Two new species of leaf-toed geckos recognized six species of Phyllodactylus and three subspecies within the Galápagos Leaf- (Phyllodactylus) from Isabela Island, toed Gecko (P. galapagensis). In 1973, Bene- Galápagos Archipelago, Ecuador detto Lanza published a detailed study of the differences in measurements and arrangement http://zoobank.org/3C330FDA-82FE-47CA- of scales among 14 populations of geckos in 8232-DEC69823FC80 Galápagos, and described three new subspe- cies.2 Recently, in 2014, Omar Torres-Carvajal Authors. Alejandro Arteaga,a Lucas Bustaman- and collaborators were the first to study the te,a Jose Vieira,a,b Washington Tapia,c Jorge evolutionary relationships among the islands’ Carrión,d and Juan M Guayasamin.b,e,f leaf-toed geckos using DNA sequences.3 These authors revealed possible colonization a b Tropical Herping (TH), Quito, Ecuador. Uni- and diversification scenarios as well as recog- versidad San Francisco de Quito (USFQ), nized ten species of endemic Phyllodactylus in Colegio de Ciencias Biológicas y Ambientales the archipelago. Of these, two were elevated (COCIBA), Instituto Biósfera, Laboratorio de from subspecies status and two were left as Biología Evolutiva, campus Cumbayá, Quito, undescribed species. Ecuador. cGalapagos Conservancy, Fairfax, United States of America. dDirección del Parque To this day, the two undescribed gecko spe- Nacional Galápagos, Puerto Ayora, Ecuador. cies, and possibly many more, remain without eGalapagos Science Center, Universidad San Francisco de Quito & University of North Caroli- formal scientific names. As a result, these en- na at Chapel Hill, Puerto Baquerizo Moreno, Isla demic Galápagos geckos have not received San Cristóbal, Galápagos, Ecuador. fCentro de formal conservation status assessment and, Investigación de la Biodiversidad y Cambio thus, have not been the focus of targeted Climático, Ingeniería en Biodiversidad y Recur- conservation actions despite facing several sos Genéticos, Universidad Tecnológica Indo- threats of extinction such as displacement by américa, Quito, Pichincha, Ecuador. introduced geckos, predation by exotic spe- cies, and even volcanic activity. Academic reviewers. Alex Pyron, Claudia Koch, and Miguel Vences. To solve this problem in conservation and tax- onomy, here we re-evaluate the validity of these ABSTRACT candidate species. First, by generating an up- dated molecular phylogeny that includes DNA In this study, we (1) describe two new spe- samples from new populations, and second, by cies of leaf-toed geckos (Phyllodactylus) from using three different species delimitation ap- Isabela Island in the Galápagos Archipelago, proaches. Within this evolutionary framework, (2) elevate the Mares Leaf-toed Gecko from we assign names (one new, one revalidated) subspecies (P. galapagensis maresi) to spe- to the two geckos previously recognized as un- cies status, (3) present an updated molecular described in Galápagos, and describe a pre- phylogeny of the archipelago’s Phyllodactylus, viously unrecognized new species from Wolf and (4) test the limits between gecko species Volcano in northern Isabela Island. in the archipelago. A unique combination of molecular and morphological characters MATERIALS AND METHODS support the validity of the new species. With these changes, the number of Phyllodactylus This study was carried out in species reported in Galápagos increases to Ethics statement. strict accordance with the guidelines for the use 12 (eleven endemic and one introduced). of live amphibians and reptiles in field research.4 All procedures with animals (see below) were INTRODUCTION reviewed by the Galápagos National Park Direc- Unlike most reptile groups in the Galápagos torate (DPNG) and the Ministerio de Ambiente Islands, the endemic leaf-toed geckos of the del Ecuador (MAE), and specifically approved genus Phyllodactylus have received little at- as part of obtaining the following permits for re- tention from researchers. Only three major sci- search and access to genetic resources: PC-31- entific works dealing with this group of lizards 17, PC-54-18, and DNB-CM-2016-0041-M-0001. have been published. Sampling. We obtained tissue samples, cre- In 1912, John Van Denburgh published the first ated photo vouchers, and generated DNA compendium of the geckos of Galápagos.1 He sequence data for ten individuals represent- 174 ing four Phyllodactylus populations in Isabela on: (1) the type series; (2) individuals released Island (Darwin Volcano and Wolf Volcano), after tail-tip sampling (labeled JMG under Marchena Island, and Gardner Islet. Appendix 1); and (3) images of life individuals that were not collected or sampled. Laboratory techniques. We extracted genom- ic DNA from 96% ethanol-preserved tissue sam- Distribution maps. We present an overview of ples (tail muscle) following protocols described the distribution of all leaf-toed geckos in Galápa- in Arteaga et al. (2018),5 and amplified target gos as well as detailed distribution maps for the regions (12S, 16S, and ND4) following proce- species described or re-described in this work. dures described Arteaga et al. (2018),5 Gamble These maps were created as described in the et al. (2008),6 and Blair et al. (2009).7 plan of the book. For some gecko species, we present binary environmental niche models DNA sequence analyses. We used a total of (ENM) to accompany or replace the dot maps. 210 DNA sequences (gene fragments 12S, These were created following parameters de- 16S, ND4, RAG1, and c-mos) to build a phylo- scribed in Arteaga et al. (2016).17 genetic tree of Ecuadorian Phyllodactylus (Fig. 1), of which 25 were generated during this work RESULTS and 185 were downloaded from GenBank. We edited and assembled new sequences Molecular phylogeny and taxonomic con- using the program Geneious ProTM 5.4.78 and sequences. The overall topology and support aligned with those downloaded from GenBank (Fig. 1) of our phylogenetic tree is similar to (Appendix 1) using MAFFT v.79 under the that obtained by Torres-Carvajal et al. (2014),3 default parameters in Geneious ProTM 5.4.7. with minor differences such as the positions Genes were combined into a single matrix with of Phyllodactylus leei and P. duncanensis, 11 partitions, one per non-coding gene and as well as those of the newly sampled gecko three per protein-coding gene corresponding populations. Our phylogeny, combined with to each codon position. The best partition morphological diagnostic traits, supports the strategies along with the best-fit models of existence of two unnamed species from Isa- evolution were obtained in PartitionFinder 210 bela Island. It also supports the validity of P. under the Bayesian information criterion. We maresi, a species currently considered a sub- assessed phylogenetic relationships under species of P. galapagensis. a Bayesian inference approach in MrBayes 3.2.011 following parameters described in DNA sequences from Marchena Island are Arteaga et al. (2018).5 GenBank accession grouped with those from Santiago Island. This numbers are listed in Appendix 1. Genetic dis- clade is distinguishable from geckos on Santa tances were calculated using the uncorrected Cruz (Phyllodactylus galapagensis) based on distance matrix in PAUP 4.0.12 DNA sequence data (Fig. 1). Genetic diver- gence in a 307 bp long fragment of the mito- Species delimitation. We tested the limits chondrial 12S gene between P. maresi and P. within potential species of Ecuadorian leaf- galapagensis is 3–4%, whereas intraspecific toed geckos using three different species distances are 0–1% in P. maresi. However, delimitation approaches following parameters geckos that inhabit Marchena and Santiago described in Koch et al. (2016)13: (1) Auto- are indistinguishable in coloration from, and matic Barcode Gap Discovery (ABGD)14; (2) overlap largely in number and arrangement of Bayesian implementation of the Poisson Tree scales with, the geckos that inhabit Mares Islet Processes model (bPTP)15; and (3) General- (see Lanza [1973]2 for comparison between ized Mixed Yule Coalescent (GMYC).16 populations of Santiago Island and Mares Islet). Morphological data. We provide maximum Mares Islet (see Fig. 3 for location of this total length, from tip of snout to tip of tail, for small islet) has an area of 4,750 m2 and is males and females of each species. These located ~900 m from the northeastern coast measurements were obtained from Lanza of Santiago Island. It is the type locality of (1973)2 or by examining comparative alcohol- Phyllodactylus galapagensis maresi.2 There- preserved specimens from the herpetology fore, we propose that P. g. maresi be elevated collections at Fundación Charles Darwin to full species status (that is, P. maresi). This is (MVECCD) and the California Academy of Sci- the most parsimonious scenario given the avail- ences (CAS). Diagnoses and descriptions of able data and general biogeographic patterns the new species of Phyllodactylus were based on the islands; however, although morphologi- 175 Figure 1. Bayesian consensus phylogeny depicting relationships among Galápagos leaf-toed geckos. The topology is derived from analysis of 2,958 bp of DNA (gene fragments 12S, 16S, ND4, RAG1, and c-mos). Voucher numbers for sequences are indicated for each terminal. Black dots indicate clades with 95–100% posterior probability values. Grey dots indicate values from 45–94%. Support values on some intraspecific branches are not shown for clarity. Colored branches repre- sent lineages occurring in Galápagos, and each color corresponds to each species’ distribution on the archipelago (see Fig. 2). Images of geckos adjacent to the labels of the tree correspond with the species indicated on the label, but not necessarily with the same voucher. Results of the spe- cies delimitations are illustrated by continuous semicircular bars. Each bar represents a species detected by one of three approaches (from the outside in, these are ABGD, GMYC, and bPTP). cal data are unambiguous, the genetic assign- within Ecuadorian Phyllodactylus recognized ment of the Mares Islet population to gecko the same number of confirmed species of populations that inhabit Bartolomé, Marchena, geckos in Galápagos as Torres-Carvajal et al. Santiago, and Rábida islands is still pending.