A morphological and molecular study of Hydrodynastes gigas (Serpentes, Dipsadidae), a widespread species from South America

Molecular analysis

We extracted the DNA from muscle, liver or scale of 27 samples of Hydrodynastes gigas, five of H. melanogigas and 12 of H. bicinctus. Samples of Hydrodynastes bicinctus were added to our analysis in order to provide a complete species representation for the genus. We used the phenol-chloroform extraction protocol (Sambrook, Fritsch & Maniatis, 1989) (Fig. 1A). We amplified the partial sequences of the mitochondrial 16S ribosomal (mtDNA) genes (16S rRNA, 326 pb) (Palumbi et al., 2002), Cytochrome b (Cytb, 618 pb) (Pook, Wüster & Thorpe, 2000), the nuclear genes (nuDNA) Oocyte maturation factor Mos (Cmos, 478 pb) (Lawson et al., 2005) and Neurotrophin-3 (NT3, 480 pb) (Townsend et al., 2008) using the standard Polymerase Chain Reaction (PCR) technique as described by (Pook, Wüster & Thorpe, 2000) and Moraes-Da-Silva et al. (2019). We visually checked all nucleotide sequences and aligned the concatenated genes (1,902 pb) using the Muscle algorithm (Edgar, 2004) in the Geneious v.9.0.5 program (https://www.geneious.com). We used the species Pseudoboa nigra and Xenopholis scalaris as outgroup of Hydrodynastes (Vidal, Dewynter & Gower, 2010; Zaher et al., 2009), and rooted the tree in Xenopholis. We used the sequences available in GenBank and deposited those generated in this study into the same database (Table 1).

We used PartitionFinder 2 to identify partitioning schemes and the most appropriate nucleotide replacement models (Lanfear et al., 2016). According to our concatenated alignment, we found five partitions evaluated by BIC (Table 2). For phylogenetic analysis, we used the Bayesian inference implemented in MrBayes v3.2.6 (Ronquist & Huelsenbeck, 2003) using the substitution models generated by PartitionFinder. We ran two independent runs of four Markov chains for 20 million generations sampling every 5,000 generations and discarding 25% as burn-in. We evaluated the stability of the analysis in Tracer v1.6, ensuring that all ESS values were above 200 (Rambaut et al., 2014). We calculated the divergence between sequences (p-distance) in Mega v10.0.5 (Tamura et al., 2013).

Morphological analysis

We examined 144 specimens of Hydrodynastes gigas and 42 specimens of H. melanogigas (Fig. 1B). Museum acronyms follow Sabaj-Pérez (2014), except for Coleção Herpetológica da Universidade Federal da Paraíba (CHUFPB), João Pessoa, PB; Coleção Zoológica Delta do Parnaíba (CZDP), Parnaíba, PI; Universidade Luterana do Brasil (MZCEULP), Palmas, TO. The specimens examined are listed in the Appendix S1.

Franco, Fernandes & Bentim (2007) described Hydrodynastes melanogigas based on 17 specimens collected in the municipalities of Palmas (type locality), Porto Nacional, and Lajeado, which are all located in the state of Tocantins, Brazil. Unfortunately, most of the type series was lost in the 2010 fire that occurred at the Instituto Butantan. Currently, only three individuals remain from the type series: two at the proper Institute in São Paulo (paratypes IBSP 65978 and IBSP 66387) and one at the Museu Nacional do Rio de Janeiro (paratype MNRJ 15101). From the remaining type-series, we analyzed all the remaining individuals.

We examined 14 meristic characters (Table 3) and eight morphometric ones (Table 4), in addition to the coloration pattern and morphology of the hemipenis. Sex was determined by the presence or absence of hemipenes through a ventral incision at the base of the tail. We measured individuals with an electronic caliper (0.01 mm) and a flexible ruler (1 mm), on their right side whenever possible. In order to test morphometric differences between H. gigas and H. melanogigas, we conducted a principal component analysis (PCA) and took the first two principal components of the ordination to create a MANOVA. We ran this analysis with adult males and females separately, and performed the analysis in R software (R Core Team, 2014) using the package Vegan (Oksanen et al., 2007). We followed the terminology of Dowling (1951) for counting the ventral scales, and Peters (1964) and Vanzolini, Ramos-Costa & Vitt (1980) for pholidosis. We surveyed the geographic coordinates of the data catalogs of zoological collections using Google Earth software.

Hemipenial morphology

We prepared a hemipenis from a topotype of Hydrodynastes melanogigas and 19 from H. gigas from different localities in the Amazon, East Brazil and La Plata hydrobasins (Appendix S1). Whenever possible, we prepared the hemipenes on the right side according to the technique originally described by Manzani & Abe (1988), as modified by Pesantes (1994), Zaher (1999), and Zaher & Prudente (2003). We stained the external calcareous structures with alizarin red, as suggested by Nunes et al. (2012), for a better visualization of microstructures in the surface of the organ. Terminology follows Dowling & Savage (1960), Zaher (1999) and Myers & Cadle (2003).

Molecular approach

We recovered the genus Hydrodynastes as monophyletic, and the topology of the concatenated gene tree showed two strongly supported clades with posterior probability ( pp = 1.00). Our concatenated dataset tree grouped Hydrodynastes melanogigas within H. gigas (Fig. 2). The uncorrected p-distance for both the mtDNA 16S and Cytb showed low genetic differences (0.01% and 0.2%, respectively) between the lineages of H. gigas and H. melanogigas. However, the genetic differences between H. gigas and H. bicinctus were 0.43% for 16S and 13% for Cytb (Table 5). Intraspecific variation in H. gigas was 0.0% to 0.04% for 16S and 0.0% to 0.17% for Cytb, while in H. bicinctus it was 0.0% for 16S and 0.0% to 0.23% for Cytb (Supplementary Material).

Morphological approach

We found overlap in all meristic and morphometric characteristics between Hydrodynastes gigas and H. melanogigas (Tables 3 and 4). The first principal components from both PCA analysis (males and females) recovered 99% of variation, and through the MANOVA of males (F = 2.2949; p = 0.1109) and females (F = 0.3463; p = 0.7095) we found no significant morphometric differences between both species (Fig. 3). In addition, we observed gradient levels of melanism in H. melanogigas (Figs. 4A–4L). We examined fully melanic specimens (Fig. 4A) to specimens with clear visible bands along the body (Fig. 4K). We also observed that some H. gigas individuals from the Amazon, La Plata and Tocantins-Araguaia basins present darker coloration overlapping the gradient of melanism found in H. melanogigas (Figs. 5A–5L). We did not find any morphological characteristics that differentiate the two species.

We did not observe coloration patterns within or between the populations of Hydrodynastes gigas (Figs. 6A–6R). We observed ontogenetic variation in the color pattern of all populations analyzed, with no distinction between males and females. Juveniles in the early stages of life have well-defined rounded dark spots all over their backs to the end of their tails; these spots are outlined by a lighter line, while in adults rounded spots may or may not be well defined, and may not present a clear outline (Figs. 7A–7J). Furthermore, we identified two young males of H. gigas (CHUNB 22053, Figs. 7I–7J); CHUNB 22068) from the type locality of H. melanogigas, as well as 18 more specimens from the Tocantins-Araguaia Basin. We provide more details in the “variation” section below.

Hemipenis morphology (Figs. 8A–8L): When fully everted and expanded, hemipenes of H. melanogigas and H. gigas are undistinguishable (Figs. 8K–8L). The hemipenis is deeply bilobed, semicaliculate and semicapitate, with three or four vertical rows of large spines arranged on each side of the body. The body of the hemipenis is covered by spikes on the sulcate and assulcate faces. The sulcus spermaticus bifurcates in the proximal region of the hemipenis body and each branch extends centrolinearly until it reaches the proximal region of the lobes, in which they follow a centrifugal position that ends at the lateral region of the tip of each. The capitulum, formed by papillate calyces and spikes, extends over most of the surface of the lobes, except in the region of the assulcate face that is occupied by two parallel rows of papillated and conspicuous body calyces that extend to the distal region of the hemipenis body, where they converge on the lobular crest and continues to the middle portion of the hemipenis. We detected low intraspecific variation among Hydrodynastes gigas populations. Some hemipenes showed little size variation in lobes and body calyces, varying from slightly visible to conspicuous in the hemipenial body.

Systematic account

Type material: syntype MNHN 3623

Type locality: Corrientes Province, Argentina.

Comments on the type series: Duméril, Bibron and Duméril’s (1854) description of Xenodon gigas did not refer to any voucher specimen. The authors only cited that M. A. d’Orbigny collected three individuals in Rio de La Plata, Corrientes Province, Argentina (without further information). The authors also mentioned a plate (Xénodon géant. Atlas, pi. 76, fig-5), which only presents a cranial picture. Overall, Wallach, Williams & Boundy (2014, p. 339) indicate that the types would be MNHN 2493 a-c, but according to Uetz et al. (2019), it would be an individual labeled MNHN 3623. Due to this conflicting information, we contacted the curator of the Herpetological Collection at the Muséum National d’Histoire Naturelle de Paris (Dr. N. Vidal) who confirmed that there is only one type specimen, a skin labeled MNHN 3623 (Fig. 9), deposited in the collection, and that the other two specimens appear to be lost . Since Duméril, Bibron & Duméril (1854) did not select any specific specimen from their type series, we therefore, designate the specimen MNHN 3623 as the lectotype of Hydrodynastes gigas.

Description of the lectotype MNHN 3623 (Fig. 9): Adult of undetermined sex; SVL 1570 mm; TL 540 mm; HL 62 mm; HW 35 mm; DN 10 mm; two internasals; nasal divided; one loreal; one preocular; three suboculars; two postoculars; temporal 2 + 2∕2 + 2 ; nine supralabials, none contacting the orbit; eleven infralabials, first to sixth contacting chin shields on the right side and first to fifth on the left side; two pairs of chin shields; dorsal 19/16/15 scales, smooth; two apical pits; ventral 153; subcaudals 74, paired and cloacal scale single.

Color of the preserved lectotype (ethanol 70%) (Fig. 9): Head brown with black ‘U’ shaped spot at the end of the parietal scale; post-ocular stripe that extends longitudinally on each side; supralabial brown with the last four scales stained black; infralabials and chin shields cream; dorsum of body brown with dark rounded spots that extend to the end of the tail; ventral body cream with three black stripes to the middle of the body.

Diagnosis: Hydrodynastes gigas is distinguished from its congener H. bicinctus by the following combination of characters: dorsal scales normally 19/19/15; ventral scales in males 150–168 and in females 152–172; subcaudal scales in males 58–88 and in females 49–84; maxillary teeth 15–17; two apical pits in the dorsal scales; post-ocular stripe that extends longitudinally (on each side); ventral body with three lines of continuous spots up to the middle of the body.

Variation: All variation in morphometric and meristic data are presented in Tables 3 and 4. Regarding coloration patterns, a considerable degree of color variation can be found in H. gigas (Figs. 4A–4L; 5A–5L; 6A–6R and 7A–7J) dorsum ranging from yellow to dark brown or completely dark in melanic populations; rounded spots on the dorsum may vary in shape and size, in some individuals they may be hollow or filled; in neonates the dark rounded spots are well defined throughout the dorsum until the end of the tail, and these spots are outlined by a lighter line; darks spot in the shape of ‘V’ or ‘U’ at the end of the parietal scale, only visible in non-melanic populations; ventral body cream/brown with three black stripes that usually go up to the middle of the body, rarely to the cloaca, some individuals do not have these stripes, in melanic populations the belly is dark without spots or when present these spots are continuous on the sides.

Distribution: Hydrodynastes gigas is widely distributed throughout South America, east of the Andes, occurring in Amazon, East Brazil, La Plata, North Brazil, Northeast South America, Parnaiba and Tocantins-Araguaia hydrobasins (Fig. 10).