Phylogeography and Population Structure of Two Brachistosternus Species (Scorpiones: Bothriuridae) from the Chilean Coastal Desert – the Perils of Coastal Living

Biological Journal of the Linnean Society, 2016, , – . With 1 ﬁgure. Biological Journal of the Linnean Society, 2017, 120 , 75–89. With 1 figures

Phylogeography and population structure of two Brachistosternus species (Scorpiones: Bothriuridae) from the Chilean coastal desert – the perils of coastal living

F. SARA CECCARELLI1*, JAIME PIZARRO-ARAYA2 and ANDRES A. OJANGUREN- AFFILASTRO1 Downloaded from https://academic.oup.com/biolinnean/article/120/1/75/2864989 by guest on 29 September 2021

1Division de Aracnologıa, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Av. Angel Gallardo 470, C1405DJR, Buenos Aires, Argentina 2Laboratorio de Entomologıa Ecologica, Departamento de Biologıa, Facultad de Ciencias, Universidad de La Serena, Casilla 599, La Serena, Chile

Received 2 May 2016; revised 22 June 2016; accepted for publication 22 June 2016

Coastal deserts are geologically dynamic areas of the Earth, affected by historical changes in sea levels and in some cases also by fault-line tectonic activity. An example of such a dynamic area is the Chilean coastal desert of the Antofagasta and Atacama regions, which harbours many endemic species, such as the bothriurid scorpion species Brachistosternus paposo and Brachistosternus roigalsinai. In this work, we carry out phylogeographic and population genetic analyses on these scorpions, using two mitochondrial (COI and cyt b) and two nuclear (Actin 5C and wingless) markers to identify species and population structuring, and link these findings to the geological history of the area. The geographical feature separating the two species is identified as the Huasco River, and distinguishing morphological features for these scorpions are presented. Population genetic and phylogeographic outcomes reflect an unstable history across this region for B. paposo and B. roigalsinai, related to sea-level changes affecting coastal habitats, including nearby islands. © 2016 The Linnean Society of London, Biological Journal of the Linnean Society,2017, 2016, 12000, 000, 75–89–000..

KEYWORDS: Chile – eustasy – population genetics – vicariant speciation.

INTRODUCTION features have been shaped by a combination of sea- level changes and tectonics (Pedoja et al., 2014). This The desert biome covers about one-fifth of the world’s means that, since the Pliocene, low-lying regions of terrestrial surface and is characterised by extreme present-day coastal areas (including low-elevation environments in which only organisms with specific islands) have been submerged numerous times and adaptations can survive (Mulroy & Rundel, 1977; areas with a shallow sea floor have repeatedly Whitford, 2002; Nagy, 2004). Deserts also provide emerged, thus also joining coastal-shelf islands to spatiotemporal habitat stability for well adapted spe- the mainland. cies (Hartley et al., 2005). Coastal deserts, however, The South American Pacific Coastal Desert (PCD) are an exception to this stability over time, as their of Chile and Peru is an example of a dynamic geo- geographical areas are prone to changes depending graphical area with an extreme environment. The on global sea levels, or eustasy (Carter, 1988). While Chilean coastal desert extends from the Tarapaca to global sea levels during the late Neogene and Qua- the Coquimbo region. The most important rivers ternary periods have been estimated to oscillate by found in this area are, from north to south, the more than 150 m (Haq, Hardenbol & Vail, 1988; Copiapo, Huasco, and the Elqui. The River Copiapo Hardenbol et al., 1998), the shorelines and their has a semi-permanent flow and is an endorheic system, meaning that it does not drain into the ocean, *Corresponding author: E-mail: [email protected] whereas the Huasco and Elqui rivers have a

© 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, , – 1 © 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2017, 120, 75–89 75 762 F.F. S. S. CECCARELLICECCARELLI ETET AL.AL. PHYLOGEOGRAPHY OF COASTAL DESERT SCORPIONS 3 permanent flow draining into the Pacific Ocean, One of the five major clades of the bothriurid scor- species (about 300 of B. roigalsinai, and 150 of B. MATERIALS AND METHODS which makes them exorheic systems. This area also pion genus Brachistosternus, named the Pacific paposo) were examined during this study to re-estab- contains several continental-shelf islands. To the Coastal Desert Clade (PCDC) (Ojanguren-Affilastro SPECIES LIMITS AND DIAGNOSTIC CHARACTERS lish and evaluate the strength of a new morphological south is the Choros archipelago, which comprises the et al., 2016), is found in the PCD, which is itself an Brachistosternus paposo and B. roigalsinai are consid- diagnostic characteristic, namely the presence of two Choros, Damas, and Gaviota islands. The farthest area of high phylogenetic diversity for this genus ered two distinct valid species based on the species dark spots in the posterodorsal margin of sternite VII from the continent is Choros, which is almost 8 km (Ceccarelli et al., 2016). The PCDC species migrated delimitation analyses of Ojanguren-Affilastro et al. and metasomal segments I–III of B. roigalsinai, which off the coast; next is Damas, about 5 km, and the and speciated along the coast in a general northward (2016; Appendix H). All available specimens of both are absent in B. paposo (see Fig. 1). closest is Gaviota, which is < 1 km off the coast; the direction, from an ancestral area on the central Chi- sea floor between these islands and the coast does lean coast (Ceccarelli et al., 2016). A sister group to not drop below 30 m. Twenty-five km north of the the remaining PCDC species, Brachistosternus Choros archipelago, is Chanaral~ island, located about paposo (Ojanguren-Affilastro & Pizarro-Araya, 2014) 8 km off the coast with an intervening seafloor depth and Brachistosternus roigalsinai (Ojanguren-Affilas- Downloaded from https://academic.oup.com/biolinnean/article/120/1/75/2864989 by guest on 29 September 2021 of about 80–120 m (Aguirre, 1967). Chanaral,~ Choros tro, 2002) inhabit a similar area of the Chilean and Damas islands belong to the Pinguino€ de Hum- coastal desert. No evidence for sympatry has been boldt National Reserve (Sistema Nacional de Areas� found so far in the two species (Ojanguren-Affilastro Silvestres Protegidas del Estado, SNASPE area; Cas- & Pizarro-Araya, 2014), with the geographical limits tro & Brignardello, 2005; see Supporting Informa- thought to be at 26° to 30° for B. roigalsinai (Ojan- tion, Figure S1 of the Supporting Information). Like guren-Affilastro, Mattoni & Prendini, 2007a; Ojan- several other coastal areas, this region has been guren-Affilastro et al., 2007b), and restricted to the affected by constant sea-level changes (Le Roux area of Paposo (approximately at 25°) for B. paposo et al., 2005) coupled with tectonic uplift and associ- (Ojanguren-Affilastro & Pizarro-Araya, 2014). Addi- ated seismic activities caused by the subduction of tionally, the presence of B. roigalsinai has also been the Nazca plate under the South American plate reported from the Choros archipelago (Pizarro-Araya (Kendrick et al., 2003; Husson, Conrad & Faccenna, et al., 2014a), and recently several specimens of this 2012). Such geological events are expected to have species have been collected from Chanaral~ island affected the local flora and fauna populations as (Alfaro, Pizarro-Araya & Mondaca, 2014; Pizarro- well. Araya et al., 2014b); however, the island specimens Population genetic and phylogeographic analyses present slight morphological differences with respect are useful tools for inferring past patterns and pro- to the continental populations, as was found for the cesses of species at the transition between intra- and tenebrionid Praocis (Praocis) spinolai (Ben�ıtez et al., inter-specific levels, which, in the case of extreme, 2014). Based on the results of a recent molecular geographically dynamic areas such as the Chilean study (Ojanguren-Affilastro et al., 2016), the distri- coastal desert, reveal the extent to which this rela- butional limits of the two species have been ques- tive instability affects its inhabitants. Furthermore, tioned, raising doubts about the diagnostic the effect of geographic barriers (such as rivers and characters of both species and the specific identity of the sea separating islands from the mainland) on their populations. genetic structuring can be assessed. Here, a more detailed study of the morphology of Among the South American desert-specialized several populations of B. roigalsinai and B. paposo fauna, several scorpions belonging to the families from their entire distributional range, combined with Bothriuridae, Buthidae, and Caraboctonidae have the data of the species delimitation analysis pre- adapted to living in this extreme environment. Par- sented by Ojanguren-Affilastro et al. (2016), sheds ticularly, the scorpion genus that is most adapted light on the identity and diagnostic characters of and diversified in these areas is Brachistosternus, these two species. Based on these newly inferred spe- belonging to the family Bothriuridae, which occurs cies limits, the phylogeography and population histo- even in the most extreme arid environments of the ries of two bothriurid scorpion species from the continent, such as the Atacama and Sechura deserts Chilean coastal desert, B. paposo and B. roigalsinai (Ochoa, 2005; Ojanguren-Affilastro & Ram�ırez, 2009; are investigated, to answer questions about specia- Kova�r�ık & Ojanguren-Affilastro, 2013; Ceccarelli tion and population structure in extreme environ- et al., 2016; Ojanguren-Affilastro et al., 2016). To ments such as coastal deserts, and to link these Figure 1. Coalescence-based ultrametric tree obtained from *BEAST using the nuclear actin and wingless, and mitochon- date, scorpion phylogeographic and population historical microevolutionary processes with the drial COI and cyt b gene fragments, with populations as terminal taxa (for codes see Table 2). Upper bar with ages (in Myr) genetic studies have been carried out on Holartic area’s geography and geological history. This project and geological epochs are shown above the tree (MI = Miocene; PO = Pliocene; PS = Pleistocene). Bayesian posterior proba- fauna of families such as Buthidae and Vaejovidae is the first phylogeographic and population genetic bilities are shown as numbers at nodes, and blue bars represent 95% highest posterior densities. Red circled terminal popu- (e.g. Mirshamsi et al., 2010; Graham, Olah-Hem-� study involving bothriurid species, or even a lations represent Brachistosternus paposo and yellow Brachistosternus roigalsinai, also shown on the map by mings & Fet, 2012; Bryson, Savary & Prendini, Neotropical scorpion, applying a wide range of meth- approximated, respectively coloured polygons and adjoining dashed lines. The map in the centre also contains blue poly- 2013; Graham et al., 2013), but never on Neotropical ods to obtain a full spectrum of structural, spatial gons marking the Copiapo, Huasco and Elquı river drainages. To the left of the map are photographs of male specimens of bothriurids. and temporal genetic dynamics. each species, each with an enlarged photograph of their respective metasomas to illustrate the difference in pigmentation.

TAXON SAMPLING AND MOLECULAR MARKER date there is no evidence for paralogues of these from recombination was used, following a conserva- values of the parameters confirmed to be > 200) SELECTION genes. Having said that, neither gene nor even tive approach. using Tracer v. 1.6 (Rambaut et al., 2014), and the To assess their population structure and phylogeo- whole-genome duplication for the scorpion species To determine whether each sampled locality con- estimated mean and standard deviation of the clock graphic histories, 56 individuals belonging to the studied here has ever been investigated. Neverthe- tained individuals that could be treated as separate rate for the two runs was noted. nominal species B. roigalsinai and B. paposo were less, based on the number and distribution of the populations, we used the Geneland v. 4.0.5 package For the *BEAST analysis on the mt and phased collected, 39 of which were collected from nine conti- nuclear genes’ heterozygous sites (see Results), the (Guillot & Santos, 2009), running the Graphical nuclear data, nucleotide substitution and clock model nental localities along the Chilean coastal desert assumption of homology is reasonable. User Interface in R v. 3.1.1 (R_core_team, 2014). priors were unlinked, setting the partitions and between the latitudes 24°S and 31°S (see Table 1 Geneland uses MCMC sampling and a spatial model nucleotide substitution models as chosen by Parti- and Supporting Information, Table S1 for details). to estimate the number of populations and their tionFinder (see Supporting Information, Table S3). DNA EXTRACTION, PCR AND SEQUENCING An additional 17 specimens of B. roigalsinai were geographic limits based on multilocus genotype data Uncorrelated lognormal relaxed clock priors were set also included from three coastal islands of Atacama – Whole genomic DNA was extracted from leg muscle and GPS coordinate inputs. The number of popula- for each molecular marker as a whole and the clock Downloaded from https://academic.oup.com/biolinnean/article/120/1/75/2864989 by guest on 29 September 2021 more specifically, two localities of the Choros archi- tissue of individual scorpions using a Qiagen DNeasy tions was set between 1 and 10 and the analysis rate for the mitochondrial fragment was set using 1 pelago (the Choros and Damas islands) and one Blood and Tissue Kit, following the manufacturer’s run for 10 million generations and a 10% burn-in, 0.0133 substitutions MyrÀ , based on the calibration locality from Chanaral~ island. One individual of protocol and digesting the tissue at 56°C for 1 h with setting the spatial correlation between allele fre- obtained in BEAST. A Yule process tree prior was Brachistosternus ochoai Ojanguren-Affilastro, 2004 proteinase K. Polymerase chain reaction (PCR) mixes quencies. To corroborate the results from Geneland, chosen and three separate MCMC runs of 20 million was used as an outgroup taxon for the tree inference contained 1.5 lL 109 PCR Buffer (Life Sciences), the program Structurama (Huelsenbeck & Andol- generations each were executed, sampling every (in BEAST, see section on COALESCENCE-BASED 10 lmol MgCl2, 0.25 lmol of each dNTP, 0.4 lmol of fatto, 2007; Huelsenbeck, Andolfatto & Huelsenbeck, 2000th generation. The convergence and ESS values TREE INFERENCE below). The analyses were based each primer, 0.1 lL Taq polymerase (5 U/lL, Life 2011) was used, running a Bayesian analysis under of the runs were verified in Tracer and the trees on molecular data pertaining to two mitochondrial Sciences), 0.5 lL (50 mg/mL) bovine serum albumin, a Dirichlet probability distribution with a gamma combined in LogCombiner, removing the first 10% as and two nuclear gene fragments in the form of DNA 1–2 lL genomic DNA and ddH2O to bring the final shape parameter and assuming no admixture. The burn-in, upon which the MCC tree with mean node sequence data. The mitochondrial markers belonged volume to 15 lL. Four gene fragments (two mito- number of populations (k) was set between 1 and heights was selected in TreeAnnotator. The BEAST to the cytochrome oxidase C subunit I (henceforth chondrial and two nuclear) were amplified using the 10, and the program was run for four replicates of and *BEAST analyses were run through the CIPRES COI) and the cytochrome B (cyt b) gene fragments, primers in Supporting Information (Table S2). All 10 million cycles with a 10% burn-in on the haplo- Science Gateway bioinformatics portal v. 3.3 (Miller, while the nuclear markers belonged to the Actin 5C amplifications were performed using the following type data from the PHASE analysis, coded as Pfeiffer & Schwartz, 2010). (actin) and wingless (wg) gene fragments. The thermal profile: 94°C for 3–5 min; 35 cycles of 95°C numbers. nuclear markers, although never used for scorpions, for 15–30 s, 42–52°C for 15–30 s, 72°C for 15–30 s; POPULATION GENETIC PARAMETER ESTIMATIONS have been employed for molecular phylogenetic stud- 72°C for 10 min. The PCR products were purified COALESCENCE-BASED TREE INFERENCE ies in other arachnids (e.g. Vink et al., 2008; Black- using ExosAP (Life Sciences) and sent for sequencing Based on the previously inferred population assign- ledge et al., 2009; Bodner & Maddison, 2012) and to to Macrogen Inc., Korea. Based on the previously inferred population limits, a ments, intra- and interpopulation genetic parame- coalescence-based analysis in *BEAST v. 1.8.2 (Heled ters were calculated in DnaSP on the complete COI & Drummond, 2010) was carried out to obtain a tree and cyt b regions as well as on the non-recombining DATA MATRICES HANDLING AND DEFINING Table 1. Sampling localities for the bothriurid scorpion with one terminal per population with a reliable regions of actin and wg. More specifically, the POPULATION LIMITS species Brachistosternus paposo and Brachistosternus topology and node age estimates, as well as a set of intrapopulation genetic diversity was calculated roigalsinai on the Chilean coast The DNA sequences of the partial COI, cyt b, actin posterior trees required for selected downstream based on the number of haplotypes (h), haplotype and wg gene fragments were edited in Sequencher analyses (see ISOLATION-WITH-MIGRATION and diversity (H), nucleotide diversity (p; Nei, 1987), Locality name Loc. # Latitude Longitude v. 4.1.4 (GeneCodes Corp.) and aligned in MAFFT BAYESIAN PHYLOGEOGRAPHIC ANALYSES sec- and the Watterson parameter (h; Watterson, 1975). 7 (Katoh & Standley, 2013) using the ‘Auto’ strat- tions below). Due to difficulties with applying node Neutrality tests (Tajima’s D [Tajima, 1989], Fu’s FS Paposo 1 25.017212 70.46711 À À egy and a gap opening penalty of 1.53. The COI age calibrations directly in *BEAST, the molecular [Fu, 1997] and R2 [Ramos-Onsins & Rozas, 2002]) Pan de Azucar� N.P. 2 26.166975 70.66353 À À and cyt b gene fragments were combined (hence- substitution (or clock) rate for the mt fragment was were also carried out to test whether all of the pop- Llanos de Challe 3 28.118129 71.09731 À À forth referred to as the ‘mt fragment’) as mitochon- estimated in BEAST v.1.8.2 (Drummond et al., ulations adhere to the neutral hypothesis. The ZnS N.P., inland drial markers are genetically linked. For the 2012), to be then used as a prior in the *BEAST (Kelly, 1997) values were calculated to infer the Llanos de Challe 4 28.129638 71.162081 À À nuclear fragments, the PHASE algorithm (Ste- analysis. The molecular rate was estimated based on levels of linkage disequilibrium (LD). All of the N.P., beach phens, Smith & Donnelly, 2001; Stephens & Don- a secondary calibration of B. roigalsinai and B. papo- parameters were calculated under the coalescent Caleta Los Burros 5 28.193802 71.154944 À À nelly, 2003) implemented in DnaSP v. 5.10.01 so’s divergence from their most recent common process (Hudson, 1990) and their statistical signifi- Chanaral~ 6 29.03388 71.577658 À À (Librado & Rozas, 2009) was used to separate ancestor (set as a normal distribution prior with a cance assessed through 10 000 simulated replicates. Island N.R. heterozygous alleles, setting 1000 MCMC iterations mean of 6 Myr and 1.5 standard deviations), esti- The D and D parameters (the average and net Damas Island N.R. 7 29.235708 71.527106 xy a À À Choros Island N.R. 8 29.263887 71.541684 (with a 10% burn-in) with the default settings for mated to have occurred between 3.88 and 8.87 Mya number of nucleotide substitutions per site between À À Punta Choros, 9 29.243571 71.434898 the ‘Recombination’ model, assuming no knowledge (Ma; 95% HPD; Ceccarelli et al., 2016). Partitioning populations, respectively) were used to assess inter- À À dunes about the specific phases. Additionally, the nuclear strategies and nucleotide substitution models for the population genetic diversity levels, applying the Choros inland 1 10 29.322137 71.233368 fragments were checked for recombination in mt fragment were then selected using PartitionFin- Jukes and Cantor correction (Jukes & Cantor, 1969). À À Choros inland 2 11 29.337366 71.224795 DnaSP, which computes the recombination parame- der v. 1.1.1 (Lanfear et al., 2012) and an uncorre- Additionally, a neighbour-joining tree was estimated À À Vicuna~ 12 30.028273 70.722026 ter R (Hudson, 1987) and provides the minimum lated lognormal clock rate was set for each partition, based on Dxy values for each marker separately À À number of recombination events as calculated by linking the tree prior and setting it to a constant-size using MEGA 6 (Tamura et al., 2013). The levels of Locality number to cross-reference with Supporting Infor- the four-gamete test (Hudson & Kaplan, 1985). coalescent process. Two independent MCMC simula- gene flow among the populations were assessed mation, Figure S1; N.P. and N.R. in locality names refer Sites containing missing data were removed and tions of 50 million generations (sampling every based on the Nst metric (Lynch & Crease, 1990) to National Park and National Reserve, respectively. only the largest portion of the gene fragment free 5000th) were run, checked for convergence (and ESS under the infinite island model (Wright, 1931).

For each population, the changes in population independent estimates of similar parameters. distribution on the standard deviation of the lognor- Table 2. Population assignments for Brachistosternus size over time were estimated by generating Migrate-n was run through the CIPRES Science mal distribution with a mean of 2.712. Four indepen- paposo and Brachistosternus roigalsinai based on Gene- extended Bayesian skyline plots (EBSP; Heled & Gateway on the nuclear and mitochondrial sequence dent replicates of the species-tree diffusion analysis land and Structurama results Drummond, 2008) in BEAST 1.8.2. Three data parti- data separately for all populations together and run- were run for 50 million generations each, logging tions were used: the mitochondrial fragment ning four MCMC simulations for 50 million genera- every 50 000 generations. To visualise the ancestral Population (COI + cyt b), the actin fragment and the non-recom- tions each with chain-swap and heating 80% HPD regions in Google Earth at 1000 year Species Locality name Loc nr. code bining portion of the wg fragment. The linear EBSP implemented (temperatures at 1, 1.5, 3, and 1 mil- intervals from 4.15 to 0 Mya, the ‘time slice’ function B. paposo Paposo 1 PP coalescent tree prior was chosen for the linked parti- lion, as per the default settings). Migration was con- of the program SPREAD v. 1.0.6 (Bielejec et al., B. paposo Pan de Azucar� N.P. 2 PA tions and an uncorrelated relaxed lognormal clock fined to occur between geographically neighbouring 2011) was used on the output of the RRW species dif- B. paposo Llanos de Challe 3 LC prior applied to each unlinked partition. Further- populations, with starting and migration rates set fusion analysis. h N.P., inland more, the GTR nucleotide substitution model was to be calculated by the program and default settings B. paposo Llanos de Challe 4 LC Downloaded from https://academic.oup.com/biolinnean/article/120/1/75/2864989 by guest on 29 September 2021 chosen as a prior for the unlinked partitions and, for used for the remaining parameters. N.P., beach the demographic population size changes, a Poisson For IMa2, separate runs were executed for RESULTS B. paposo Caleta Los Burros 5 LC prior was applied. The MCMC chains were run for Brachistosternus paposo and B. roigalsinai popula- B. roigalsinai Chanaral~ 6 IC DATA MATRICES AND COALESCENCE-BASED TREE 20 million generations, sampling every 2000th gener- tions, setting the input topologies for the populations Island N.R. INFERENCE ation. Graphs of population size changes over time as inferred by *BEAST, in each case combining mito- B. roigalsinai Damas Island N.R. 7 CH were then generated from the output using Python chondrial and nuclear DNA data while providing the The details for the data matrices of the COI, cyt b, B. roigalsinai Choros Island N.R. 8 CH scripts available online (https://code.google.com/ inheritance scalar (0.25 for the mtDNA as compared actin, and wg gene fragments used in this study can B. roigalsinai Punta Choros, 9 CH archive/p/beast-mcmc/downloads). with the nucDNA). In the absence of known genera- be found in Supporting Information (Table S4). Prior dunes tion times for these scorpions, the value was set at 3, to their reduction based on inferred recombination B. roigalsinai Choros inland 1 10 CH an average value for commonly known generation events, the nuclear gene fragments, actin and wing- B. roigalsinai Choros inland 2 11 CH SPATIAL POPULATION ANALYSES times in scorpions (e.g. Polis & Farley, 1980; Boulton less, contained no polymorphic sites for 24/34 and 22/ B. roigalsinai Vicuna~ 12 VI The program Alleles In Space (Miller, 2005) was & Polis, 1990; Polis, 1990), and the option of adding 51 (homozygous) individuals, respectively. Further- used to detect potential barriers to gene flow across an unsampled ‘ghost’ population was activated. Four more, seven and 19 individuals contained only one N.P. and N.R. in locality names refer to National Park space (Manel et al., 2003) by running an Allelic separate MCMC runs of 1 million generations were dimorphic site for actin and wingless, respectively. and National Reserve, respectively. Aggregation Index Analysis, which quantifies the executed for each species to compare ESS values and The maximum number of dimorphic sites for a single spatial patterns of individual haplotypes, setting chain mixing. For each run, 60 chains were set with individual was three for actin and four for wingless. 5000 randomised replicates. To help visualize the heating terms of 0.975 and 0.75, and the uniform pri- No individual had more than two nucleotidic vari- populations and all markers, Fu’s Fs values were all genetic distance patterns across the distributional ors for the migration, population size and splitting ants at one site for either marker. positive, but non-significant, while R2 values were range of B. paposo and B. roigalsinai, the program’s time were set at maximum values of 5 each upon Based on the *BEAST tree inference, a clear sepa- significantly different than expected under a neutral ‘Genetic Landscape Shape’ interpolation was applied revising the histograms of preliminary runs. The ration is noted between the three populations of model. Similarly, significant results were obtained on a 50 9 100 grid, obtaining a landscape shape plot Nielsen & Wakeley (2001) likelihood ratio test imple- Brachistosternus paposo and B. roigalsinai (see for all markers and populations for the ZnS statistic where the surface plot height represents genetic dis- mented in IMa2 was used to assess significant levels Fig. 1). Age estimates were of 4.06 Ma (2.5–5.58 95% measuring LD. tance across space (Miller, 2005; Miller et al., 2006). of migration. HPD) for the MRCA of the species B. paposo and B. The results of the Fst and Dxy/Da calculations, used Further spatial visualisation, this time for haplo- roigalsinai, and of 1.36 Ma (0.32–2.84 95% HPD) to determine structuring within vs. structuring types, was obtained by running a TCS (Templeton, and 1.27 Ma (0.22–2.52 95% HPD) for the MRCAs of between populations, are shown in Supporting Infor- Crandall & Sing, 1992) haplotype network analysis BAYESIAN PHYLOGEOGRAPHIC ANALYSIS the B. paposo and B. roigalsinai populations, respec- mation (Table S6). The lowest structuring was found implemented in the program popART (Leigh & Bry- Bayesian phylogeographic analyses combine both tively. between populations of B. roigalsinai of Chanaral~ ant, 2015), which also facilitates visualization of hap- spatial and temporal information to trace the most island (IC) and the Choros region (CH; Choros island lotype representations on a map. Lastly, the probable movements of populations through space and Punta Choros) based on the mitochondrial and regression significance of the previously calculated and time. A relaxed random walk (RRW) Bayesian POPULATION DYNAMICS wingless gene fragments, while based on these same pairwise genetic distances (Dxy) was evaluated and phylogeographic approach (Lemey et al., 2010) was The analyses carried out in Geneland and Struc- two markers, there is genetic structuring between isolation by distance tested, using a Mantel test therefore used to infer the movements of B. roigalsi- turama to estimate the number of populations both the northern populations of B. paposo (Paposo [PP], (Mantel, 1967) with 30 000 randomizations for the nai and B. paposo populations across geographic recovered five populations from the 12 sampled local- Pan de Azucar� National Park [PA] and Llanos de natural log-transformed geographical distances pro- space through time. The approach developed by ities (see Supporting Information, Figures S2 and Challe National Park [LC]) and the coastal popula- vided by the Isolation By Distance Web Service (Jen- Nylinder et al. (2014) was applied using the program S3). Even though the individuals from Paposo (Loc tions of B. roigalsinai of Chanaral~ island/Choros sen, Bohonak & Kelley, 2005). BEAST v. 1.8.2. For this approach, the last 2000 spe- 1) and Pan de Azucar� National Park (Loc 2) were region. The actin fragment appears to confound this cies trees from the *BEAST analysis described ear- grouped together, we decided to treat them as dis- general pattern. A graphical representation of lier were used, after pruning the outgroup taxon in tinct populations for subsequent analyses since they genetic structuring (or lack thereof) for each marker ISOLATION-WITH-MIGRATION ANALYSES Mesquite v. 3.04 (Maddison & Maddison, 2015). Goo- are separated by more than 120 km. The final popu- can be found in the neighbour-joining trees from the

To determine the extent of genetic interchange gle Earth v. 7.1.5 was used to create polygons of the lation assignment by locality can be found in Dxy calculations in Supporting Information, Fig- between the populations of this study, current and approximate extant distributions for each population, Table 2. ure S4. historical migration rates and gene ﬂow were and used with the posterior distribution of species For the intrapopulation DNA sequence variation The extended Bayesian skyline plots (EBSP) for assessed using Bayesian analyses in both the trees for the species-tree diffusion analysis under the and neutrality tests, the calculated values are pre- the different populations show no change in popula- migrate-n v. 3.6.11 (Beerli, 2006, 2009) and the IMa2 RRW process. Following Nylinder et al. (2014), the sented in Supporting Information (Table S5). For tion size (LC and PA) or a slight increase (PP) dur- (Hey & Nielsen, 2007; Hey, 2010) programs, as same priors were used, including a prior exponential Tajima’s D, negative values were obtained for all ing the last 50 000 years (50 kyr) for B. paposo. In

6 Downloaded from https://academic.oup.com/biolinnean/article/120/1/75/2864989 by guest on 29 September 2021 DISCUSSION Information, Figure S6, which is the Genetic Land- IC and CH = 9.25 9 10À , B. roigalsinai’s CH and levels of the Andes began receiving a greater amount 6 scape Shape plot, the peaks represent the genetic VI = 6.12 9 10À ), but lower between the populations The outcomes of this work redefine the geographic of snowfall coming from the humid air masses of the distance between scorpions from different areas. This from LC (B. paposo) and IC (B. roigalsinai) limits of the two species studied, with B. paposo Pacific anticyclone. The melting of these snow 6 information indicates that the lowest genetic dis- (mtDNA = 1.74 9 10À ). This pattern is slightly dif- being found in the northern localities of PP, PA and masses increased the water flow of rivers such as the tance is found in the south-western part of B. ferent based on the wingless marker, where the low- LC, and B. roigalsinai in IC, the adjacent mainland, Huasco, which became permanent and exorheic, roigalsinai’s distributional range (between Chanaral~ est mean migration rate is between B. roigalsinai’s CH and VI (Fig. 1). The new morphological diagnos- therefore creating an effective barrier for most of the 6 Island and the Choros region), with moderate genetic CH and VI populations (4.63 9 10À ), followed by LC tic character presented here and used to definitively epigeic arthropod fauna. A similar pattern of separa- 6 distances between populations towards the north, (B. paposo) and IC (B. roigalsinai) (3.99 9 10À ), separate B. roigalsinai and B. paposo could be ques- tion between coastal scorpion species is found while the highest genetic distance can be found while the highest migration rates are between the tioned, since the pigment pattern presents high vari- between Brachistosternus sciosciae Ojanguren-Affi- 6 between Vicuna~ (VI) and neighbouring populations conspecific populations PP and PA (7.49 9 10À ), IC ability in scorpions with wide distributions (such as lastro, 2002 and Brachistosternus cepedai Ojan- 6 6 for B. roigalsinai and between neighbouring popula- and CH (7.29 9 10À ), and PA and LC (6.67 9 10À ). B. roigalsinai and B. paposo) (Ojanguren-Affilastro, guren-Affilastro, Agusto, Pizarro-Araya & Mattoni tions of B. paposo and B. roigalsinai (Llanos de The nuclear actin fragment shows no discernible pat- 2001, 2004); however, despite the overall high pig- 2007, both with very similar morphology and habitat Challe and Chanaral~ Island/Choros region). Consid- tern regarding inter- vs. intraspecific differences in ment variability of both species, in this particular requirements (Ojanguren-Affilastro, 2002; Ojan- ering the haplotype networks and distribution across mutation rates. The graphical outputs from the case this character proved to be a strong and easily guren-Affilastro et al., 2007a, b), and both also sepa- space (Supporting Information, Figure S7), one can migrate-n runs can be found in Supporting Informa- recognizable diagnostic character in all specimens rated by the Huasco river. The Copiapo river, on the see that there are no shared mitochondrial haplo- tion, Figure S9. examined. other hand, is endorheic and with a comparatively types between the populations and that there is a The isolation-with-migration analyses in IMa2 In terms of geography, the two species appear to reduced flow compared to the Huasco and Elqui riv- clear separation between the two scorpion species estimated a very recent split between the B. roigalsi- be separated by the Huasco River, with B. roigalsi- ers, and has therefore not acted as a barrier to gene regarding the similarities of the haplotype nai populations from IC and CH (13 ka), and a nai’s distributional range limited to the south by the flow for scorpions in the recent past. sequences. Within species the populations are gener- 2.5 Ma split between these populations and VI, with Elqui River and B. paposo’s northern range by the An assessment of the genetic information used in ally structured as well, with a few exceptions such estimated smaller ancestral than current popula- Absolute Desert (Gajardo, 1993). This finding read- this study reveals a general lack of structuring and as the Choros region, where certain haplotypes are tions. Also, migration between ancestral populations justs the distributional limits previously established, poor phylogenetic resolution for the nuclear gene more similar to those from other populations than to was not recovered as a possibility, estimating that where B. roigalsinai was thought to extend further fragments, whereas there is greater resolution pro- their own. The nuclear haplotype networks display the isolation between the population from VI and north (up to the northern limit of Atacama), and B. vided by the mitochondrial markers. This is probably star-shaped patterns, reflecting a general lack of those from CH and IC occurred at their point of paposo to be endemic to the area of Paposo (Ojan- due to the weaker phylo- and population genetic sig- genetic structuring, especially for the actin fragment, divergence. For B. paposo, on the other hand, the guren-Affilastro, 2002; Ojanguren-Affilastro et al., nal in nuclear than in mitochondrial markers, since where one haplotype is shared between four (non- ancestral population size was estimated to be similar 2007a, b; Ojanguren-Affilastro & Pizarro-Araya, the latter presumably have a higher mutation and adjacent) populations. Conversely, for wingless, one to current population sizes of PA and LC, while the 2014). coalescence rate, so they segregate sooner than the haplotype is present in all populations, a second hap- population from PP is estimated to be smaller. The divergence time estimates for the species and nuclear alleles. The overall low structuring may be lotype is found only in B. paposo populations, and B. Ancestral migrations were estimated between the each of their three populations varied, depending on due to past hybridisation events between the two roigalsinai’s population from Vicuna~ has a unique MRCA of PA + LC and PP, as well as unsampled the methods used, with the IMa2 age estimates species, if the barrier to gene flow (hypothesised as haplotype, indicating a certain degree of geographi- ‘ghost’ populations, although they were found not to being older than ages obtained with *BEAST, even the River Huasco) disappeared during drier spells in cal structuring. Similarly, from the graphs showing be significant by the Nielsen & Wakeley test. The though the confidence intervals in both cases were geological history. This would be supported by the isolation-by-distance (Supporting Information, Fig- divergence between the LC and PA populations is large and thus overlapped. Although the molecular phylogeographic analysis, which only truly detects a 1 1 ure S8), the overwhelming pattern is that genetic estimated at 430 kya and at 2.5 Mya for the diver- clock rate of 0.0133 substitutions siteÀ MyrÀ separation of distributional ranges for the two spe- structuring by locality and geographic distance is gence of PP from LC+PA, with current migration obtained for the mitochondrial fragment is higher cies approximately 400 kya. only truly detectable in the mitochondrial gene frag- rates similar to ancestral estimates (0.17, but not than estimates previously obtained for COI in scorpi- The relatively early point of separation between ment and, to a much lesser extent, in the wingless significant; see Supporting Information, Figure S10). ons (e.g. 0.00925: Ceccarelli et al., 2016; ; 0.007: Gan- populations of B. roigalsinai from VI and CH fragment, but not for actin. tenbein et al., 2005), the fact that a faster-evolving obtained here by IMa2 (about 2.5 Ma, albeit with The mutation-scaled effective population sizes (h) fragment (cyt b) was included here means that the large confidence intervals) can be explained by the and the mutation-scaled immigration rates (M) BAYESIAN PHYLOGEOGRAPHY rates obtained in this study are bound to be higher, fact that Vicuna~ is located in the Precordillera, a low obtained from migrate-n are shown in Supporting The RRW phylogeographic analyses delimited the and are therefore plausible. mountain range west of the Andes, whereas the Information (Table S7). Considering the mutation ancestral area of both B. roigalsinai and B. paposo The temporal congruence of the divergence of B. Choros populations are found mainly in the coastal rates of the markers and assuming a generation time approximately 4 Ma as almost the entire present-day paposo and B. roigalsinai’s MRCA during the Plio- littoral plains. The intermediate area between these of 3 years, the effective population sizes were lowest area of distribution of the two species with 85% cene, with the appearance of greater river flow from two populations is mostly covered by low mountains

6 DISCUSSION Downloaded from https://academic.oup.com/biolinnean/article/120/1/75/2864989 by guest on 29 September 2021 Information, Figure S6, which is the Genetic Land- IC and CH = 9.25 9 10À , B. roigalsinai’s CH and levels of the Andes began receiving a greater amount 6 scape Shape plot, the peaks represent the genetic VI = 6.12 9 10À ), but lower between the populations The outcomes of this work redefine the geographic of snowfall coming from the humid air masses of the distance between scorpions from different areas. This from LC (B. paposo) and IC (B. roigalsinai) limits of the two species studied, with B. paposo Pacific anticyclone. The melting of these snow 6 information indicates that the lowest genetic dis- (mtDNA = 1.74 9 10À ). This pattern is slightly dif- being found in the northern localities of PP, PA and masses increased the water flow of rivers such as the tance is found in the south-western part of B. ferent based on the wingless marker, where the low- LC, and B. roigalsinai in IC, the adjacent mainland, Huasco, which became permanent and exorheic, roigalsinai’s distributional range (between Chanaral~ est mean migration rate is between B. roigalsinai’s CH and VI (Fig. 1). The new morphological diagnos- therefore creating an effective barrier for most of the 6 Island and the Choros region), with moderate genetic CH and VI populations (4.63 9 10À ), followed by LC tic character presented here and used to definitively epigeic arthropod fauna. A similar pattern of separa- 6 distances between populations towards the north, (B. paposo) and IC (B. roigalsinai) (3.99 9 10À ), separate B. roigalsinai and B. paposo could be ques- tion between coastal scorpion species is found while the highest genetic distance can be found while the highest migration rates are between the tioned, since the pigment pattern presents high vari- between Brachistosternus sciosciae Ojanguren-Affi- 6 between Vicuna~ (VI) and neighbouring populations conspecific populations PP and PA (7.49 9 10À ), IC ability in scorpions with wide distributions (such as lastro, 2002 and Brachistosternus cepedai Ojan- 6 6 for B. roigalsinai and between neighbouring popula- and CH (7.29 9 10À ), and PA and LC (6.67 9 10À ). B. roigalsinai and B. paposo) (Ojanguren-Affilastro, guren-Affilastro, Agusto, Pizarro-Araya & Mattoni tions of B. paposo and B. roigalsinai (Llanos de The nuclear actin fragment shows no discernible pat- 2001, 2004); however, despite the overall high pig- 2007, both with very similar morphology and habitat Challe and Chanaral~ Island/Choros region). Consid- tern regarding inter- vs. intraspecific differences in ment variability of both species, in this particular requirements (Ojanguren-Affilastro, 2002; Ojan- ering the haplotype networks and distribution across mutation rates. The graphical outputs from the case this character proved to be a strong and easily guren-Affilastro et al., 2007a, b), and both also sepa- space (Supporting Information, Figure S7), one can migrate-n runs can be found in Supporting Informa- recognizable diagnostic character in all specimens rated by the Huasco river. The Copiapo river, on the see that there are no shared mitochondrial haplo- tion, Figure S9. examined. other hand, is endorheic and with a comparatively types between the populations and that there is a The isolation-with-migration analyses in IMa2 In terms of geography, the two species appear to reduced flow compared to the Huasco and Elqui riv- clear separation between the two scorpion species estimated a very recent split between the B. roigalsi- be separated by the Huasco River, with B. roigalsi- ers, and has therefore not acted as a barrier to gene regarding the similarities of the haplotype nai populations from IC and CH (13 ka), and a nai’s distributional range limited to the south by the flow for scorpions in the recent past. sequences. Within species the populations are gener- 2.5 Ma split between these populations and VI, with Elqui River and B. paposo’s northern range by the An assessment of the genetic information used in ally structured as well, with a few exceptions such estimated smaller ancestral than current popula- Absolute Desert (Gajardo, 1993). This finding read- this study reveals a general lack of structuring and as the Choros region, where certain haplotypes are tions. Also, migration between ancestral populations justs the distributional limits previously established, poor phylogenetic resolution for the nuclear gene more similar to those from other populations than to was not recovered as a possibility, estimating that where B. roigalsinai was thought to extend further fragments, whereas there is greater resolution pro- their own. The nuclear haplotype networks display the isolation between the population from VI and north (up to the northern limit of Atacama), and B. vided by the mitochondrial markers. This is probably star-shaped patterns, reflecting a general lack of those from CH and IC occurred at their point of paposo to be endemic to the area of Paposo (Ojan- due to the weaker phylo- and population genetic sig- genetic structuring, especially for the actin fragment, divergence. For B. paposo, on the other hand, the guren-Affilastro, 2002; Ojanguren-Affilastro et al., nal in nuclear than in mitochondrial markers, since where one haplotype is shared between four (non- ancestral population size was estimated to be similar 2007a, b; Ojanguren-Affilastro & Pizarro-Araya, the latter presumably have a higher mutation and adjacent) populations. Conversely, for wingless, one to current population sizes of PA and LC, while the 2014). coalescence rate, so they segregate sooner than the haplotype is present in all populations, a second hap- population from PP is estimated to be smaller. The divergence time estimates for the species and nuclear alleles. The overall low structuring may be lotype is found only in B. paposo populations, and B. Ancestral migrations were estimated between the each of their three populations varied, depending on due to past hybridisation events between the two roigalsinai’s population from Vicuna~ has a unique MRCA of PA + LC and PP, as well as unsampled the methods used, with the IMa2 age estimates species, if the barrier to gene flow (hypothesised as haplotype, indicating a certain degree of geographi- ‘ghost’ populations, although they were found not to being older than ages obtained with *BEAST, even the River Huasco) disappeared during drier spells in cal structuring. Similarly, from the graphs showing be significant by the Nielsen & Wakeley test. The though the confidence intervals in both cases were geological history. This would be supported by the isolation-by-distance (Supporting Information, Fig- divergence between the LC and PA populations is large and thus overlapped. Although the molecular phylogeographic analysis, which only truly detects a 1 1 ure S8), the overwhelming pattern is that genetic estimated at 430 kya and at 2.5 Mya for the diver- clock rate of 0.0133 substitutions siteÀ MyrÀ separation of distributional ranges for the two spe- structuring by locality and geographic distance is gence of PP from LC+PA, with current migration obtained for the mitochondrial fragment is higher cies approximately 400 kya. only truly detectable in the mitochondrial gene frag- rates similar to ancestral estimates (0.17, but not than estimates previously obtained for COI in scorpi- The relatively early point of separation between ment and, to a much lesser extent, in the wingless significant; see Supporting Information, Figure S10). ons (e.g. 0.00925: Ceccarelli et al., 2016; ; 0.007: Gan- populations of B. roigalsinai from VI and CH fragment, but not for actin. tenbein et al., 2005), the fact that a faster-evolving obtained here by IMa2 (about 2.5 Ma, albeit with The mutation-scaled effective population sizes (h) fragment (cyt b) was included here means that the large confidence intervals) can be explained by the and the mutation-scaled immigration rates (M) BAYESIAN PHYLOGEOGRAPHY rates obtained in this study are bound to be higher, fact that Vicuna~ is located in the Precordillera, a low obtained from migrate-n are shown in Supporting The RRW phylogeographic analyses delimited the and are therefore plausible. mountain range west of the Andes, whereas the Information (Table S7). Considering the mutation ancestral area of both B. roigalsinai and B. paposo The temporal congruence of the divergence of B. Choros populations are found mainly in the coastal rates of the markers and assuming a generation time approximately 4 Ma as almost the entire present-day paposo and B. roigalsinai’s MRCA during the Plio- littoral plains. The intermediate area between these of 3 years, the effective population sizes were lowest area of distribution of the two species with 85% cene, with the appearance of greater river flow from two populations is mostly covered by low mountains

© 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, , – © 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, , – © 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2017, 120, 75–89 8410 F. S. S. CECCARELLI CECCARELLI ETET AL. AL. PHYLOGEOGRAPHY OF COASTAL DESERT SCORPIONS 11 and hills, which provide a suitable environment for the mainland and with lower sea levels allowing the environmental conditions and landmasses, and/or by This study was funded by the DIULS PR13121 and B. roigalsinai only in some of its lowest, flattest islands to reconnect. Such has been the fate also of the effect of different ecological communities present PR15121/VACDDI001 projects of the University of areas. Therefore, this spatial fragmentation is likely Chanaral~ Island and the Choros archipelago, sepa- on the islands. Another point to note is that while La Serena, La Serena, Chile (JPA), PICT 2010-764, to have promoted the isolation between the Choros rated from the mainland by a present-day maximum Chanaral~ island harbours one endemic species of spi- PICT 2011-1007 and iBol (Arg.) 2012 to AAOA, and and Vicuna~ populations. These events would also seafloor depth of between 80–120 and 20–30 m, der of the family Zodariidae (Grismado & Pizarro- a postdoctoral grant from CONICET (Consejo Nacio- explain the high genetic distances detected in the respectively. At times when the sea levels were Araya, 2016), and the Choros archipelago contains nal de Investigaciones Cient�ıficas y Tecnicas,� Argen- Landscape Shape Plot of Alleles in Space between therefore lower than these maximum depths, the one endemic coleopteran of the family Tenebrionidae, tina) to FSC. We also thank A. Sanchez-Gracia,� A. populations from these two localities. islands were connected to the mainland and the pop- Gyriosomus granulipennis (Pizarro-Araya & Flores, Mart�ınez-Aqu�ıno and N. Trujillo-Arias for advice on The general population genetic trend revealed in ulations of B. roigalsinai were able to interbreed in a 2004; Alfaro, Pizarro-Araya & Flores, 2009) there is population genetic analyses and E. Gonzalez-� this study, when comparing each of the three popula- generally larger area. The lack of genetic differences a lack of endemic scorpion species on these islands. Santillan� and three anonymous referees for com- tions of B. paposo and B. roigalsinai, is that the between the populations of B. roigalsinai of the This is probably a result of the high vagility of the ments which helped improve the manuscript. We are three populations of the former show more genetic Choros archipelago and the nearby mainland sug- Downloaded from https://academic.oup.com/biolinnean/article/120/1/75/2864989 by guest on 29 September 2021 scorpions of the area and their lower ecological/ge- especially indebted to Ferm�ın Alfaro, Juan Enrique structuring and isolation, earlier divergence, higher gests that a connection of these islands to the conti- netic constraints compared to other epigeic arthro- Barriga-Tun~on,� Ricardo Botero-Trujillo, Luis Com- mutation rates and less migration than B. roigalsi- nent existed until very recently. This is also reflected pods. In fact, most scorpions from these islands can pagnucci, Camilo Mattoni, Pablo Agusto, Jose Mon- nai populations. Nevertheless, it is difficult to say by the scarcity of morphological differences between be considered stenotopic species, that can survive in daca, Hernan� Iuri, Carolina Cuezzo, Alberto Castex, whether B. paposo populations underwent recent island and continental specimens, and by the pres- a wide range of environments (Agusto et al., 2006; Juan Campusano, Cristian Rivera (CONAF-Atacama, expansions after a bottleneck (as suggested by the ence of similar scorpion communities in both areas Pizarro-Araya et al., 2014a). Another plausible, and Chanaral~ Island), Patricio Ortiz (Aljavig), Paula population genetic parameters Tajima’s D, Fu’s FS (Pizarro-Araya et al., 2014a). possibly concurrent, explanation could be the differ- Martinez and Pablo Arrospide� (CONAF-Coquimbo) and R2, and by IMa2), since the extended Bayesian Regarding the population from Chanaral~ island, ent reproductive strategies presented by the endemic for their invaluable help during the collecting trips. skyline plots (EBSP) showed stable population sizes the low structuring and lack of genetic diversity may arthropods compared to scorpions. Most arthropods during the last 500 kyr. However, given the shifts in be the result of a recent bottleneck following the are typical r strategists, presenting a large number suitable habitats due to sea-level changes, it is possi- island’s separation from the mainland. The esti- of offspring, high growth rate and short generation REFERENCES ble that populations may have been reduced in size mated 13 kyr of separation between the Chanaral~ times (i.e. life span), all of which favour higher speci- by restricted available distributional ranges, thus island and Choros region populations coincides with ation rates. Conversely, field data (J. Pizarro pers. Aguirre L. 1967. Geolog�ıa de las islas Choros, Damas y de undergoing genetic bottlenecks. In the case of B. the end of the last glacial maximum, during which obs.) reveal that both scorpion species studied here Punta Choros, Provincia de Coquimbo. Revista Minerales roigalsinai, recent population expansion appears to the sea-level was about 100 m lower than today, thus are typical K strategists (Wallwork, 1982), like many (Chile) 22: 73–83. have taken place in the population from VI, based on exposing the intermittent seafloor. Despite the other desert scorpions (Warburg & Polis, 1990), with Agusto P, Mattoni CI, Pizarro-Araya J, Cepeda-Pizarro several independent lines of evidence (Tajima’s D, apparently short time span of separation between fewer offspring, a lower growth rate, and longer gen- J, Lopez-Cort� es� F. 2006. Comunidades de escorpiones (Arachnida: Scorpiones) del desierto costero transicional de Fu’s FS and R2, and EBSP). For both species, the the Chanaral~ and continental populations of B. eration times (hence lower speciation rates) meaning markers tested also revealed LD, which may be roigalsinai, island specimens are about 30% smaller that it would take longer for scorpions to speciate Chile. Revista Chilena de Historia Natural 79: 407–421. caused by mutation, random genetic drift, selection than continental specimens, and much more densely into island endemics. Alfaro FM, Pizarro-Araya J, Flores GE. 2009. Epigean and/or gene flow, all of which would indicate some pigmented (Pizarro-Araya et al., MS in prep.). This In summary, the Chilean coastal desert, including tenebrionids (Coleoptera: Tenebrionidae) from the Choros archipelago (Coquimbo Region, Chile). Entomological News form of disturbance to the populations, most likely reduction in size has been documented in other insu- its continental-shelf islands, harbours species which 120: 125–130. brought about by past environmental instability. lar organisms (especially well studied in mammals) have had to adapt to a coastline habitat that is con- Alfaro FM, Pizarro-Araya J, Mondaca J. 2014. New insu- Recent demographic expansion has been found in which commonly display the phenomenon of gigan- stantly changing due to sea-level oscillations and tec- lar records of Germarostes (Germarostes) posticus (Germar) another desert scorpion (Graham , 2013), sug- et al. tism or dwarfism, also termed the ‘island rule’ (Van tonics, by moving. This geological instability is (Coleoptera, Hybosoridae, Ceratocanthinae) for the chilean gesting that scorpions respond relatively quickly to Valen, 1973). Williams (1980) and later Polis (1990) reflected in the present population genetic and phylo- transitional coastal desert. Coleopterist Bulletin 68: 387– localised environmental changes by moving their refer to some cases of gigantism in scorpions in geographic studies by the general low genetic diver- 390. ranges. This is in contrast to other arachnid groups islands of Baja California; Williams (1980) states sity and apparent recent diversification of the Beerli P. 2006. Comparison of Bayesian and maximum-like- such as opilionids, which have been found to contain that ‘these size variations are probably best populations. The insular populations, although only lihood inference of population genetic parameters. Bioinfor- genetically highly structured populations, presum- explained by differences in prey availability in vari- recently separated, already show some degree of dif- matics 22: 341–345. ably due to their low vagility (Bragagnolo et al., ous habitats’. This finding could also explain the ferentiation, while perhaps more striking is the Beerli P. 2009. How to use MIGRATE or why are Markov 2015; Clouse et al., 2015). smaller average size of B. roigalsinai of Chanaral~ vicariant effect of the Huasco River on the two spe- chain Monte Carlo programs difficult to use. In: Bertorelle The molecular evidence for low structuring and for island, where in a recent biological inventory of the cies, B. paposo and B. roigalsinai. So while this Giorgio, Bruford MW, Hauffe, Heidi C, Rizzoli A, Vernesi events leading to depleted and unequal allelic diver- epigeic insects, Pizarro-Araya et al. (2014a, b) discov- river, and to a certain degree the inland hills have C, eds. Population Genetics for Animal Conservation. Cam- sity (as shown by the population genetic parameters ered a remarkably lower species richness compared played a role in separating and structuring popula- bridge, UK: Cambridge University Press, 42–79. as well as the clumped distribution detected by the to the continent. This represents an impoverishment tions, eustasy is likely to have had a destabilizing Ben�ıtez HA, Pizarro-Araya J, Bravi R, Sanzana MJ, Allelic Aggregation Index Analysis), further suggests in prey availability for scorpions on the island, since effect on coastal populations. Alfaro FM. 2014. Morphological variation on isolated pop- relatively recent environmental instability, especially they feed mainly on epigeic arthropods. ulations of Praocis (Praocis) spinolai. Journal of Insect for B. roigalsinai, but also for B. paposo. In contrast Comparing and contrasting Chanaral~ island and Science 14: 11. to the apparent climatic stability of deserts such as Bielejec F, Rambaut A, Suchard MA, Lemey P. 2011. the Choros archipelago, it is noteworthy that B. ACKNOWLEDGEMENTS the Atacama (Hartley et al., 2005), coastal areas and roigalsinai is the only scorpion species present on SPREAD: spatial phylogenetic reconstruction of evolution- habitats are affected by sea-level changes and in the former, in contrast to the rich scorpion communi- Our acknowledgements to CONAF (Corporacion� ary dynamics. Bioinformatics 27: 2910–2912. some cases tectonism (Pedoja et al., 2014), immers- ties found on the latter. Differences in both island Nacional Forestal, Chile) for helping us obtain per- Blackledge TA, Scharff N, Coddington JA, Szuts€ T, Wenzel JW, Hayashi CY, Agnarsson I. 2009. Recon- ing and emersing extended lowland areas, at times communities could be explained either by their dif- mission and providing facilities to work in the structing web evolution and spider diversification in the forming islands by immersing intermittent land to ferent histories, since both present almost identical SNASPE areas (Projects No. 006/2014 and 028/2015).

© 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, , – © 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, , – © 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2017, 120, 75–89 10 F. S. CECCARELLI ET AL. PHYLOGEOGRAPHY OF COASTAL DESERT SCORPIONS 1185 and hills, which provide a suitable environment for the mainland and with lower sea levels allowing the environmental conditions and landmasses, and/or by This study was funded by the DIULS PR13121 and B. roigalsinai only in some of its lowest, flattest islands to reconnect. Such has been the fate also of the effect of different ecological communities present PR15121/VACDDI001 projects of the University of areas. Therefore, this spatial fragmentation is likely Chanaral~ Island and the Choros archipelago, sepa- on the islands. Another point to note is that while La Serena, La Serena, Chile (JPA), PICT 2010-764, to have promoted the isolation between the Choros rated from the mainland by a present-day maximum Chanaral~ island harbours one endemic species of spi- PICT 2011-1007 and iBol (Arg.) 2012 to AAOA, and and Vicuna~ populations. These events would also seafloor depth of between 80–120 and 20–30 m, der of the family Zodariidae (Grismado & Pizarro- a postdoctoral grant from CONICET (Consejo Nacio- explain the high genetic distances detected in the respectively. At times when the sea levels were Araya, 2016), and the Choros archipelago contains nal de Investigaciones Cient�ıficas y Tecnicas,� Argen- Landscape Shape Plot of Alleles in Space between therefore lower than these maximum depths, the one endemic coleopteran of the family Tenebrionidae, tina) to FSC. We also thank A. Sanchez-Gracia,� A. populations from these two localities. islands were connected to the mainland and the pop- Gyriosomus granulipennis (Pizarro-Araya & Flores, Mart�ınez-Aqu�ıno and N. Trujillo-Arias for advice on The general population genetic trend revealed in ulations of B. roigalsinai were able to interbreed in a 2004; Alfaro, Pizarro-Araya & Flores, 2009) there is population genetic analyses and E. Gonzalez-� this study, when comparing each of the three popula- generally larger area. The lack of genetic differences a lack of endemic scorpion species on these islands. Santillan� and three anonymous referees for com- tions of B. paposo and B. roigalsinai, is that the between the populations of B. roigalsinai of the This is probably a result of the high vagility of the ments which helped improve the manuscript. We are three populations of the former show more genetic Choros archipelago and the nearby mainland sug- scorpions of the area and their lower ecological/ge- especially indebted to Ferm�ın Alfaro, Juan Enrique Downloaded from https://academic.oup.com/biolinnean/article/120/1/75/2864989 by guest on 29 September 2021 structuring and isolation, earlier divergence, higher gests that a connection of these islands to the conti- netic constraints compared to other epigeic arthro- Barriga-Tun~on,� Ricardo Botero-Trujillo, Luis Com- mutation rates and less migration than B. roigalsi- nent existed until very recently. This is also reflected pods. In fact, most scorpions from these islands can pagnucci, Camilo Mattoni, Pablo Agusto, Jose Mon- nai populations. Nevertheless, it is difficult to say by the scarcity of morphological differences between be considered stenotopic species, that can survive in daca, Hernan� Iuri, Carolina Cuezzo, Alberto Castex, whether B. paposo populations underwent recent island and continental specimens, and by the pres- a wide range of environments (Agusto et al., 2006; Juan Campusano, Cristian Rivera (CONAF-Atacama, expansions after a bottleneck (as suggested by the ence of similar scorpion communities in both areas Pizarro-Araya et al., 2014a). Another plausible, and Chanaral~ Island), Patricio Ortiz (Aljavig), Paula population genetic parameters Tajima’s D, Fu’s FS (Pizarro-Araya et al., 2014a). possibly concurrent, explanation could be the differ- Martinez and Pablo Arrospide� (CONAF-Coquimbo) and R2, and by IMa2), since the extended Bayesian Regarding the population from Chanaral~ island, ent reproductive strategies presented by the endemic for their invaluable help during the collecting trips. skyline plots (EBSP) showed stable population sizes the low structuring and lack of genetic diversity may arthropods compared to scorpions. Most arthropods during the last 500 kyr. However, given the shifts in be the result of a recent bottleneck following the are typical r strategists, presenting a large number suitable habitats due to sea-level changes, it is possi- island’s separation from the mainland. The esti- of offspring, high growth rate and short generation REFERENCES ble that populations may have been reduced in size mated 13 kyr of separation between the Chanaral~ times (i.e. life span), all of which favour higher speci- by restricted available distributional ranges, thus island and Choros region populations coincides with ation rates. Conversely, field data (J. Pizarro pers. Aguirre L. 1967. Geolog�ıa de las islas Choros, Damas y de undergoing genetic bottlenecks. In the case of B. the end of the last glacial maximum, during which obs.) reveal that both scorpion species studied here Punta Choros, Provincia de Coquimbo. Revista Minerales roigalsinai, recent population expansion appears to the sea-level was about 100 m lower than today, thus are typical K strategists (Wallwork, 1982), like many (Chile) 22: 73–83. have taken place in the population from VI, based on exposing the intermittent seafloor. 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Recon- ing and emersing extended lowland areas, at times communities could be explained either by their dif- mission and providing facilities to work in the structing web evolution and spider diversification in the forming islands by immersing intermittent land to ferent histories, since both present almost identical SNASPE areas (Projects No. 006/2014 and 028/2015).

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Atlantic Rain Forest reveals poor dispersal, low diversity species, and description of the male of Cyrioctea cruz Plat- New York: Mammalian Protein Metabolism Academic 145–159. Downloaded from https://academic.oup.com/biolinnean/article/120/1/75/2864989 by guest on 29 September 2021 and extensive mitochondrial introgression. Invertebrate Sys- nick (Araneae, Zodariidae). Zootaxa 4107: 267–276. Press, 21–132. Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the tematics 29: 386–404. Guillot G, Santos F. 2009. A computer program to simulate Katoh K, Standley DM. 2013. MAFFT multiple sequence CIPRES Science Gateway for inference of large phyloge- Bryson RW Jr, Savary WE, Prendini L. 2013. Biogeogra- multilocus genotype data with spatially auto-correlated alignment software version 7: improvements in perfor- netic trees. Pp. 1–8 in Proceedings of the Gateway Comput- phy of scorpions in the Pseudouroctonus minimus complex allele frequencies. Molecular Ecology Resources 9: 1112– mance and usability. Molecular Biology and Evolution 30: ing Environments Workshop (GCE), 14 Nov. 2010, New (Vaejovidae) from south-western North America: implica- 1120. 772–780. Orleans, LA. tions of ecological specialization for pre-quaternary diversi- Haq BU, Hardenbol J, Vail PR. 1988. Mesozoic and Ceno- Kelly JK. 1997. A test of neutrality based on interlocus asso- Mirshamsi O, Sari A, Elahi E, Hosseini S. 2010. Phyloge- fication. Journal of Biogeography 40: 1850–1860. zoic chronostratigraphy and cycles of sea level change. In: ciations. Genetics 146: 1197–1206. netic relationships of Mesobuthus eupeus (C.L. Koch, 1839) Carter RWG. 1988. Coastal Environments: An Introduction Wilgus CK, Hastings BS, Kendall CGSt, Posamentier HW, Kendrick E, Bevis M, Smalley RJ, Brooks B, Vargas inferred from COI sequences (Scorpiones: Buthidae). Jour- to the Physical. Ecological and Cultural Systems of Coastli- Ross CA, Van Wagoner JC, eds. Sea level changes: an inte- RB, Laur�ıa E, Fortes LPS. 2003. The Nazca-South Amer- nal of Natural History 44: 2851–2872. nes: Academic Press, London, UK. grated approach. Tulsa, OK: Society of Economic Paleontol- ica Euler vector and its rate of change. Journal of South Mulroy TW, Rundel PW. 1977. Adaptations to desert envi- Castro C, Brignardello L. 2005. Geomorfolog�ıa aplicada a ogists and Mineralogists Special Publication 42: 71–108. American Earth Sciences 16: 125–131. ronments. BioScience 27: 109–114. la ordenacion� territorial de litorales arenosos. Orientaciones Hardenbol J, Thierry J, Farley MB, Jacquin T, Gra- Kovar��ık F, Ojanguren-Affilastro AA. 2013. Illustrated cata- Nagy KA. 2004. Water economy of free-living desert animals. para la proteccion,� usos y aprovechamiento sustentables del ciansky P-C, Vial PR 1998. Mesozoic and Cenozoic log of scorpions. Part II. Bothriuridae; Chaerilidae; Buthidae International Congress Series 1275: 291–297. sector de Los Choros, Comuna de La Higuera, IV Region.� sequence chronostratigraphic framework of European I., genera Compsobuthus, Hottentotta, Isometrus, Lychas, and Nei M. 1987. Molecular evolutionary genetics. New York: Revista de Geografıa Norte Grande 33: 33–58. basins. In: Graciansky P-C, Hardenbol J, Jacquin T, Vail Sassanidotus. Czech Republic: Jakub Rol�c�ık Publisher, 400. Columbia University Press. Ceccarelli FS, Ojanguren-Affilastro AA, Ram�ırez MJ, PR eds., Mesozoic and Cenozoic Sequence Stratigraphy of Lanfear R, Calcott B, Ho SYW, Guindon S. 2012. Parti- Nielsen R, Wakeley J. 2001. Distinguishing migration from Ochoa JA, Mattoni CI, Prendini L 2016. Andean uplift European Basins SEPM, Tulsa, OK: Special Publication 60: tionFinder: combined selection of partitioning schemes and isolation: a Markov chain Monte Carlo approach. Genetics drives diversification of the bothriurid scorpion genus Chart 1, 3–13. substitution models for phylogenetic analyses. Molecular 158: 885–896. Brachistosternus. Journal of Biogeography. doi:10.1111/ Hartley AJ, Chong G, Houston J, Mather AE. 2005. 150 Biology and Evolution 29: 1695–1701. Nylinder S, Lemey P, De Bruyn M, Suchard MA, Pfeil jbi.12760. million years of climatic stability: evidence from the Ata- Le Roux JP, Gomez� C, Venegas C, Fenner J, Middleton BE, Walsh N, Anderberg AA. 2014. On the biogeography Clouse RM, Sharma PP, Stuart JC, Davis LR, Giribet cama Desert, northern Chile. Journal of the Geological H, Marchant M, Buchbinder B, Frassinetti D, Mar- of Centipeda: a species-tree diffusion approach. Systematic G, Boyer SL, Wheeler WC. 2015. Phylogeography of the Society, London 162: 421–424. quardt C, Gregory-Wodzicki KM, Lavenu A. 2005. Neo- Biology 63: 178–191. harvestman genus Metasiro (Arthropoda, Arachnida, Opil- Heled J, Drummond AJ. 2008. 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SUPPORTING INFORMATION Additional Supporting Information may be found online in the supporting information tab for this article: Figure S1. Map showing the SNASPE Pinguino€ de Humboldt National Reserve. Figure S2. Geneland heat maps.