A genomic view on the adaptation and diversification of natural populations

Earlier studies in evolutionary genetics focused on a few model organisms such as fruit flies or mice that are limited when it comes to answering evolutionary and ecological questions. In contrast, genetic studies of natural populations have now become common and can provide a more realistic understanding of how natural selection, genetic drift, mutation, and gene flow shape the patterns of phenotypic and genetic diversity, as well as adaptation and diversification across a range of environmental conditions. This dissertation illustrates how current genomic tools can be effectively used to (1) study the evolutionary history of species along altitudinal gradients, and (2) understand the genetic basis of adaptive phenotypes in populations inhabiting high-altitude habitats. In the first research paper, we compared whole-genome resequencing data of Eastern honey bee (Apis cerana) populations from high and low altitudes in southwestern China. We identified several regions of the genome that appeared to have been under positive selection in highland bee populations. Candidate loci in these genomic regions included genes related to reproduction and feeding behavior. In the second paper, we generated a transcriptome reference for the Neotropical frogs of the genus Oreobates by sequencing RNA from one individual of the La Paz robber frog (Oreobates cruralis). In the third paper, we used that transcriptome to selectively target and enrich ~18,000 genes across species of Oreobates collected along the Andean Mountains, in South America. We found that highland species have smaller effective populations and accumulate nonsynonymous mutations faster than species sampled at lower altitudes. These mutations can be targeted by natural selection and contribute to the adaptation and differentiation of populations in mountain environments. In the fourth and final paper, we pointed out that genomics has to be integrated with other sources of evidence to understand evolutionary and ecological processes more deeply than was thought in the past

Población introducida de tritón alpino (Mesotriton alpestris) en el Prepirineo catalán

Peer reviewe

Unexpectedly high levels of cryptic diversity uncovered by a complete DNA barcoding of reptiles of the Socotra Archipelago

Few DNA barcoding studies of squamate reptiles have been conducted. Due to the signifi- cance of the Socotra Archipelago (a UNESCO Natural World Heritage site and a biodiversity hotspot) and the conservation interest of its reptile fauna (94% endemics), we performed the most comprehensive DNA barcoding study on an island group to date to test its applicability to specimen identification and species discovery. Reptiles constitute Socotra ' s most impor- tant vertebrate fauna, yet their taxonomy remains under-studied. We successfully DNA-bar-coded 380 individuals of all 31 presently recognized species. The specimen identification success rate is moderate to high, and almost all species presented local barcoding gaps.The unexpected high levels of intra-specific variability found within some species suggest cryptic diversity. Species richness may be under-estimated by 13.8-54.4%. This has implications in the species ranges and conservation status that should be considered for conservation planning. Other phylogenetic studies using mitochondrial and nuclear markers are congruent with our results. We conclude that, despite its reduced length (663 base pairs), cytochrome c oxidase 1, COI, is very useful for specimen identification and for detecting intra-specific diversity, and has a good phylogenetic signal. We recommend DNA barcoding to be applied to other biodiversity hotspots for quickly and cost-efficiently flagging species discovery, preferentially incorporated into an integrative taxonomic framework

A genomic view on the adaptation and diversification of natural populations

A dissertation submitted in fulfillment of the requirements for the degree of Doctor of Philosophy at University of Seville, 2019Earlier studies in evolutionary genetics focused on a few model organisms such as fruit flies or mice that are limited when it comes to answering evolutionary and ecological questions. In contrast, genetic studies of natural populations have now become common and can provide a more realistic understanding of how natural selection, genetic drift, mutation, and gene flow shape the patterns of phenotypic and genetic diversity, as well as adaptation and diversification across a range of environmental conditions. This dissertation illustrates how current genomic tools can be effectively used to (1) study the evolutionary history of species along altitudinal gradients, and (2) understand the genetic basis of adaptive phenotypes in populations inhabiting high-altitude habitats. In the first research paper, we compared whole-genome resequencing data of Eastern honey bee (Apis cerana) populations from high and low altitudes in southwestern China. We identified several regions of the genome that appeared to have been under positive selection in highland bee populations. Candidate loci in these genomic regions included genes related to reproduction and feeding behavior. In the second paper, we generated a transcriptome reference for the Neotropical frogs of the genus Oreobates by sequencing RNA from one individual of the La Paz robber frog (Oreobates cruralis). In the third paper, we used that transcriptome to selectively target and enrich ~18,000 genes across species of Oreobates collected along the Andean Mountains, in South America. We found that highland species have smaller effective populations and accumulate nonsynonymous mutations faster than species sampled at lower altitudes. These mutations can be targeted by natural selection and contribute to the adaptation and differentiation of populations in mountain environments. In the fourth and final paper, we pointed out that genomics has to be integrated with other sources of evidence to understand evolutionary and ecological processes more deeply than was thought in the pastPeer reviewe

Another record or a new taxon? A candidate species of Chalcides Laurenti, 1768, in north Africa

The first author is funded by an FPI (Formación de Personal Investigador) grant from the Ministerio de Economía y Competitividad, Spain (BES-2014-069006).N

Phylogenomics and evolutionary history of Oreobates (Anura: Craugastoridae) Neotropical frogs along elevational gradients

Mountain ranges offer opportunities for understanding how species evolved and diversified across different environmental conditions. Neotropical frogs of the genus Oreobates (Anura: Craugastoridae) are adapted to highland and lowland habitats along the Andes, but many aspects of their evolution remain unknown. We studied their evolutionary history using ~18,000 exons enriched by targeted sequence-capture. Since capture success was very variable across samples, we evaluated to what degree differing data filtering produced robust inferences. The inferred evolutionary framework evidenced phylogenetic discordances among lowland species that can be explained by taxonomic misidentification or admixture of ancestral lineages. Highland species showed smaller effective populations than lowland frogs, probably due to greater habitat fragmentation in montane environments. Stronger genetic drift likely decreased the power of purifying selection and led to an increased proportion of nonsynonymous mutations in highland populations that could play an important role in their adaptation. Overall, our work sheds light on the evolutionary history and diversification of this group of Neotropical frogs along elevational gradients in the Andes as well as on their patterns of intraspecific diversity.This research was supported by grants from the Spanish Government (Ministerio de Economía y Competitividad) to C.V. (CGL2013-47547-P and CGL2016-75227-P), I.D.l.R (CGL2011-30393), and an FPI (Formación de Personal Investigador) fellowship (BES‐2014‐069006) as well as three travel grants (EEBB‐I‐16‐10576, EEBB‐I‐17‐12168, and EEBB‐I‐18‐12878) to S.M.-M.Peer reviewe

Phylogenomics and evolutionary history of Oreobates (Anura: Craugastoridae) Neotropical frogs along elevational gradients

Mountain ranges offer opportunities for understanding how species evolved and diversified across different environmental conditions. Neotropical frogs of the genus Oreobates (Anura: Craugastoridae) are adapted to highland and lowland habitats along the Andes, but many aspects of their evolution remain unknown. We studied their evolutionary history using ~18,000 exons enriched by targeted sequence-capture. Since capture success was very variable across samples, we evaluated to what degree differing data filtering produced robust inferences. The inferred evolutionary framework evidenced phylogenetic discordances among lowland species that can be explained by taxonomic misidentification or admixture of ancestral lineages. Highland species showed smaller effective populations than lowland frogs, probably due to greater habitat fragmentation in montane environments. Stronger genetic drift likely decreased the power of purifying selection and led to an increased proportion of nonsynonymous mutations in highland populations that could play an important role in their adaptation. Overall, our work sheds light on the evolutionary history and diversification of this group of Neotropical frogs along elevational gradients in the Andes as well as on their patterns of intraspecific diversity.This research was supported by grants from the Spanish Government (Ministerio de Economía y Competitividad) to C.V. (CGL2013-47547-P and CGL2016-75227-P), I.D.l.R (CGL2011-30393), and an FPI (Formación de Personal Investigador) fellowship (BES‐2014‐069006) as well as three travel grants (EEBB‐I‐16‐10576, EEBB‐I‐17‐12168, and EEBB‐I‐18‐12878) to S.M.-M.Peer reviewe

Eleutherodactylus cattus Rodríguez, Dugo-Cota, Montero-Mendieta, Gonzalez-Voyer, Bosch, Vences & Vilà, 2017, sp. n.

<i>Eleutherodactylus cattus</i> sp. n. <p>(Fig. 5 A,B)</p> <p> <b>Holotype.</b> CZACC14.14152, adult male collected while vocalizing in the trail to Pico El Gato, Sierra del Cobre, 20.01364 N, 76.04809 W, 844 m a.s.l, by A. Rodríguez and R. Alonso in May 2010.</p> <p> <b>Paratypes.</b> CZACC 14.14150–51, 14.14153–60, adult males with the same data as the holotype.</p> <p> <b>Etymology.</b> The species name is an invariable noun in apposition to the genus name, derived from Latin <i>cattus</i> = cat. It refers to the type locality Loma del Gato (Cat Mountain Ridge) in the Sierra Maestra Mountains, a locality that was neglected for long time in herpetological explorations of Cuba and that surely deserves further attention.</p> <p> <b>Diagnosis.</b> A small species of <i>Eleutherodactylus</i> that can be assigned to the subgenus <i>Eleutherodactylus</i> based on its genetic, acoustic, and morphological similarities with members of the <i>E. auriculatus</i> species group (Hedges, <i>et al.</i>, 2008; Rodríguez, <i>et al.</i>, 2010b). It is most closely related to <i>E. glamyrus</i> with which it shares several morphological, ecological, and behavioral features. However, males of <i>Eleutherodactylus cattus</i> can be readily differentiated from <i>E. glamyrus</i> by their larger size (SVL = 23.1–24.7 mm in <i>E. cattus</i> vs 17.4–21.3 mm in <i>E. glamyrus</i>) and the following combination of advertisement call features: longer duration (call duration = 128.4– 243.1 ms in <i>E. cattus</i> vs 47.2–112 ms in <i>E. glamyrus</i>), longer rise time (call rise time = 16–173.7 ms in <i>E. cattus</i> vs 1.9–32.2 ms in <i>E. glamyrus</i>), lower dominant frequency (dominant frequency = 3036.2–3337.6 Hz in <i>E. cattus</i> vs 3006–3799.6 Hz in <i>E. glamyrus</i>), and greater frequency modulation (frequency modulation = 140.9–332.8 Hz in <i>E. cattus</i> vs 73.2–262.3 Hz in <i>E. glamyrus</i>). Additionally, <i>E. cattus</i> differs from <i>E. glamyrus</i> in the studied mitochondrial DNA sequences by 8.2% (<i>cob</i>) and 3.2% (<i>16S</i>) and by one substitution in the nuclear <i>Rag-1</i> gene (see results).</p> <p> <b>Description.</b> Head as wide as body, width smaller than length; snout subacute in dorsal and lateral views overhanging the jaw; narines laterally oriented and moderately protuberant; rostral canthus rounded and straight, loreal region smooth and abruptly tilted, lips not enlarged. Superior eyelids with conical and small tubercles also present but less evident in the interorbital area. Tympanum present without supratympanic fold, postrictal tubercles present. Coanae oval, partially concealed by the maxillary arch in ventral view. Vomerine odontophores short, straight, separated and nearly perpendicular to the longitudinal body axis. Tongue rounded, longer than wide with notched posterior edge, fixed by the anterior edge and free in its 3/4 of length. Vocal slits and enlarged vocal sac present in males.</p> <p>Dorsal skin slightly tuberculated, without dorsolateral folds, and less tuberculated in the lower half of flanks. Ventral skin slightly areolated but without folds. Cloacae not expanded. Glandular regions not evident. Hands with ulnar tubercle subconical, palmar tubercle simple and of similar size as the thenar; thenar tubercle oval and pronounced. Supernumerary tubercles absent; fingers with rounded and subconical subarticular tubercles and without lateral expansions. Finger tips rounded and expanded in all cases with the ventral surface forming a circular digital pad bordered by a circumferential groove in 2/3 of its distal edge. Width of the largest toe pad (finger III) roughly similar to tympanum diameter. Finger order III> II> IV> I. One moderate tubercle present on heel, no tubercles on the outer edge of the tarsal, metatarsal tubercles subconical and smaller than subarticular tubercles; internal oval, same size as external; plantar supernumerary tubercles absent; subarticular tubercles rounded and subconical. Toes not webbed, toe tips rounded and expanded in all cases with the ventral surface forming a circular digital pad bordered by a circumferential groove in 2/3 of its distal edge. Heels overlap when thighs are placed perpendicularly to the longitudinal body axis. Toe size order IV> III> V> II> I.</p> <p> <b>Color in alcohol.</b> Color pattern was similar among the individuals of the type series. Dorsum varies from brownish to beige with darker mottled and two mostly well-defined dark brownish or black anterior dorsolateral marks; the flanks show a similar pattern, lighter from dorsum to belly, a relatively wide dark brown strip from the insertion of the forelimb to the eye; belly pale and throat (vocal sac) from yellowish to light brown, underside of the limbs slightly yellowish (see Figure 5 A–B for dorsal and ventral views of the holotype).</p> <p> <b>Color in life.</b> Dorsum color varies from brownish to greenish tan, with an interocular bar of darker brown color followed by an X shaped mark both darker than the background color. A distinctive pattern of chevron-like bands is evident in the sacral region extending to the hind legs. Eyelids with a greenish wash, pupils creamy to coopery colored with horizontal slit, iris black. The flanks are lighter colored than the dorsum, tympanum creamy white. Loreal region color varies from tan to greenish with an obvious black stripe that extends from snout to supratympanic fold and becomes progressively diffuse towards the insertion of the forelimb. Venter whitish to translucent, vocal sac yellow. A photograph of a living paratype is presented in figure 5 C.</p> <p> <b>Measurements of the holotype (mm).</b>: (see Methods for abbreviations) SVL 23.9; HW 9.0; HL 9.1; IN 2.1; EN 2.9; IO 4.2; EL 3.0; TyL 1.2; FaL 5.7; HaL 1.8; F1 2.7; F2 3.0; F3 4.6; F4 2.9; FP1 0. 6; FP2 0.7; FP3 0.9; FP4 1.0; ThL 10.7; TL 11.6; FL 7.1; T1 3.6; T2 4.2; T3 6.1; T4 9.4; T5 7.3; TP1 0.7; TP2 0.6; TP3 0.6; TP4 0.7; TP5 0.6.</p> <p> <b>Remarks.</b> Although the proposed diagnostic features allow a clear discrimination between adult males of <i>Eleutherodactylus cattus</i> and <i>E. glamyrus</i>, a straightforward classification of non-vocal juveniles and females is so far impossible without genetic analyses. It is likely that adult females of <i>E. cattus</i> oversize those of <i>E. glamyrus</i> but no female specimens are available for <i>E. cattus</i> and a proper test of this hypothesis will require the collection of a sufficient number of female specimens of both taxa. It can also be hypothesized that additional diagnostic features for both taxa will be recovered after a detailed examination of osteological and soft tissue examinations are conducted.</p> <p> <b>Distribution.</b> This species is only known from the type locality but assuming it has specialized to high elevations like its sister taxon, <i>Eleutherodactylus glamyrus</i>, it could well be found in neighboring areas above 800 m a.s.l..</p> <p> <b>Natural history.</b> Field data indicate that <i>Eleutherodactylus cattus</i> is a nocturnal species that inhabits the montane rainforests and elfin woodlands above 800 m a.s.l., in areas of Loma del Gato-Monte Líbano Ecological Reserve, in the Sierra del Cobre massif, Santiago de Cuba province, in eastern Cuba. In this region, the mean monthly air temperature is 18.4 °C with minimal and maximum mean values around to 15.7 °C and 22.4 °C, respectively. Mean monthly relative humidity is high year round, and ranges between 87–92%, the mean annual precipitation is 1.220 mm, with May and October being the rainiest months (Potrony, <i>et al.</i>, 1994; Reyes, 1999). Males of <i>Eleutherodactylus cattus</i> were observed calling in the vegetation at 1.16 ± 0.47m (mean ± SD; range: 0.50–2.20 m) above the ground in the understory (Figure 5 C). This forest stratum is dense and rich in shrubs and herbaceous plants, with ferns, liverworts, mosses and terrestrial orchids (Figure 5 D). Calling males were heard in three nearby localities in Loma El Gato, between 844–1070 m a.s.l (ascent trail on the northern slope of Pico El Gato, 844 m a.s.l; surroundings of Loma de la Cruz, 1070 m a.s.l.; and Loma de La Juana, 900 m a.s.l). <i>E. cattus</i> showed an apparent acoustic activity peak at dusk (between 19:00–21:00 hrs) and shortly before dawn (5:00– 6:30hrs). Calling males appear to show preference for exposed surfaces of leaves and ferns, but they can call at different orientations, facing down or horizontally.</p> <p> At least another eight species of <i>Eleutherodactylus</i> are known to occur in sympatry with <i>E. cattus</i> in the surroundings of the type locality: <i>Eleutherodactylus atkinsi</i> Dunn, <i>E. auriculatus,</i> <i>E. cuneatus</i> (Cope), <i>E. dimidiatus</i> (Cope), <i>E. gundlachi</i> Schmidt, <i>E. ionthus</i> Schwartz, <i>E. limbatus</i> (Cope), <i>E. ricordii</i> (Duméril and Bibron), plus the hylid <i>Osteopilus septentrionalis</i> (Duméril and Bibron). Two species of the <i>Eleutherodactylus auriculatus</i> species group, <i>E. ionthus</i> and <i>E. auriculatus</i> (Hedges, <i>et al.</i>, 2008; Padial, <i>et al.</i>, 2014) were heard and observed vocalizing simultaneously with <i>E. cattus</i> at the type locality. The first species, <i>E. ionthus</i>, vocalizes from high perches (above three meters) on the arboreal stratum often on epiphytic plants (Bromeliaceae). This species produces a very different advertisement call in terms of temporal and spectral structure (Hedges <i>et al.</i>, 1992; Díaz and Cádiz, 2008). The second species, <i>E. auriculatus</i>, uses similar calling sites (substrates and heights) in the understory as <i>E. cattus</i>, but the acoustic features of its advertisement call are distinctly different (e.g. faster call rate, shorter call duration and higher dominant frequency), as already noted by Estrada and Hedges (1997).</p>Published as part of <i>Rodríguez, Ariel, Dugo-Cota, Álvaro, Montero-Mendieta, Santiago, Gonzalez-Voyer, Alejandro, Bosch, Roberto Alonso, Vences, Miguel & Vilà, Carles, 2017, Cryptic within cryptic: genetics, morphometrics, and bioacoustics delimitate a new species of Eleutherodactylus (Anura: Eleutherodactylidae) from Eastern Cuba, pp. 501-522 in Zootaxa 4221 (5)</i> on pages 509-512, DOI: 10.11646/zootaxa.4221.5.1, <a href="http://zenodo.org/record/253468">http://zenodo.org/record/253468</a&gt

Cryptic within cryptic: genetics, morphometrics, and bioacoustics delimitate a new species of Eleutherodactylus (Anura: Eleutherodactylidae) from Eastern Cuba

We studied the variation in genetics, bioacustics, and morphology in Eleutherodactylus glamyrus, a regionally endemic frog species restricted to high elevations in the Sierra Maestra Massif, Western Cuba that was originally described as a cryptic species hidden under the name E. auriculatus. Genetic analysis of mtDNA sequences of the 16S and cob genes identify two allopatric and strongly supported mitochondrial clades (phylogroups) which also showed no haplotype sharing in the nuclear Rag-1 gene. Bioacustic, and morphological comparisons concordantly identify these two phylogroups as independent evolutionary lineages. Therefore, we herein restrict the name Eleutherodactylus glamyrus Estrada and Hedges to populations represented in our analyses as the western phylogroup (Cordillera del Turquino to Pico La Bayamesa) and consider specimens from the eastern phylogroup (Sierra del Cobre) to represent a new species described and named as Eleutherodactylus cattus. Our results add to the growing list of Eleutherodactylus species endemic to Cuba and highlight the importance of combining different sources of evidence for obtaining robust assessments of species limits in amphibians.Fieldwork was partially funded by the Deutsche Gesellschaft für Herpetologie und Terrarienkunde (DGHT). The IdeaWild foundation provided important recording equipment. A substantial part of the work of AR was supported by a Georg Forster Fellowship from the Alexander von Humboldt Foundation. Analyses partially funded by a grant from the Spanish government, Ministerio de Economía y Competitividad, to C.V. (CGL2013-47547-P)