Deconstructing a Species-Complex: Geometric Morphometric and Molecular Analyses Define Species in the Western Rattlesnake (Crotalus viridis)

Abstract

Morphological data are a conduit for the recognition and description of species, and their acquisition has recently been broadened by geometric morphometric (GM) approaches that co-join the collection of digital data with exploratory ‘big data’ analytics. We employed this approach to dissect the Western Rattlesnake (Crotalus viridis) species-complex in North America, currently partitioned by mitochondrial (mt)DNA analyses into eastern and western lineages (two and seven subspecies, respectively). The GM data (i.e., 33 dorsal and 50 lateral head landmarks) were gleaned from 2,824 individuals located in 10 museum collections. We also downloaded and concatenated sequences for six mtDNA genes from the NCBI GenBank database. GM analyses revealed significant head shape differences attributable to size and subspecies-designation (but not their interactions). Pairwise shape distances among subspecies were significantly greater than those derived from ancestral character states via squared-change parsimony, with the greatest differences separating those most closely related. This, in turn, suggests the potential for historic character displacement as a diversifying force in the complex. All subspecies, save one, were significantly differentiated in a Bayesian discriminant function analysis (DFA), regardless of whether our priors were uniform or informative (i.e., mtDNA data). Finally, shape differences among sister-clades were significantly greater than expected by chance alone under a Brownian model of evolution, promoting the hypothesis that selection rather than drift was the driving force in the evolution of the complex. Lastly, we combine head shape and mtDNA data so as to derived an integrative taxonomy that produced robust boundaries for six OTUs (operational taxonomic units) of the C. viridis complex. We suggest these boundaries are concomitant with species-status and subsequently provide a relevant nomenclature for its recognition and representation.

Extremely Divergent Haplotypes in Two Toxin Gene Complexes Encode Alternative Venom Types within Rattlesnake Species

“…This appears to be a very plausible explanation in light of the present range of neurotoxic C. helleri being very close to the present extent of the range of C. scutulatus [8, 14], and viable hybrids having been obtained between C. scutulatus and C. helleri in captivity [40]. We note that these two species are estimated to have shared a common ancestor 2–4 mya [31].…”

“…The C. oreganus dataset contained multiple species, including C. helleri, and was processed to show the known C. helleri range that has been described previously [14]. …”

“…For example, the widely dispersed, predominantly neurotoxic Mojave rattlesnake (C. scutulatus) includes geographically restricted, non-neurotoxic, hemorrhagic populations (Figure 1A [red dots on light blue]) [10–12], whereas the Southern Pacific rattlesnake (C. helleri [14]) is predominantly hemorrhagic but includes a geographically restricted neurotoxic population (Figure 1A [blue dot on yellow]) [8, 9] and the timber rattlesnake ( C. horridus ) includes widely dispersed individuals of both types (Figure 1A [blue dots on green]) [13]. In general, neurotoxic individuals express high levels of a heterodimeric neurotoxin (composed of acidic and basic phospholipase A2 [PLA2] polypeptides) and have been designated type A venoms [11], while hemorrhagic individuals produce undetectable levels of neurotoxin [11, 15] and express high levels of venom metalloproteinases (MPs) [15, 16], an abundant component of hemorrhagic venoms [17, 18].…”

Congruent Molecular and Morphological Diversity of Macaronesian Limpets: Insights into eco-evolutionary Forces and Tools for Conservation

“…The development of geometric morphometric techniques has considerably advanced morphological studies (Davis et al, 2016) and in comparison to traditional distance-based methods permits a more detailed description of phenotypic variation. For both P. aspera and P. rustica, Macaronesian and continental populations were observed to be highly genetically diverged and reciprocally monophyletic.…”

DNA Barcodes from Snake Venom: a Broadly Applicable Method for Extraction of DNA from Snake Venoms

“…Further, because venom components vary taxonomically, geographically, ontogenetically and with dietary preference [12,13], venomic analysis alone is unlikely to provide definitive species determination of unknown samples. Recent revision or clarification of taxonomic relationships within venomous snakes has led to the invalidation of previous species delineations, with the concomitant complication of identification of venom samples collected without voucher reference specimens of the snakes [1423,24,25]. Because snake higher taxonomy is still disputed and warrants futher investigation, it is imperative to couple phenotypic data (in this case, venom components and protein variation) with genotypic data [15,21].…”