Phylogenomics reveals novel relationships among Neotropical crocodiles (Crocodylus spp.).

Extant species in the order Crocodylia are remnants of an ancient lineage of large-bodied archosaur reptiles. Despite decades of systematic studies, phylogenetic relationships among members of the genus Crocodylus (true crocodiles) in the Neotropics are poorly understood. Here we estimated phylogenomic relationships among the four extant Crocodylus species in the Americas. Species-tree reconstructions using genotypic data from 17,538 SNPs collected for 33 individuals spanning six Crocodylus species (four ingroup and two outgroup) revealed novel relationships for all Neotropical species. For the first time, C. acutus, the American crocodile, was recovered as monophyletic when individuals from Antillean and continental populations were analyzed together. Our results also contradict previous inferences based on mitochondrial DNA data and a limited number of nuclear markers by robustly grouping Morelet's crocodile (C. moreletii) as the sister species to C. acutus, suggesting a novel phylogeographic hypothesis for the group. The present study punctuates the importance of using nuclear genome-wide information and representative sampling for resolving phylogenetic relationships, especially in broadly distributed species and those with complex evolutionary histories.

Molecular data from contemporary and historical collections reveal a complex story of cryptic diversification in the Varanus (Polydaedalus) niloticus Species Group.

Previous studies of color pattern, tongue pigmentation, and scale counts have been used to distinguish two species of semiaquatic varanids in Africa, but these findings have yet to be tested with molecular data. The Varanus (Polydaedalus) niloticus Species Group is comprised of the Nile monitor (V. niloticus) and the Ornate monitor (V. ornatus). Due to the high rate of exploitation of both species for bushmeat, the leather industry, and the pet trade, a clear understanding of the taxonomy and genetic partitioning is necessary for effective management. Here we utilize a multilocus approach, consisting of mitochondrial and nuclear markers, totaling 4251 bp, as well as microsatellite loci to assess the taxonomic validity and intraspecific evolutionary patterns within the V. niloticus Species Group. By incorporating historical specimens from museum collections as well as contemporary samples, we obtained range-wide coverage for both species across Africa. Concordant results from various approaches all suggest that V. ornatus does not represent a distinct monophyletic group. Our analyses recovered three genetic clades within V. niloticus, representing western, northern, and southern lineages. The western clade was found to diverge first, around 7.7 mya (95% HPD: 4.6-11.0 mya) and exhibits 8.4% and 8.7% uncorrected sequence divergence between the northern and southern V. niloticus clades, respectively. This geographically separate lineage corresponds to previous descriptions of Tupinambis stellatusDaudin (1802). These findings not only call for taxonomic revision of this species group, but also shed light on the biogeographic history of Africa as well as aid in the management planning of varanids and other co-distributed African species.

Fine-scale genetic analysis of the exploited Nile monitor (Varanus niloticus) in Sahelian Africa.

BACKGROUND: Overexploitation of wildlife populations results in direct consequences, such as extinction and local extirpation, as well as indirect effects including genetic diversity loss and changes in genetic structure. A clear understanding of the underlying genetic patterns of harvested species is necessary for sustainable management. The Nile monitor (Varanus niloticus) is a commercially valuable species in the international leather industry, with the highest levels of exploitation concentrated throughout Sahelian Africa. In this study, we examined the fine-scale genetic patterns of V. niloticus populations in the Sahel, with the expectation that the genetic structure would correspond to riverine drainage basins. The analyses were based on genotypes at 11 microsatellite loci for 318 individuals, spanning three separate watersheds throughout the Sahel. RESULTS: Our analyses identified four genetic clusters throughout the region, one of which (the westernmost population) exhibited very high levels of genetic differentiation (FST = 0.47). Contrary to our expectation, the largest genetic break occurred within a single watershed, the Niger basin, rather than between watersheds. However, other localities displayed evidence of reduced gene flow between watershed boundaries. Across methods, the westernmost population exhibited lower estimates of N e as well as lower levels of genetic diversity compared to the other inferred populations. While we did not detect evidence for recent population bottlenecks, our analyses indicated historic population declines around 1,000-1,800 years ago. CONCLUSION: We found that the underlying genetic structure of Varanus niloticus across Sahelian Africa reflects historic as well as present-day patterns of riverine drainages. The high degree of differentiation found for the westernmost population indicates the presence of a separate lineage, and should be taken into consideration when setting harvest limits. The historic population decline for two of the populations corresponds to a drastic expansion of an ancient human civilization in the region, suggesting that human exploitation of V. niloticus has a longer history than previously thought.

Life on the edge-a changing genetic landscape within an iconic American pika metapopulation over the last half century.

Declines and extirpations of American pika (Ochotona princeps) populations at historically occupied sites started being documented in the literature during the early 2000s. Commensurate with global climate change, many of these losses at peripheral and lower elevation sites have been associated with changes in ambient air temperature and precipitation regimes. Here, we report on a decline in available genetic resources for an iconic American pika metapopulation, located at the southwestern edge of the species distribution in the Bodie Hills of eastern California, USA. Composed of highly fragmented habitat created by hard rock mining, the ore dumps at this site were likely colonized by pikas around the end of the 19th century from nearby natural talus outcrops. Genetic data extracted from both contemporary samples and archived natural history collections allowed us to track population and patch-level genetic diversity for Bodie pikas across three distinct sampling points during the last half- century (1948-1949, 1988-1991, 2013-2015). Reductions in within-population allelic diversity and expected heterozygosity were observed across the full time period. More extensive sampling of extant patches during the 1988-1991 and 2013-2015 periods revealed an increase in population structure and a reduction in effective population size. Furthermore, census records from the last 51 years as well as archived museum samples collected in 1947 from a nearby pika population in the Wassuk range (Nevada, USA) provide further support of the increasing isolation and genetic coalescence occurring in this region. This study highlights the importance of museum samples and long-term monitoring in contextualizing our understanding of population viability.

An ancient icon reveals new mysteries: mummy DNA resurrects a cryptic species within the Nile crocodile.

The Nile crocodile (Crocodylus niloticus) is an ancient icon of both cultural and scientific interest. The species is emblematic of the great civilizations of the Nile River valley and serves as a model for international wildlife conservation. Despite its familiarity, a centuries-long dispute over the taxonomic status of the Nile crocodile remains unresolved. This dispute not only confounds our understanding of the origins and biogeography of the 'true crocodiles' of the crown genus Crocodylus, but also complicates conservation and management of this commercially valuable species. We have taken a total evidence approach involving phylogenetic analysis of mitochondrial and nuclear markers, as well as karyotype analysis of chromosome number and structure, to assess the monophyletic status of the Nile crocodile. Samples were collected from throughout Africa, covering all major bioregions. We also utilized specimens from museum collections, including mummified crocodiles from the ancient Egyptian temples at Thebes and the Grottes de Samoun, to reconstruct the genetic profiles of extirpated populations. Our analyses reveal a cryptic evolutionary lineage within the Nile crocodile that elucidates the biogeographic history of the genus and clarifies long-standing arguments over the species' taxonomic identity and conservation status. An examination of crocodile mummy haplotypes indicates that the cryptic lineage corresponds to an earlier description of C. suchus and suggests that both African Crocodylus lineages historically inhabited the Nile River. Recent survey efforts indicate that C. suchus is declining or extirpated throughout much of its distribution. Without proper recognition of this cryptic species, current sustainable use-based management policies for the Nile crocodile may do more harm than good.

A phylogenetic hypothesis for Crocodylus (Crocodylia) based on mitochondrial DNA: evidence for a trans-Atlantic voyage from Africa to the New World.

The phylogenetic relationships among extant species of Crocodylus (Crocodylia) have been inconsistently resolved by previous systematic studies. Here we used nearly complete mitochondrial (mt) genomes (∼16,200 base pairs) for all described Crocodylus species, eight of which are new to this study, to derive a generally well-supported phylogenetic hypothesis for the genus. Model-based analyses support monophyly of all Asian+Australian species and paraphyly of Crocodylus niloticus (Nile crocodile) with a monophyletic New World clade nested within this species. Wild-caught Nile crocodiles from eastern populations group robustly with the four New World species to the exclusion of Nile crocodiles from western populations, a result that is also favored by parsimony analyses and by various subpartitions of the overall mt dataset. The fossil record of Crocodylus extends back only to the Late Miocene, while the earliest fossils assigned to C. niloticus and to New World Crocodylus are Pliocene. Therefore, in combination with paleontological evidence, mt DNA trees imply a relatively recent migration of Crocodylus from Africa to the Americas, a voyage that would have covered hundreds of miles at sea.

Multilocus Metabarcoding of Terrestrial Leech Bloodmeal iDNA Increases Species Richness Uncovered in Surveys of Vertebrate Host Biodiversity.

Leech-derived invertebrate DNA (iDNA) has been successfully leveraged to conduct surveys of vertebrate host biodiversity across the Indo Pacific. However, this technique has been limited methodologically, typically only targeting mammalian 16S rDNA, or both 16S and vertebrate 12S rDNA for leech host determination. To improve the taxonomic richness of vertebrate host species in iDNA surveys, we re-analyze datasets from Bangladesh, Cambodia, China, and Madagascar through metabarcoding via next generation sequencing (NGS) of 12S, 16S (2 types, one designed to target mammals and the other, residual eDNA), nicotinamide adenine dinucleotide hydride dehydrogenase 2 (ND2), and cytochrome c oxidase subunit 1 (COI). With our 5 primer sets, we identify 41 unique vertebrate hosts to the species level, among 1,200 leeches analyzed, along with an additional 13 taxa to the family rank. Within our 41 taxa, we note that adding ND2 and COI loci increased species richness detection by 25%. NGS has emerged as more efficient than Sanger sequencing for large scale metabarcoding applications and, with the decline in cost of NGS, our pooled sample multilocus protocol is an attractive option for iDNA biodiversity surveys.

High levels of population genetic differentiation in the American crocodile (Crocodylus acutus).

The American crocodile (Crocodylus acutus) is a widely distributed species across coastal and brackish areas of the Neotropical region of the Americas and the Greater Antilles. Available information on patterns of genetic differentiation in C. acutus shows a complex structuring influenced by interspecific interactions (mainly hybridization) and anthropogenic actions (mostly historical hunting, recent poaching, habitat loss and fragmentation, and unintentional translocation of individuals). In this study, we used data on mitochondrial DNA control region and 11 nuclear polymorphic microsatellite loci to assess the degree of population structure of C. acutus in South America, North America, Central America and the Greater Antilles. We used traditional genetic differentiation indices, Bayesian clustering and multivariate methods to create a more comprehensive picture of the genetic relationships within the species across its range. Analyses of mtDNA and microsatellite loci show evidence of a strong population genetic structure in the American crocodile, with unique populations in each sampling locality. Our results support previous findings showing large degrees of genetic differentiation between the continental and the Greater Antillean C. acutus. We report three new haplotypes unique to Venezuela, which are considerably less distant from the Central and North American haplotypes than to the Greater Antillean ones. Our findings reveal genetic population differentiation between Cuban and Jamaican C. acutus and offer the first evidence of strong genetic differentiation among the populations of Greater Antillean C. acutus.

Ideating iDNA: Lessons and limitations from leeches in legacy collections.

Indirect methods for conducting faunal inventories present great promise, and genomic inventories derived from environmental sources (eDNA) are improving. Invertebrate ingested DNA (iDNA) from terrestrial leeches in the family Haemadipsidae has shown potential for surveying vertebrates and biodiversity monitoring in protected areas. Here we present an initial, and critical, evaluation of the limitations and biases of current iDNA protocols for biodiversity monitoring using both standard and NGS barcoding approaches. Key findings include the need for taxon relevant multi-locus markers and reference databases. In particular, the limitations of available reference databases have profound potential to mislead and bias eDNA and iDNA results if not critically interpreted. Nevertheless, there is great potential for recovery of amplifiable DNA from gut contents of invertebrate museum specimens which may reveal both temporal patterns and cryptic diversity in protected areas with increased efficiency. Our analyses of ingested DNA (iDNA) from both freshly stored and previously collected (legacy) samples of terrestrial leeches successfully identified vertebrates from Myanmar, Australia and Madagascar and indicate the potential to characterize microbial communities, pathogen diversity and interactions at low cost.

Historical mitochondrial diversity in African leopards (Panthera pardus) revealed by archival museum specimens.

Once found throughout Africa and Eurasia, the leopard (Panthera pardus) was recently uplisted from Near Threatened to Vulnerable by the International Union for the Conservation of Nature (IUCN). Historically, more than 50% of the leopard's global range occurred in continental Africa, yet sampling from this part of the species' distribution is only sparsely represented in prior studies examining patterns of genetic variation at the continental or global level. Broad sampling to determine baseline patterns of genetic variation throughout the leopard's historical distribution is important, as these measures are currently used by the IUCN to direct conservation priorities and management plans. By including data from 182 historical museum specimens, faecal samples from ongoing field surveys, and published sequences representing sub-Saharan Africa, we identify previously unrecognized genetic diversity in African leopards. Our mtDNA data indicates high levels of divergence among regional populations and strongly differentiated lineages in West Africa on par with recent studies of other large vertebrates. We provide a reference benchmark of genetic diversity in African leopards against which future monitoring can be compared. These findings emphasize the utility of historical museum collections in understanding the processes that shape present biodiversity. Additionally, we suggest future research to clarify African leopard taxonomy and to differentiate between delineated units requiring monitoring or conservation action.

Genetic evidence supports a distinct lineage of American crocodile (Crocodylus acutus) in the Greater Antilles.

Four species of true crocodile (genus Crocodylus) have been described from the Americas. Three of these crocodile species exhibit non-overlapping distributions-Crocodylus intermedius in South America, C. moreletii along the Caribbean coast of Mesoamerica, and C. rhombifer confined to Cuba. The fourth, C. acutus, is narrowly sympatric with each of the other three species. In this study, we sampled 113 crocodiles across Crocodylus populations in Cuba, as well as exemplar populations in Belize and Florida (USA), and sequenced three regions of the mitochondrial genome (D-loop, cytochrome b, cytochrome oxidase I; 3,626 base pair long dataset) that overlapped with published data previously collected from Colombia, Jamaica, and the Cayman Islands. Phylogenetic analyses of these data revealed two, paraphyletic lineages of C. acutus. One lineage, found in the continental Americas, is the sister taxon to C. intermedius, while the Greater Antillean lineage is most closely related to C. rhombifer. In addition to the paraphyly of the two C. acutus lineages, we recovered a 5.4% estimate of Tamura-Nei genetic divergence between the Antillean and continental clades. The reconstructed paraphyly, distinct phylogenetic affinities and high genetic divergence between Antillean and continental C. acutus populations are consistent with interspecific differentiation within the genus and suggest that the current taxon recognized as C. acutus is more likely a complex of cryptic species warranting a reassessment of current taxonomy. Moreover, the inclusion, for the first time, of samples from the western population of the American crocodile in Cuba revealed evidence for continental mtDNA haplotypes in the Antilles, suggesting this area may constitute a transition zone between distinct lineages of C. acutus. Further study using nuclear character data is warranted to more fully characterize this cryptic diversity, resolve taxonomic uncertainty, and inform conservation planning in this system.

Return of a giant: DNA from archival museum samples helps to identify a unique cutthroat trout lineage formerly thought to be extinct.

Currently one small, native population of the culturally and ecologically important Lahontan cutthroat trout (Oncorhynchus clarkii henshawi, LCT, Federally listed) remains in the Truckee River watershed of northwestern Nevada and northeastern California. The majority of populations in this watershed were extirpated in the 1940s due to invasive species, overharvest, anthropogenic water consumption and changing precipitation regimes. In 1977, a population of cutthroat trout discovered in the Pilot Peak Mountains in the Bonneville basin of Utah, was putatively identified as the extirpated LCT lacustrine lineage native to Pyramid Lake in the Truckee River basin based on morphological and meristic characters. Our phylogenetic and Bayesian genotype clustering analyses of museum specimens collected from the large lakes (1872-1913) and contemporary samples collected from populations throughout the extant range provide evidence in support of a genetically distinct Truckee River basin origin for this population. Analysis of museum samples alone identified three distinct genotype clusters and historical connectivity among water bodies within the Truckee River basin. Baseline data from museum collections indicate that the extant Pilot Peak strain represents a remnant of the extirpated lacustrine lineage. Given the limitations on high-quality data when working with a sparse number of preserved museum samples, we acknowledge that, in the end, this may be a more complicated story. However, the paucity of remnant populations in the Truckee River watershed, in combination with data on the distribution of morphological, meristic and genetic data for Lahontan cutthroat trout, suggests that recovery strategies, particularly in the large lacustrine habitats should consider this lineage as an important part of the genetic legacy of this species.

Fine scale patterns of genetic partitioning in the rediscovered African crocodile, Crocodylus suchus (Saint-Hilaire 1807).

Landscape heterogeneity, phylogenetic history, and stochasticity all influence patterns of geneflow and connectivity in wild vertebrates. Fine-scale patterns of genetic partitioning may be particularly important for the sustainable management of widespread species in trade, such as crocodiles. We examined genetic variation within the rediscovered African crocodile, Crocodylus suchus, across its distribution in West and Central Africa. We genotyped 109 individuals at nine microsatellite loci from 16 sampling localities and used three Bayesian clustering techniques and an analysis of contemporary gene flow to identify population structure across the landscape. We identified up to eight genetic clusters that largely correspond to populations isolated in coastal wetland systems and across large distances. Crocodile population clusters from the interior were readily distinguished from coastal areas, which were further subdivided by distance and drainage basin. Migration analyses indicated contemporary migration only between closely positioned coastal populations. These findings indicate high levels of population structure throughout the range of C. suchus and we use our results to suggest a role for molecular tools in identifying crocodile conservation units for this species. Further research, including additional sampling throughout the Congo and Niger drainages, would clarify both the landscape connectivity and management of this species.

Genetic diversity and structure in the Endangered Allen Cays Rock Iguana, Cyclura cychlura inornata.

The Endangered Allen Cays Rock Iguana (Cyclura cychlura inornata) is endemic to the Allen Cays, a tiny cluster of islands in the Bahamas. Naturally occurring populations exist on only two cays (<4 ha each). However, populations of unknown origin were recently discovered on four additional cays. To investigate patterns of genetic variation among these populations, we analyzed nuclear and mitochondrial markers for 268 individuals. Analysis of three mitochondrial gene regions (2,328 bp) and data for eight nuclear microsatellite loci indicated low genetic diversity overall. Estimates of effective population sizes based on multilocus genotypes were also extremely low. Despite low diversity, significant population structuring and variation in genetic diversity measures were detected among cays. Genetic data confirm the source population for an experimentally translocated population while raising concerns regarding other, unauthorized, translocations. Reduced heterozygosity is consistent with a documented historical population decline due to overharvest. This study provides the first range-wide genetic analysis of this subspecies. We suggest strategies to maximize genetic diversity during ongoing recovery including additional translocations to establish assurance populations and additional protective measures for the two remaining natural populations.

Combining genetic and distributional approaches to sourcing introduced species: a case study on the Nile monitor (Varanus niloticus) in Florida.

Three separate breeding populations of the Nile monitor (Varanus niloticus) have been identified in Florida, USA, located in Cape Coral, West Palm Beach and Homestead Air Reserve Base. This large, predatory lizard could have negative effects on Florida's native wildlife. Here, we infer the source of the introduced populations using genetic and statistical approaches, as well as estimate the potential non-native distribution of V. niloticus in North America. We collected genetic data from 25 Florida individuals as well as utilized genetic datasets from reference individuals spanning the full native distribution throughout sub-Saharan Africa. Using occurrence data from the inferred source population and the full species range, we built ecological niche models (ENMs) and projected them onto North America to determine regions with suitable climate. Our results indicated that the introduced populations resulted from three separate introduction events, and all originated from the southern coastal region of West Africa. The ENM built from the West African source population predicted only the southernmost portions of North America to be suitable. Conversely, the model derived from the full species' range predicted suitable climates across a large portion of the United States. This information can be used to focus management and eradication efforts.

Native fishes in the Truckee River: Are in-stream structures and patterns of population genetic structure related?

In-stream structures are recognized as significant impediments to movement for freshwater fishes. Apex predators such as salmonids have been the focus of much research on the impacts of such barriers to population dynamics and population viability however much less research has focused on native fishes, where in-stream structures may have a greater impact on long term population viability of these smaller, less mobile species. Patterns of genetic structure on a riverscape can provide information on which structures represent real barriers to movement for fish species and under what specific flow conditions. Here we characterize the impact of 41 dam and diversion structures on movement dynamics under varying flow conditions for a suite of six native fishes found in the Truckee River of California and Nevada. Microsatellite loci were used to estimate total allelic diversity, effective population size and assess genetic population structure. Although there is spatial overlap among species within the river there are clear differences in species distributions within the watershed. Observed population genetic structure was associated with in-stream structures, but only under low flow conditions. High total discharge in 2006 allowed fish to move over potential barriers resulting in no observed population genetic structure for any species in 2007. The efficacy of in-stream structures to impede movement and isolate fish emerged only after multiple years of low flow conditions. Our results suggest that restricted movement of fish species, as a result of in-stream barriers, can be mitigated by flow management. However, as flow dynamics are likely to be altered under global climate change, fragmentation due to barriers could isolate stream fishes into small subpopulations susceptible to both demographic losses and losses of genetic variation.

Integrating molecular, phenotypic and environmental data to elucidate patterns of crocodile hybridization in Belize.

The genus Crocodylus comprises 12 currently recognized species, many of which can be difficult to differentiate phenotypically. Interspecific hybridization among crocodiles is known to occur in captivity and has been documented between some species in the wild. The identification of hybrid individuals is of importance for management and monitoring of crocodilians, many of which are Convention on International Trade in Endangered Species (CITES) listed. In this study, both mitochondrial and nuclear DNA markers were evaluated for their use in confirming a suspected hybrid zone between American crocodile (Crocodylus acutus) and Morelet's crocodile (Crocodylus moreletii) populations in southern Belize where individuals and nests exhibiting atypical phenotypic features had previously been observed. Patterns observed in both phenotypic and molecular data indicate possible behavioural and ecological characteristics associated with hybridization events. The results of the combined analyses found that the majority of suspected hybrid samples represent crosses between female C. acutus and male C. moreletii. Phenotypic data could statistically identify hybrids, although morphological overlap between hybrids and C. moreletii reduced reliability of identification based solely on field characters. Ecologically, C. acutus was exclusively found in saline waters, whereas hybrids and C. moreletii were largely absent in these conditions. A hypothesized correlation between unidirectional hybridization and destruction of C. acutus breeding habitats warrants additional research.