale counts and measurements in sp. n. is presented in Table 2. Color in life of paratypes UTA R-60205 and DHMECN 6753 (Fig. 5) is similar to color in preservative of holotype; iris copper brown. Middorsal blotches from head to cloaca vary between 55–66; one specimen (UTA R-60205) had one middorsal blotch covering five vertebral scales.

Figure 5.

Paratypes of sp. n. UTA R-60205 (top), DHMECN 6753 (bottom). Photographs by ENS and MYM.

Distribution and ecology.

inhabits Chocoan rainforests on the Pacific coast in northern Ecuador (Fig. 6). It occurs in lowland evergreen forest (Cerón et al. 1999) at elevations of 115–260 m in the provinces of Carchi and Esmeraldas. This new species has been collected in sympatry with in Esmeraldas, PageBreakand most likely also shares its distribution with . Other colubrid snakes collected in Tobar Donoso (Carchi) are , , , , sp., , ,PageBreak , , , , and . The known localities of lie in close proximity to the Ecuador-Colombia border and we expect for it to be found in neighboring Colombia.

Figure 6.

Distribution of sp. n. (circles) and its sister species (squares) in Ecuador.

Etymology.

The specific epithet chocoensis is an adjective derived from Chocó, the very humid tropical region comprising the Pacific coast of northern Ecuador, Colombia and Panama (Morrone 2001). This region is part of the 274,597 km2 Tumbes-Chocó-Magdalena hotspot as defined by Conservation International, which includes more than 320 species of reptiles.

Phylogenetic relationships.

Selected models of evolution for sampled fragments of ND4 and cyt-b genes were HKY+I+G and TPM2uf+I+G, respectively. The resulting 50% majority rule consensus tree (Fig. 7) supports strongly (PP=1) a sister taxon relationship between sp. n. and , as well as the exclusivity (Rieppel 2010) of both species. Similarly, and are recovered as PageBreakexclusive clades with strong support (PP=1). Noteworthy the clade includes samples from Guatemala, Costa Rica, Panama, Brasil, and Colombia, Ecuador and Peru on both sides of the Andes. In agreement with previous hypotheses, is paraphyletic; of three samples included in this study, one from Guatemala is sister to the clade with strong support (PP=1), whereas the other two, from Mexico, are weakly supported as sister to the sp. n. and clade.

Figure 7.

Majority rule (50%) consensus tree of 18,000 trees obtained from a Bayesian analysis of two mitochondrial genes (cyt-b, ND4) and 29 specimens. Asterisks correspond to posterior probability values > 0.99. Voucher numbers followed by country of collection are indicated for each terminal. E: east of the Andes, W: west of the Andes.

Discussion

Myers (1982) distinguished two monophyletic groups within – lentiferus and cenchoa – based on hemipenial characters, maxillary dentition, relative tongue length, and coloration. According to Myers, the lentiferus group included and as sister taxa, as well as , whereas the cenchoa group included , and . Since the phylogenetic tree presented in this paper does not include all species of , we cannot rigorously test Myers’ hypothesis of phylogenetic relationships within . Nonetheless, two major differences are worth noting. First, in our phylogenetic tree is sister to all other species of (but see below). Second, in agreement with previousPageBreak work (Daza et al. 2009), we recover a paraphyletic , with specimens from Guatemala closely related to as postulated by Myers (1982), and specimens from Mexico in a clade with and the sp. n. (Fig. 7).

Monophyly of remains controversial, but we refrained from testing it without better taxon and character sampling. Previous phylogenetic studies based on DNA sequence data have failed to support the monophyly of as currently circumscribed (Daza et al. 2009; Mulcahy 2007). Except for a tree including only two species of (Fig. 6 in Daza et al. 2009) and a Maximum Parsimony tree (Fig. 5 in Mulcahy 2007), these studies suggest that is sister to a clade containing and as sister taxa. Furthermore, the phylogenetic tree presented in this paper is congruent with this hypothesis (Fig. 7), suggesting that might belong to a clade different from . In fact, this species differs from other in several morphological (e.g., no prominent dorsal blotches, or conspicuously enlarged vertebral scales; Fig. 3) and behavioral (e.g., head-flaring) features (Mulcahy 2007; Myers 1982).

1	Longitudinal scale rows at midbody 17	2
–	Longitudinal scale rows at midbody 15	Imantodes lentiferus
2	Vertebral scales 2.5–4 times wider than adjacent dorsal scales; dorsal color pattern with conspicuous dark blotches(Fig. 3)	3
–	Vertebral scales similar in size or slightly wider than adjacent dorsal scales; dorsal color pattern with dark spots and speckles (Fig. 3)	Imantodes inornatus
3	Loreal present; dorsal blotches include more than two vertebral scales and extend onto edge of ventrals (Fig. 3)	Imantodes cenchoa
–	Loreal absent; dorsal blotches include two or less vertebral scales and do not extend laterally onto ventrals (Fig. 3)	Imantodes chocoensis