Research Article |
Corresponding author: Angele Martins ( angelemartins@gmail.com ) Academic editor: Uwe Fritz
© 2023 Angele Martins, Manuella Folly, Guilherme Nunes Ferreira, Antônio Samuel Garcia da Silva, Claudia Koch, Antoine Fouquet, Alessandra Machado, Ricardo Tadeu Lopes, Roberta Pinto, Miguel Trefaut Rodrigues, Paulo Passos.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Martins A, Folly M, Ferreira GN, Garcia da Silva AS, Koch C, Fouquet A, Machado A, Lopes RT, Pinto R, Rodrigues MT, Passos P (2023) An evolutionary paradox on threadsnakes: Phenotypic and molecular evidence reveal a new and remarkably polymorphic species of Siagonodon (Serpentes: Leptotyphlopidae: Epictinae) from Amazonia. Vertebrate Zoology 73: 345-366. https://doi.org/10.3897/vz.73.e98170
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Threadsnakes are known for their conserved external morphology and historically controversial systematics, challenging taxonomic, biogeographic and evolutionary researches in these fields. Recent morphological studies—mostly based on µCT data of the skull and lower jaw—have resolved systematic issues within the group, for instance leading to the description of new taxa or re-positioning little-known scolecophidian taxa in the tree of life. Herein we describe a new polymorphic species of the genus Siagonodon from Amazonia based on morphological (external, osteology and hemipenis) and molecular data, and provide the first hemipenial description for the genus. We also reassign Siagonodon acutirostris to the genus Trilepida based on osteological data in combination with molecular evidence. The new species described represents an evolutionary paradox for scolecophidians because the species displays a remarkable variation in the shape of the snout region that is otherwise always highly conserved in this clade. Finally, this study reinforces the importance of protected areas as essential in maintaining vertebrate populations, including those that are not yet formally described.
Hemipenial morphology, intraspecific variation, Neotropics, osteology, phylogeny, polymorphism, Squamata
The fossorial snakes known as ‘Scolecophidia’ Cope, 1864 are divided in three major lineages—Anomalepididae Taylor, 1939, Leptotyphlopidae Stejneger, 1892, and Typhlopoidea Romer, 1945—altogether comprising about 460 species that occur throughout the globe, except for Antarctica (
The threadsnakes of the family Leptotyphlopidae currently comprise about 140 species that inhabit the Americas, sub-Saharan Africa, the Arabian peninsula and southwestern Asia (
The genus Siagonodon currently comprises four species—S. acutirostris Pinto and Curcio, 2011, S. borrichianus (Degerbøl 1923), S. cupinensis (Bailey and Carvalho 1946), and S. septemstriatus (Schneider 1801)—that occur along the cis-Andean region of South America from northern French Guiana and Suriname, to its southernmost record in Argentina (
Herein we provide the description of a new polymorphic species of the genus Siagonodon based on combined morphological and molecular data, and provide the first hemipenial description for the genus. We also reassign Siagonodon acutirostris to the genus Trilepida based on osteological (skull and lower jaw) data in combination with molecular evidence.
We examined specimens housed in the following institutions (see Appendix
We follow
Aiming to assess the phylogenetic position of Siagonodon spp. within Leptotyphlopidae, we included representatives of all genera currently included in the Subfamily Epictinae Hedges, Adalsteinsson & Branch, 2009, improving the representativeness of Trilepida spp., and representatives of two other basal snake families (Boidae Gray, 1825 and Typhlopidae Merrem, 1820). We selected specific out-group terminals trying to maximize character coverage (i.e., homologous sequences available from GenBank) and phylogenetic structure according to the trees of the two most densely sampled leptotyphlopid phylogenies (
We obtained tissue samples from 17 individuals representing five nominal and one undescribed species: Siagonodon acutirostris, S. cupinensis, Siagonodon sp., Trilepida dimidiata, Trilepida jani, and Trilepida salgueiroi. The new samples were acquired through field work, loans and donations. We checked identifications from all new tissue samples by direct examination of the voucher specimens, comparing such material with type specimens, topotypes and additional comparative material from previously recognized species for accurate identifications. We extracted DNA sequence data by performing PCRs using a PCR Master Mix and a pair of primers for each segment: BDNF forward and BDNF-reverse; NTF3_f1, and NTF3_r1 (
Thermocycling for DNA amplification for the first partition began with a denaturation at 94°C (5 min), followed by 40 cycles of denaturation at 94°C (30 s), annealing at 43–48°C (30 s), extension at 70°C (1 min), and a final step at 72°C after the final cycle (7 min). PCR products were visualized in 1% agarose gels and sent to Macrogen Inc. (Seoul, Republic of Korea) for purification and sequencing reactions. PCR products of Trilepida spp. were purified with Exosap and sent to Genoma USP — Centro de Estudos do Genoma Humano e Células-Tronco. Resulting electropherograms for both DNA strands were analyzed using Chromas Lite 2.01 and edited using MEGA 6.0 and adjusted manually to generate consensus sequences for each specimen. Sequences were checked with basic local alignment search tool (
All sequences were aligned using MAFFT version 7 (
We performed partitioned Bayesian Inference (BI) using MrBayes 3.2.2 (
In this study, we followed the general lineage species concept of
The authors declare that this contribution represents an original work and that no experiment was done with live animals. All specimens pertaining to Siagonodon sp. nov. were collected under the permits Abio No 1146/2019, Ofício 848/2020/COMIP/CGTEF/DILIC (Brazil) and APA no. 973-23-1 (French Guiana).
The topology recovered based on BI shows that the genus Siagonodon (as currently defined) is paraphyletic (Fig.
Furthermore, the new species of Siagonodon is strongly supported (pp > 0.95) as the sister species of S. cupinensis, and this clade represents the sister-group of S. septemstriatus (Fig.
Adult female,
Six specimens, two females (
The putative autapomorphy of Siagonodon exiguum sp. nov. may be represented by lateral pointed projections on the snout or with inconspicuous lateral projection by presenting a slight concavity on the mid-rostral portion of snout (see below in comparison the opposite states for snout condition).
Siagonodon exiguum sp. nov. can be distinguished from all congeners by the following combination of external characters: midbody scale rows 14; midtail scale rows 14; supralabials two (1+1); infralabials four; subcaudals 15–18; middorsal scale rows 255–289; total number of precloacal vertebrae 239–263; supraocular scales absent; frontal scale distinct, not fused with rostral; body dorsum and vent uniformly beige; eye totally covered by ocular scale, eye spot unconspicuous and reduced; caudal scales not fused; terminal spine absent; cloacal vertebrae 3–5, caudal vertebrae 18–20, the last one composed of 3 fused vertebrae; snout concave with conspicuous lateral tapered projections, without tapered projections, and slightly concave anteriorly or without projections and truncate anteriorly.
Siagonodon exiguum is distinguished from all congeners by the presence (in most specimens) of lateral pointed projections on the snout, or by a slightly concave snout (when present; vs. acuminate in S. borrichianus; truncate in S. cupinensis and S. septemstriatus); by having 15–18 subcaudal scales (vs. 10–13 in S. borrichianus, 12–16 in S. cupinensis, and 10–12 in S. septemstriatus); by having a pair of optic nerve foramina (vs. single foramen in S. septemstriatus); from S. cupinensis and S. septemstriatus by the fusion of the prootic+otooccipital bones (vs. distinct bones); from S. borrichianus and S. septemstriatus by having 255–289 middorsal scales (vs. 163–285 in S. borrichianus and 215–244 in S. septemstriatus); from S. borrichianus and S. septemstriatus by having 230–269 midventral scales (vs. 245–272 in S. borrichianus and 196–224 in S. septemstriatus); from S. borrichianus and S. septemstriatus by having 14 scales around midtail (vs. 10 in S. borrichianus and 12 in S. septemstriatus); from S. septemstriatus and S. borrichianus by its uniform beige dorsum and vent color (vs. striped pattern in S. septemstriatus, and uniformly dark brown in S. borrichianus).
Species | Middorsal scales | Midventral scales | Subcaudal scales | Infralabial scales | Midtail scales | Source |
S. borrichianus | 163–285 | 245–272 | 10–13 | 3 | 10 |
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S. cupinensis | 225–293 | 224–270 | 12–16 | 3–4 | 14 |
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S. exiguum sp. nov. | 255–289 | 230–269 | 15–18 | 4 | 14 | This study |
S. septemstriatus | 213–247 | 196–237 | 10–12 | 4 | 12 | Pinto et al. (2011); |
Adult female, Total length (TL) 186 mm, Tail length (TAL) of 8.0 mm; midbody diameter (MB) 3.2 mm; midtail diameter 2.7 mm; TL/TAL 23.2; TL/MB 5.8; rostral width 1.2 mm; inconspicuous eye spot covered by ocular scale; head length 4.9 mm, head width 2.9 mm; head subcylindrical, markedly truncate anteriorly; body subcylindrical, slightly tapered caudally near the tail; head not enlarged, indistinguishable from neck. Snout truncate in lateral and ventral views; rostral hexagonal in frontal views, and trapezoidal in ventral view; dorsal apex concave, not reaching the anterior limit of oculars; rostral contacting supranasal and infranasal laterally, and frontal dorsally; nasal completely divided horizontally by oblique suture crossing nostril; nostril roughly semicircular, positioned mostly dorsal of nasal suture; supranasal twice as high as long, contacting rostral anteriorly, infranasal ventrally, first supralabial ventrally, ocular posteriorly, and frontal dorsally; supranasal ventral boundary slightly longer than upper border of infranasal scale; infranasal twice as high as long; upper lip border formed by rostral, infranasal, anterior supralabial, ocular, and posterior supralabial scales; temporal scale not distinct in size from dorsal scales of lateral rows; two supralabials (1+1) entirely separated from each other by ocular; first supralabial about 1.5 times higher than long, not reaching the nostril level or the eye spot; second supralabial roughly semicircular, slightly longer than high, twice as high as first supralabial, its posterior margin in broad contact with temporal; ocular high, dorsal apex acuminate, anterior border straight and vertical, about 3 times higher than long, contacting posterior margins of supranasal and first supralabial anteriorly, parietal posteriorly, frontal and postfrontal dorsally; eye indistinct, covered by ocular scale; eye spot positioned in central area of expanded upper part of ocular, displaced far above nostril level; frontal subcircular, slightly wider than the other middorsal head plates (postfrontal, interparietal, and interoccipital); middorsal head plates (postfrontal, interparietal, and interoccipital) subequal in size, trapezoidal in dorsal view, weakly imbricate; frontal enlarged, about twice as wide as long, contacting rostral, supranasals, oculars, and postfrontal; postfrontal about twice as wide as long, contacting frontal, oculars, parietals, and interparietal; interparietal twice as wide as long, contacting postfrontal, parietals, occipitals, and interoccipital; interoccipital about as wide as long, contacting interparietal, occipitals, and first dorsal scale of vertebral row; parietal hexagonal, about as wide as long; lower margin contacting upper border of second supralabial, posterior margin contacting temporal, occipital, and interparietal, anterior margin in broad contact with ocular and postfrontal; occipital almost twice wider than long, its lower limit attaining level of upper margin of second supralabial, although separated from the latter by temporal; symphysial subcircular, anterior and posterior borders respectively straight and slightly convex, about twice wider than long; four infralabials; first infralabial small, subtriangular; second and third infralabials subequal, wide, somewhat higher than long, not pigmented; fourth infralabial twice longer than high high, distinctively longer than others, as high as second and third supralabial, not pigmented; dorsal scales homogeneous, cycloid, smooth, slightly imbricate, arranged in 14 scale rows around midbody and in the middle of the tail; middorsal scales 289; midventral scales 266; cloacal shield short and semicircular, almost twice as wide as long; 16 subcaudals; fused caudals absent; terminal spine indistinct or absent.
Coloration of holotype relatively faded after preservation; dorsal, paraventral and ventral scales uniformly beige; dorsal and ventral head shields creamish white; cloacal shield creamish white.
Skull (Fig.
Three-dimensional reconstruction of the skull of Siagonodon exigumm sp. nov. based on µ-CT data in dorsal (A, D, G), lateral (b, e, h), and ventral (c, f, i) views.
Dentary supports series of five long teeth ankylosed to inner surface of medial margin of dental concha; mental foramen nearly under fifth tooth; dorsoposterior process of dentary with rounded enlargement (n = 1), possibly to provide attachment for Musculus ceratomandibularis and/or to Musculus cervicomandibularis (
Three-dimensional reconstruction of the lower jaw of Siagonodon exigumm sp. nov. based on µ-CT data in lateral (a, c, e) and medial (b, d, f) views.
Atlas composed of neural arches, not fused dorsally or ventrally; ventral element (intercentrum I sensu
Three-dimensional reconstruction of the atlas (first to third column) and axis (fourth to sixth column) of Siagonodon exigumm sp. nov. in anterior, lateral and posterior views based on µ-CT data.
Composed of ilium, ischium, femur, and pubis. Ilium and pubis rod-like; ischium approximately rectangular and fused to pubis (n = 1) or ischium rod-like and not fused to pubis (n = 2*); ilium represents longest bone of pelvic girdle, being fused to pubis (n = 2*) or not (n = 1); femur approximately rectangular and curved with dorsolateral claw-like process (n = 2*) or without such process (n = 1).
Hyoid Y-shaped, extending from 8–11th (
Fully everted and almost maximally expanded hemipenis (examined ex-situ) renders a unilobed organ, about 2 mm long, slightly broadened at base, followed by a short tapered area that posteriorly expands towards apex; basal portion not ornamented on proximal half; hemipenial body with no ornamentation on either sulcate or asulcate sides; sulcus spermaticus single, entering organ on basal surface and extending toward apex of organ; sulcal folds raised and not ornamented; distal half of hemipenial body covered by three inconspicuous transverse flounces; organ apex convex and not ornamented; single and tapered projection develops from apex of asulcate side.
Middorsal scales 285–289 (287 ± 2.8; n = 2) in females and 255–280 (269 ± 12.5; n = 4) in males; midventral scales 266–267 (266.5 ± 0.7; n = 2) in females and 230–269 (253 ± 16.4; n = 4) in males; subcaudals 15–16 (15.5 ± 0.7; n = 2) in females and 16–18 (17 ± 0.8; n = 4) in males; TL 184–204.2 (195.1 ± 12.9; n = 2) in females and 171.1–200.3 (187.1 ± 12; n = 4) in males; TL/TAL ratio 20.0–23.2 (21.6 ± 2.2; n = 2) in females and 19.4–22.5 (20.6 ± 1.5; n = 4) in males; TAL 4.3–4.9% of TL (4.6 ± 0.5; n = 2) in females and 4.4–5.1% (4.9 ± 0.3; n = 4) in males; TL/midbody diameter 2.4–3.1 (2.7 ± 0.5; n = 2) in females and 2.3–2.8 (2.6 ± 0.6; n = 4) in males; TAL/midtail diameter 2.8–3.5 (3.1 ± 0.4; n = 2) in females and 2.5–3.4 (3 ± 0.3; n = 4) in males.
Color pattern of paratypes mostly resembles that of holotype except for the following: dorsum and vent uniformly light brown in three paratypes (
Precloacal vertebrae 248–260 (254 ± 8.5; n = 2) in females and 239–263 (253 ± 10; n = 4) in males; cloacal vertebrae 4–5 (4.5 ± 0.7; n = 2) in females and 3–4 (3.5 ± 0.6; n = 4) in males; caudal vertebrae 18 in females (n = 2) and 18–20 (19.2 ± 0.9; n = 4) in males.
The specific epithet exiguum is a Latin word meaning little, short, scanty and/or poor. Although readers might think that the name was chosen in allusion to the small size of the species, we dedicate the species to all minorities in science including gender, race/color, ethnicity, and sexual orientation that are considerably underrepresented in the academia throughout the world, especially in higher positions. Although several worldwide initiatives have been implemented in the past few years, the scientific environment is still biased, discouraging and, sometimes noxious, largely because of persisting systemic social, economic and cultural aspects.
Siagonodon exiguum is currently known from the type locality in Pará State (Brazil) and French Guiana (Mitaraka, Nouragues – A. Fouquet pers. obs and Angouleme –
The X-ray images of two specimens (
Siagonodon acutirostris
Pinto & Curcio, 2011:58 (
Siagonodon acutirostris – Wallach et al. 2014: 665.
Siagonodon acutirostris – Nogueira et al. 2019.
Trilepida acutirostris is distinguished from all Neotropical leptotyphlopids by the following unique combination of characters: snout slightly acuminate in lateral and ventral views; absence of supraocular scale; middorsal cephalic plates distinctively enlarged; ocular scale subheptagonal, dorsal apex acuminate and anterior border straight, roughly vertical at eye level; first and second supralabial scales not reaching eye level; two supralabials (1+1); fused caudals absent; temporal scale not distinct; rostral subcircular in dorsal view; middorsal scales 169–183; midventral scales 161–173; subcaudal scales 9–11; 12 scales around the middle of the tail; dorsum uniformly pale copper on five dorsal scale rows, contrasting with the whitish cream tonality covering nine scale rows of venter, and thereby reaching the paraventral region of trunk. Osteology characters: the basioccipital participating in the formation of the foramen magnum, paired nasals and fused supraoccipitals.
The fossorial snakes of the Family Leptotyphlopidae (approximately 140 nominal extant species;
By contrast, other generic allocations (e.g., Siagonodon borrichianus and Rena spp.) still require to be confirmed through congruence between new anatomical approaches (
Besides the phenotypic and molecular evidence herein provided for the allocation of S. acutirostris in Trilepida, additional meristic characters also seem to support the distinctiveness between Siagonodon and Trilepidia. For instance, Siagonodon spp. (except for S. borrichianus) present a higher middorsal scale count (196–293; see Table
The burrowing lifestyle has been repeatedly associated with phenotype conservativeness in fossorial lineages in general, including ‘Scolecophidia’ (
Conversely, specimens from the same locality in Brazil show a low mtDNA variation (<2%), but exhibit different snout shapes (pointed vs. not pointed). Even though recent studies (e.g.,
We provide the first hemipenial description for any of the currently known species of the genus Siagonodon. The organ morphology does not resemble any of the known hemipenes amongst Epictinae (see Wallach, 2016,
Finally, Siagonodon exiguum is probably endemic to the Eastern Guiana Shield (Guiana region of
The conservativeness of the external morphological characters of scolecophidians represent a challenge when trying to understand their evolution. Therefore, the combination of morphological (external and internal) and molecular data appears to be crucial in resolving systematic issues within this group. These data also reinforce the utility of osteological and hemipenial data in recognizing new taxa, especially amongst threadsnakes (Leptotyphlopidae). Even though scolecophidians are known for displaying a very conserved external morphology, polymorphic taxa—although rare—might still be unknown to science, therefore representing a true paradigm in the evolution of threadsnakes. Therefore, external morphological data might not be disregarded when considering the phenotypic evolution of the group and may as well be significant in identifying new taxa, including those that exhibit many autapomorphies.
We are thankful to the following persons for allowing us to examine specimens under their care: A.L.C. Prudente (
* Specimens with one asterisk were µCT-scanned for skull comparisons.
** Specimens with double asterisk were analyzed based on dry or cleared and stained specimens
Epictia ater. EL SALVADOR: San Miguel: KU 183846**; NICARAGUA: CHINANDEGA: Volcan Chongo: KU 194336, 200596; ESTELLI: KU 174119, 174120–21, 174134–35.
Epictia goudotii. VENEZUELA: Aragua: Girardot: Cuyagua:
Epictia magnamaculata. HONDURAS: Islas de la Bahía: Isla de Utila:
Epictia munoai. URUGUAY: Tacuarembo: Tambores: Pozo Hondo:
Epictia phenops. MEXICO: Yucatán:
Epictia rufidorsa. PERU: Lima: Rimac Valley:
Epictia tenella. BRAZIL: AMAZONAS: Urucá:
Habrophallos collaris. FRENCH GUIANA: Itoupe:
Siagonodon cupinensis. BRAZIL: Mato Grosso: Barra do Tapirapes:
Siagonodon exiguum. BRAZIL: Pará: Monte Branco, Oriximiná:
Siagonodon septemstriatus. BRAZIL: Amazonas: Manaus: Negro River: Negro Rover Bank (
Trilepida acutirostris. BRAZIL: Tocantins: Almas:
Trilepida brasiliensis. BRAZIL: Mato Grosso do Sul: Corumbá (
Trilepida dimidiata. BRAZIL: Roraima: Boa Vista (
Trilepida fuliginosa. BRAZIL: Goiás: Caldas Novas (
Trilepida jani. BRAZIL: Minas Gerais: Ouro Preto (LZV 813S**); Ouro Branco (LZV 778S*); without locality (
Trilepida joshuai. COLOMBIA: Antioquia: Jericó (IBSP 8919*); Valle del Cauca: Tocota: (
Trilepida koppesi. BRAZIL: Aporé (
Trilepida macrolepis. BRAZIL: Pará: Paraupebas: Floresta Nacional de Carajás (
Trilepida pastusa. ECUADOR: Carchi: Tulcán (
Trilepida salgueiroi. BRAZIL: Bahia: São José do Macuco (currently São José da Vitória) (
Table S1
Data type: .xlsx
Explanation note: GenBank accession number for specimens used in the current study.
Table S2
Data type: .xlsx
Explanation note: Genetic distances of Siagonodon spp. in comparison to other Epictinae genera based on 16s sequences.
X-Ray Images
Data type: .zip
Explanation note: X-Ray Images of specimens of Siagonodon exiguum sp. nov.