Edgar Benavides

DNA from the Past Informs Ex Situ Conservation for the Future: An “Extinct” Species of Galápagos Tortoise Identified in Captivity

Background Although not unusual to find captive relicts of species lost in the wild, rarely are presumed extinct species rediscovered outside of their native range. A recent study detected living descendents of an extinct Galápagos tortoise species (Chelonoidis elephantopus) once endemic to Floreana Island on the neighboring island of Isabela. This finding adds to the growing cryptic diversity detected among these species in the wild. There also exists a large number of Galápagos tortoises in captivity of ambiguous origin. The recently accumulated population-level haplotypic and genotypic data now available for C. elephantopus add a critical reference population to the existing database of 11 extant species for investigating the origin of captive individuals of unknown ancestry. Methodology/Findings We reanalyzed mitochondrial DNA control region haplotypes and microsatellite genotypes of 156 captive individuals using an expanded reference database that included all extant Galápagos tortoise species as well as the extinct species from Floreana. Nine individuals (six females and three males) exhibited strong signatures of Floreana ancestry and a high probability of assignment to C. elephantopus as detected by Bayesian assignment and clustering analyses of empirical and simulated data. One male with high assignment probability to C. elephantopus based on microsatellite genotypic data also possessed a “Floreana-like” mitochondrial DNA haplotype. Significance Historical DNA analysis of museum specimens has provided critical spatial and temporal components to ecological, evolutionary, taxonomic and conservation-related research, but rarely has it informed ex situ species recovery efforts. Here, the availability of population-level genotypic data from the extinct C. elephantopus enabled the identification of nine Galápagos tortoise individuals of substantial conservation value that were previously misassigned to extant species of varying conservation status. As all captive individuals of C. elephantopus ancestry currently reside at a centralized breeding facility on Santa Cruz, these findings permit breeding efforts to commence in support of the reestablishment of this extinct species to its native range.

Lineage fusion in Galápagos giant tortoises.

Although many classic radiations on islands are thought to be the result of repeated lineage splitting, the role of past fusion is rarely known because during these events, purebreds are rapidly replaced by a swarm of admixed individuals. Here, we capture lineage fusion in action in a Galápagos giant tortoise species, Chelonoidis becki, from Wolf Volcano (Isabela Island). The long generation time of Galápagos tortoises and dense sampling (841 individuals) of genetic and demographic data were integral in detecting and characterizing this phenomenon. In C. becki, we identified two genetically distinct, morphologically cryptic lineages. Historical reconstructions show that they colonized Wolf Volcano from Santiago Island in two temporally separated events, the first estimated to have occurred ~199 000 years ago. Following arrival of the second wave of colonists, both lineages coexisted for approximately ~53 000 years. Within that time, they began fusing back together, as microsatellite data reveal widespread introgressive hybridization. Interestingly, greater mate selectivity seems to be exhibited by purebred females of one of the lineages. Forward‐in‐time simulations predict rapid extinction of the early arriving lineage. This study provides a rare example of reticulate evolution in action and underscores the power of population genetics for understanding the past, present and future consequences of evolutionary phenomena associated with lineage fusion.

Description of a New Galapagos Giant Tortoise Species (Chelonoidis; Testudines: Testudinidae) from Cerro Fatal on Santa Cruz Island

The taxonomy of giant Galapagos tortoises (Chelonoidis spp.) is currently based primarily on morphological characters and island of origin. Over the last decade, compelling genetic evidence has accumulated for multiple independent evolutionary lineages, spurring the need for taxonomic revision. On the island of Santa Cruz there is currently a single named species, C. porteri. Recent genetic and morphological studies have shown that, within this taxon, there are two evolutionarily and spatially distinct lineages on the western and eastern sectors of the island, known as the Reserva and Cerro Fatal populations, respectively. Analyses of DNA from natural populations and museum specimens, including the type specimen for C. porteri, confirm the genetic distinctiveness of these two lineages and support elevation of the Cerro Fatal tortoises to the rank of species. In this paper, we identify DNA characters that define this new species, and infer evolutionary relationships relative to other species of Galapagos tortoises.

Lineage Identification and Genealogical Relationships Among Captive Galápagos Tortoises

Genetic tools have become a critical complement to traditional approaches for meeting short- and long-term goals of ex situ conservation programs. The San Diego Zoo (SDZ) harbors a collection of wild-born and captive-born Galápagos giant tortoises (n = 22) of uncertain species designation and unknown genealogical relationships. Here, we used mitochondrial DNA haplotypic data and nuclear microsatellite genotypic data to identify the evolutionary lineage of wild-born and captive-born tortoises of unknown ancestry, to infer levels of relatedness among founders and captive-born tortoises, and assess putative pedigree relationships assigned by the SDZ studbook. Assignment tests revealed that 12 wild-born and five captive-born tortoises represent five different species from Isabela Island and one species from Santa Cruz Island, only five of which were consistent with current studbook designations. Three wild-born and one captive-born tortoise were of mixed ancestry. In addition, kinship analyses revealed two significant first-order relationship pairs between wild-born and captive-born tortoises, four second-order relationships (half-sibling) between wild-born and captive tortoises (full-sibs or parent-offspring), and one second-order relationship between two captive-born tortoises. Of particular note, we also reconstructed a first-order relationship between two wild-born individuals, violating the founder assumption. Overall, our results contribute to a worldwide effort in identifying genetically important Galápagos tortoises currently in captivity while revealing closely related founders, reconstructing genealogical relationships, and providing detailed management recommendations for the SDZ tortoises.

Naturally rare versus newly rare: demographic inferences on two timescales inform conservation of Galapagos giant tortoises.

Long-term population history can influence the genetic effects of recent bottlenecks. Therefore, for threatened or endangered species, an understanding of the past is relevant when formulating conservation strategies. Levels of variation at neutral markers have been useful for estimating local effective population sizes (Ne) and inferring whether population sizes increased or decreased over time. Furthermore, analyses of genotypic, allelic frequency, and phylogenetic information can potentially be used to separate historical from recent demographic changes. For 15 populations of Galápagos giant tortoises (Chelonoidis sp.), we used 12 microsatellite loci and DNA sequences from the mitochondrial control region and a nuclear intron, to reconstruct demographic history on shallow (past ∽100 generations, ∽2500 years) and deep (pre-Holocene, >10 thousand years ago) timescales. At the deep timescale, three populations showed strong signals of growth, but with different magnitudes and timing, indicating different underlying causes. Furthermore, estimated historical Ne of populations across the archipelago showed no correlation with island age or size, underscoring the complexity of predicting demographic history a priori. At the shallow timescale, all populations carried some signature of a genetic bottleneck, and for 12 populations, point estimates of contemporary Ne were very small (i.e., < 50). On the basis of the comparison of these genetic estimates with published census size data, Ne generally represented ∽0.16 of the census size. However, the variance in this ratio across populations was considerable. Overall, our data suggest that idiosyncratic and geographically localized forces shaped the demographic history of tortoise populations. Furthermore, from a conservation perspective, the separation of demographic events occurring on shallow versus deep timescales permits the identification of naturally rare versus newly rare populations; this distinction should facilitate prioritization of management action.

Phylogenetic inference of nuptial trait evolution in the context of asymmetrical introgression in North American darters (teleostei).

Introgressive hybridization and incomplete lineage sorting complicate the inference of phylogeny, and available species‐tree methods do not simultaneously account for these processes. Both hybridization and ancestral polymorphism have been invoked to explain divergent phylogenies inferred from different datasets for Stigmacerca, a clade of 11 North American darter species. Species of Stigmacerca are characterized by a mating system involving parental care with males guarding nesting territories and fertilized eggs. Males of four species of Stigmacerca develop egg‐mimic nuptial structures on their second dorsal fins during the breeding season. Previous phylogenies suggest contrasting scenarios for the evolution of this nuptial trait. Using a combination of coalescent‐based methods, we analyzed a dataset comprising a mitochondrial gene and 15 nuclear loci to estimate relationships and simultaneously test for introgressive hybridization. Our analyses identified several instances of interspecific gene flow involving both cytoplamsmic haplotypes and nuclear alleles. The new phylogeny was used to infer a single origin and recent loss of egg‐mimic structures in Stigmacerca and led to the discovery of a phylogenetically distinct species. Our results highlight the limited strategies available to account for introgressive hybridization in the inference of species relationships and the likely effects of this process on reconstructing trait evolution.

From species divergence to population structure: A multimarker approach on the most basal lineage of Salamandridae, the spectacled salamanders (genus Salamandrina) from Italy

The Apennine Peninsula is one of Europes main glacial refugial areas and harbors a large number of lineages and species. Here, a pattern of higher genetic diversity in the south compared to that of the north is characteristic of most vertebrates; however, most studies that have produced these results have relied only on inferences based on mitochondrial DNA. The spectacled salamanders (genus Salamandrina) are endemic to the Apennine Peninsula and have diverged into two sibling species: S. terdigitata (in the south) and S. perspicillata (in the north), presumably in the late Miocene or early Pliocene. By sequencing one mitochondrial (cytb) and two nuclear genes (RAG1 and POMC) and genotyping 10 microsatellite loci, we traced the evolution of these sibling species from their divergence to their contemporary population structure at a fine scale. Using a multilocus coalescent-based approach, we estimated the temporal divergence of both species at approximately 2.25 mya (million years ago), which, hence, is much younger than previous estimates. The classical pattern of high genetic diversity in the south and lower diversity in the north was confirmed only for some markers, and the demographic histories of the two species differed substantially. Whereas S. perspicillata (north) expanded from a single major refugium in the center of the Apennine Peninsula, populations of S. terdigitata (south) persisted through cooler periods in multiple refugia. Further, the fine-scale population genetic structure of 16 S. perspicillata populations revealed significant genetic differentiation, even across short geographic distances. The results of our study stress that for a better understanding of phylogeographic patterns and past demographic processes, both mitochondrial and multiple nuclear loci should be analyzed to avoid gene-specific, and possibly biased results.

Genetic Differentiation between Marine Iguanas from Different Breeding Sites on the Island of Santa Fé (Galápagos Archipelago)

We studied patterns of genetic diversity within and among 5 populations (318 individuals) of Galápagos marine iguanas (Amblyrhynchus cristatus) from the island Santa Fé. Populations were separated by distances of 0.2 to 9.9 km. We sequenced 1182 base pairs of the mitochondrial control region and screened 13 microsatellite loci for variability. We also added data from 5 populations (397 individuals) sampled on 4 neighboring islands (Santa Cruz, Floreana, Espanola, and San Cristobal). The 5 Santa Fé populations, revealed as genetically distinct from populations on other islands, present relatively low levels of genetic diversity, which are similar for both microsatellite (average observed heterozygosity from 0.7686 to 0.7773) and mitochondrial DNA (mtDNA) markers (haplotypic and nucleotide diversity from 0.587 to 0.728 and from 0.00079 to 0.00293, respectively), and comparable with those observed in similar-sized sampling sites on other islands. There was frequency-based evidence of genetic structure between northern and southern sites on Santa Fé (F(st) of 0.0027-0.0115 for microsatellite and 0.0447-0.2391 for mtDNA), but the 4 southern sites showed little differentiation. Most of the intra-island genetic variation was allocated within rather than between sites. There was no evidence of sex-biased dispersal or population substructuring due to lek-mating behavior, suggesting that these 2 observed behaviors are not strong enough to leave an evolutionary signal on genetic patterns in this species.

Small size does not restrain frugivory and seed dispersal across the evolutionary radiation of Galápagos lava lizards

Abstract Frugivory in lizards is often assumed to be constrained by body size; only large individuals are considered capable of consuming fruits, with the potential of acting as seed dispersers. However, only one previous study has tested the correlation of frugivory with body and head size at an archipelago scale across closely related species. All nine lava lizards (Microlophus spp.) were studied on the eleven largest Galápagos islands from 2010 to 2016 to investigate whether frugivory is related to body and head size. We also tested whether fruit abundance influences fruit consumption and explored the effect of seed ingestion on seedling emergence time and percentage. Our results showed that across islands, lava lizards varied considerably in size (64–102 mm in mean snout–vent length) and level of frugivory (1–23%, i.e., percentage of droppings with seeds). However, level of frugivory was only weakly affected by size as fruit consumption was also common among small lizards. Lava lizards consumed fruits throughout the year and factors other than fruit abundance may be more important drivers of fruit selection (e.g., fruit size, energy content of pulp). From 2,530 droppings, 1,714 seeds of at least 61 plant species were identified, 76% of the species being native to the Galápagos. Most seeds (91%) showed no external structural damage. Seedling emergence time (44 versus 118 days) and percentage (20% versus 12%) were enhanced for lizard-ingested seeds compared to control (uningested) fruits. De-pulping by lizards (i.e., removal of pulp with potential germination inhibitors) might increase the chances that at least some seeds find suitable recruitment conditions. We concluded that lizards are important seed dispersers throughout the year and across the whole archipelago, regardless of body size.

Phylogenetic and Morphological Diversity of the Etheostoma zonistium Species Complex with the Description of a New Species Endemic to the Cumberland Plateau of Alabama

Abstract We provide a description of the Blueface Darter, Etheostoma cyanoprosopum, which is distributed in the upper Sipsey Fork of the Mobile Basin and the upper portion of the Bear Creek system in the Tennessee River Drainage. The distinctiveness of Etheostoma cyanoprosopum is assessed through analysis of morphological variation and molecular phylogenetic diversity within the Etheostoma zonistium species complex. In addition to analyzing disparity of morphometric and meristic traits, we present phylogenetic analyses of a mitochondrial gene and two nuclear genes and identify genetic clusters through analysis of 25 microsatellite loci. In the mitochondrial DNA (mtDNA) gene tree, Etheostoma cyanoprosopumis resolved as the sister lineage to a clade containing all other species of the Etheostoma zonistium complex. Etheostoma zonistium is paraphyletic with respect to both Etheostoma pyrrhogaster and Etheostoma cervus, which do not resolve as sister species in the mtDNA gene tree. The two nuclear gene trees are much less resolved, but the S7 ribosomal protein intron 1 (S7) gene tree resolves Etheostoma cyanoprosopum and all sampled populations of Etheostoma zonistium as a clade with strong Bayesian posterior node support. Etheostoma cyanoprosopum is morphologically differentiated from Etheostoma zonistium by a shallower body, a more elongate nape, a higher number of lateral line scales, a higher number of transverse scale rows, and differences in coloration.