Montane and coastal species diversification in the economically important Mexican grasshopper genus Sphenarium (Orthoptera: Pyrgomorphidae)

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Carlos Pedraza-Lara a, Ludivina Barrientos-Lozano b, Aurora Y. Rocha-Sánchez b, Alejandro Zaldívar-Riverón a,⇑ a Colección Nacional de Insectos, Instituto de Biología, Universidad Nacional Autónoma de México, 3er. Circuito exterior s/n Cd. Universitaria, Copilco, Coyoacán, A.P. 70-233, C.P. 04510, D.F., México, Mexico b Instituto Tecnológico de Cd. Victoria, Blvd. Emilio Portes Gil No. 1301, C.P. 87010, Ciudad Victoria, Tamaulipas, Mexico

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Montane and coastal species diversification in the economically important Mexican grasshopper genus Sphenarium (Orthoptera: Pyrgomorphidae)

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Article history: Received 2 August 2014 Revised 31 December 2014 Accepted 2 January 2015 Available online xxxx

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Keywords: Barcoding Species limits Mesoamerica Neotropics Introgression

a b s t r a c t The genus Sphenarium (Pyrgomorphidae) is a small group of grasshoppers endemic to México and Guatemala that are economically and culturally important both as a food source and as agricultural pests. However, its taxonomy has been largely neglected mainly due to its conserved interspecific external morphology and the considerable intraspecific variation in colour pattern of some taxa. Here we examined morphological as well as mitochondrial and nuclear DNA sequence data to assess the species boundaries and evolutionary history in Sphenarium. Our morphological identification and DNA sequence-based species delimitation, carried out with three different approaches (DNA barcoding, general mixed Yule-coalescent model, Bayesian species delimitation), all recovered a higher number of putative species of Sphenarium than previously recognised. We unambiguously delimit seven species, and between five and ten additional species depending on the data/method analysed. Phylogenetic relationships within the genus strongly support two main clades, one exclusively montane, the other coastal. Divergence time estimates suggest late Miocene to Pliocene ages for the origin and most of the early diversification events in the genus, which were probably influenced by the formation of the Trans-Mexican Volcanic Belt. A series of Pleistocene events could have led to the current species diversification in both montane and coastal regions. This study not only reveals an overlooked species richness for the most popular edible insect in Mexico, but also highlights the influence of the dynamic geological and climatic history of the region in shaping its current diversity. Ó 2015 Elsevier Inc. All rights reserved.

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1. Introduction

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The Mexican territory has complex geological (FerrusquíaVillafranca, 1998) and palaeoclimatic histories (Ramamoorthy et al., 1993; Vidal-Zepeda, 2005), which have played a large role in shaping its striking current biodiversity. This region contains four main mountain ranges, whose origin dates from different geological periods, either ancient and relatively recent (FerrusquíaVillafranca, 1998; Vidal-Zepeda, 2005). Moreover, a series of glacial climatic cycles and changes in the isometric sea levels occurred during the Pleistocene led to various events of expansion and contraction of the range of both coastal and highland habitats (Ferrari et al., 2000; McDonald, 1993; Moreno-Letelier and Piñero, 2009).

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⇑ Corresponding author.

A number of studies carried out with various animal taxa distributed across Mexico have highlighted the influence that the above geological and palaeoclimatic events had in their evolutionary history, revealing distribution patterns with strong phylogeographic structure both in high altitude (Bryson et al., 2011a,b) and lowland (e.g., Mulcahy and Mendelson III, 2000; ZaldivarRiveron et al., 2004; Zarza et al., 2008) regions. Only few of these studies, however, have focused on arthropods distributed through Middle America (e.g. Anducho-Reyes et al., 2008). The grasshopper genus Sphenarium Charpentier (Pyrgomorphidae) is a small group of orthopterans that currently comprises six described species, two of which have two recognised subspecies each. Members of this genus are distributed both in high altitude and coastal regions from Nayarit in western Mexico to Guatemala (Eades et al., 2014; Kevan and Akbar, 1964) (Fig. 1A–F). These grasshoppers are of considerable economic interest since they have

E-mail address: [email protected] (A. Zaldívar-Riverón). http://dx.doi.org/10.1016/j.ympev.2015.01.001 1055-7903/Ó 2015 Elsevier Inc. All rights reserved.

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been eaten by humans since prehispanic times (Ramos-Elorduy, 1993). Moreover, some of its species can be serious crop pests (e.g. S. p. purpurascens Charpentier), causing considerable damage to agricultural fields (Cerritos and Cano-Santana, 2008). Sphenarium p. purpurascens has been by far the most studied taxon of the genus in terms of its reproductive biology and ecology. This subspecies is univoltine and highly abundant, with nymphs emerging during the rainy season in late spring to early summer and reaching sexual maturity in August (Cueva del Castillo et al., 1999). A similar reproductive biology, including a high reproductive capacity, also appears to occur in the remaining members of the group (Zaldívar-Riverón, pers. obs.). Despite its economic relevance, the taxonomy of Sphenarium has been largely neglected. Most species descriptions and taxonomic arrangements for this genus were published more than a century ago, and the actual status, morphological characterisation and geographic distribution of most of its currently recognised forms remain unclear. The most relevant taxonomic studies carried out for Sphenarium in the last decades reported a conserved interspecific external morphology as well as considerable intraspecific colour pattern variation in some taxa (Boyle, 1975; Kevan and Akbar, 1964). However, these and a previous work (Márquez, 1962) showed that features from male and female genitalia are informative for delimiting species in the group.

Sphenarium p. purpurascens appears to be the taxon with the widest geographic distribution, having been reported to occur along the Trans Mexican Volcanic Belt (TMVB) and Sierra Madre del Sur (Márquez, 1962). The remaining recognised forms, on the other hand, have been recorded to have more restricted distributions (Boyle, 1975; Kevan, 1977), though these remain largely unknown. In addition, recent collecting by the authors has found various populations of uncertain species assignation. The conserved morphology in Sphenarium and its wide range of habitats in a territory with an intricate geological and palaeoclimatic past make it an attractive to study different aspects of its evolutionary history. However, the number of speciation events that have occurred in the genus can be difficult to assess due to its apparent recent origin and diversification and by the presumably similar reproductive biology and of its species, which can lead to incomplete lineage sorting and/or introgression of DNA by hybridization, respectively. Here we assess the number of species divergence processes in Sphenarium based on mitochondrial (mt) and nuclear DNA sequence data. For this, we examined more than 150 specimens covering its eight recognised forms and most of the known geographic distribution for the genus. We first carried out separate analyses for the mt and nuclear markers to detect cases of mt introgression and/or species lineage sorting. We then carried out

Fig. 1. Dorsal colour pattern of males belonging to selected species of Sphenarium. (A) S. sp. Gro. 6, San Jeronimito, Guerrero; (B) S. m. mexicanum Saussure, 1859, Alvarado, Veracruz; (C) Sphenarium sp. Oax. 9, Mpio. Mixtepec, near Puerto Escondido, Oaxaca; (D) S. p. purpurascens Charpentier, 1842, UNAM, Ciudad Universitaria, D. F.; (E) Sphenarium sp. Gro. 7, México-Acapulco highway, km. 314, Guerrero; (F) S. variabile Kenan, Highway 190 La Reforma-Yautepec, Oaxaca. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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groups members of other two pyrgomorphid tribes, Sphenacris crassicornis Bolívar (Ichthiacridini) and Ichthyotettix mexicanus (Saussure) (Ichthyotetitginni), using the latter species to root all the trees. A list with the specimens examined, their locality data and GenBank accession numbers for the three gene markers examined is shown in Supplementary material 1. This information is also available in the project file ‘Species limits in Sphenarium (Orthoptera)’ (SPHEN project), which is contained in the projects section of the Barcode of Life Data Systems (www.boldsystems. org).

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three different DNA sequence-based species delimitation approaches, and the congruence between these analyses and information obtained from genitalia and dorsal pattern of males was employed to establish the number of species involved following an integrative taxonomic approach by congruence (sensu Padial et al., 2010). Our results have not only revealed an overlooked species richness within the most popular edible insect group in Mexico, but also support previous studies that highlight the influence of the geological and climatic history of this region in shaping its biodiversity.

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2. Methods

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2.1. Taxon sampling

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A total of 153 specimens assigned to Sphenarium collected from 37 localities situated in various high altitude and coastal regions of Mexico were examined (Fig. 2, Supplementary material 1). These sampled localities covered most part of the known geographic distribution of each recognised form, including, when known, their type localities. Moreover, some specimens of uncertain species identity were collected. Most members of the tribe Sphenariini occur in the Old World, except for the species of the small subtribe Sphenariina, which is represented by Sphenarium, Prosphena Bolívar and Jaragua PerezGelabert, Dominici & Hierro, which are restricted to Middle America and the Caribean. Prosphena is only known by one species from Guatemala, whereas Jaragua has two described species from Dominican Republic (Eades et al., 2014). We could not obtain tissue samples of the latter two genera, and therefore we included as out-

DNA sequences of two mt and one nuclear gene markers were examined. The two mt markers were 634 bp of the cytochrome oxidase I (COI) gene corresponding to the barcoding locus (Hebert et al., 2003b), and 419 bp of the cytochrome b (cyt b) gene. The nuclear marker comprised 632 bp of the second and third domain regions of the ribosomal (r) 28S gene. COI sequences were obtained for two to eight specimens of each of the sampled populations, whereas cyt b and 28S sequences were generated for a subset of specimens according to their observed COI variation. Genomic DNA was obtained from muscular tissue dissected from the hind leg of each specimen using the Exgene Tissue SV (GeneAllÒ) extraction kit and following the manufacturer’s protocol, except by eluting the DNA template in a final volume of 150 ll of ddH2O. Amplification of the above three markers was performed in 15 ll total volume reactions containing: 1 PCR buffer, 0.5 lM of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl2,

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Fig. 2. Localities and taxa included in this study. Circles = sampled localities from high altitude locations, Squares = samples from lowland locations, colours refer to the taxa assignations based on the phylogenetic tree showed in Fig. 4. Numbers correspond to locality records listed in Supplementary material 1. Black squares = outgroups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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1 U of Platinum Taq DNA polymerase (INVITROGEN), and 10–50 ng of template DNA. COI was amplified with the universal LCO-1490 and HCO-2198 primers designed by (Folmer et al., 1994) and the PCR conditions described in (Hebert et al., 2003a). Amplification of cyt b employed the CB1_5 and CB2_5 primers and the PCR conditions mentioned in (Simon et al., 1994). For 28S, we designed the following primers specific for orthopterans: 28S_ORT_F (GTT CAA AGT ACG TGA AAC CGT TC) and 28_ORT_R (CTC GCA ATG AGG ACG AGA CG). The PCR program for 28S included an initial cycle of 3 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 52° and 60 s at 72 °C, and a final extension of 4 min at 72 °C. PCR products were sent for purification and sequencing to the High-Throughput Genomics Unit, University of Washington (http://www.htseq.org/index.html). Sequences were edited with Sequencher version 5.1 (GeneCodesCorporation). None of the gene markers showed any sequence length variation, and thus their alignment was performed manually. Corrected genetic distances for COI were calculated using the K2P evolutionary model (Kimura, 1980) with the program MEGA version 6.0 (Tamura et al., 2013).

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2.3. Gene genealogies

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Mitochondrial introgression and incomplete lineage sorting are two phenomena that can lead to erroneous species tree reconstructions (Funk and Omland, 2003; Willis et al., 2014). We therefore first carried out separate phylogenetic analyses for the mt and nuclear markers in order to detect whether they occur in our samples. Specimens that were placed in discordant sequence clusters in our mt and nuclear gene genealogies were assumed to represent cases of mitochondrial introgression (Bryson et al., 2014) or incomplete lineage sorting (Leaché et al., 2013), and therefore were excluded from the species delimitation and interspecific phylogenetic analyses. Bayesian partitioned and maximum likelihood (ML) analyses were carried out for the separate mt and nuclear data sets using the programs MrBayes version 3.2 (Ronquist et al., 2012) and RAxML version 7.2.8 (Stamatakis et al., 2008), respectively. Bayesian analyses consisted of two individual runs of 20 million generations each, had uniform priors and sampled trees every 1000 generations. Partitions in the Bayesian analyses were delimited according to their codon positions for the mt markers, whereas the 28S data set was considered as a single partition. The evolutionary models selected for the above partitions were chosen based on the Bayesian information criterion implemented in JModeltest version 2.1.3 (Darriba et al., 2012) and are listed in Supplementary material 2. Burn-in in all runs was established after 10 million generations to ensure convergence of the standard deviation of split frequencies, and posterior probabilities P 0.95 were regarded as significantly supported (Hillis and Bull, 1993). The ML analyses on the other hand used the rapid bootstrap algorithm (Stamatakis et al., 2008), and nodes P 70 were considered as highly supported (Hillis and Bull, 1993). All partitions in the ML analyses employed the GTR + G evolutionary model. A previous molecular study with orthopterans has reported the existence of nuclear integration of mtDNA (i.e. nuclear mt pseudogenes) based on the presence of indels, internal stop codons and nucleotide composition (Song et al., 2008). These pseudogenes, when they are not detected, lead to the inclusion of paralogous sequences, and therefore result in incorrect estimates of phylogenies. In our study, COI-like sequences were obtained instead of the barcoding locus for specimens belonging to the Gro. 9 and Oax. 2 populations. These paralogous sequences, which were excluded from all analyses, were detected by the presence of internal stop-codons.

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2.4. DNA sequence-based species delimitation

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247 In recent years, various DNA sequence-based approaches that 248 implement coalescent methods for delimiting species have been 249 developed both for single and multilocus data (Fujita et al., 250 2012). Species boundaries in Sphenarium were therefore 251 assessed using one single and one multilocus-based coalescent 252 method. 253 We first used the widely employed 2% COI distance approach 254 (2%D; (Hebert et al., 2003a) which is automated by the Barcode 255 index number (BIN) criterion (Ratnasingham and Hebert, 2013) 256 and implemented in the BOLD web page (www.boldsystem.org). 257 The BIN system has been argued to represent a reliable source of 258 evidence for the rapid comparison of presumptive species, which 259 is coupled with an informatics platform that maps each newly 260 acquired sequence to an existing animal COI data base 261 (Ratnasingham and Hebert, 2013). 262 The single locus-based General Mixed Yules-Coalescent (GMYC) 263 method (Pons et al., 2006) was performed for the separate COI and 264 cyt b markers using in each case a subset of sequences (32 ingroup 265 specimens), as well as for a COI + cyt b data set that only included 266 specimens with sequences for the above two markers (37 ingroup 267 specimens). A Bayesian relaxed clock analysis was performed for 268 each matrix with the program BEAST version 1.7.4 (Drummond 269 et al., 2012), running the MCMC chains for 10 million generations, 270 using a lognormal distribution parameter for clock rate, a coales271 cent prior and the above evolutionary models (or their nearest 272 available option) for each partition. Burn-in was established in all 273 analyses after five million generations. The reconstructed ultra274 metric trees were subsequently employed for GMYC analyses using 275 the single-threshold approach with the SPLIT package (http://rforge.r-project.org/projects/splits/) implemented in R version Q5 276 277 2.10.1 (R Core Development Team, 2009). 278 We also employed the multilocus-based Bayesian species 279 delimitation (BSD) method described by (Rannala and Yang, 280 2003) and implemented in the program Bayesian Phylogenetics 281 and Phylogeography version 2.2 (Rannala and Yang, 2003; Yang 282 and Rannala, 2010). This method estimates the posterior distribu283 tion for species delimitation models differing by the number of 284 species using a multilocus data set (Yang and Rannala, 2010). Four 285 species delimitation models were tested, two considering the spe286 cies recovered by the 2%D and GMYC species delimitation analyses 287 (models 1 and 2, respectively), a third one considering the morpho288 species delimitation (model 3), and the remaining one being simi289 lar to the latter model except by dividing S. rugosum and the 290 population Gro. 4 into two separate species (model 4). All models 291 employed the fully resolved tree topology of the Bayesian COI + cyt 292 b + 28S analysis using BEAST (see below). 293 Prior distributions on ancestral population sizes (h) and root age 294 in the species tree (s0) can have a strong impact on the posterior 295 probabilities for species delimitation (Leaché and Fujita, 2010; 296 Yang and Rannala, 2010). In our analyses, a gamma prior G (a, b) 297 was used on the distribution of the (hs) and (s0) parameters, both 298 with a prior mean = a/b and a prior variance = a/b2, and the Dirich299 let was assigned for the remaining divergence time parameters 300 (Rannala and Yang, 2003; Yang and Rannala, 2010). We followed 301 previous studies (Leaché and Fujita, 2010; Leaché et al., 2013) 302 and evaluated three prior combinations differing in their gamma 303 G (a, ß) distribution parameter: (1) assuming large ancestral popu304 lation sizes h  G (1, 10) and deep divergences among species 305 s0  G (1, 10), both with a prior mean = 0.1, variance = 0.01; (2) 306 assuming large ancestral population sizes h  G (1, 10) and shallow 307 divergences among species s0  G (1, 1000); and (3) assuming 308 small ancestral population sizes h  G (1, 1000) and shallow diver309 gences among species s0  G (1, 1000), both with prior 310 mean = 0.001 and variance = 5  10 7.

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Each rjMCMC analysis was run twice for 200,000 generations to confirm consistency between runs (sampling interval of one), using the algorithm 0 with the automatic fine-tuning parameter. Burn-in was determined to occur after 20,000 generations. Speciation probability values P 0.95 were regarded as strong support for a speciation event (Leaché and Fujita, 2010; Leaché et al., 2013).

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2.5. Integrative taxonomy

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Characters from male genitalia have proven to be taxonomically informative in various groups of acridomorphans (Eades, 2000). Moreover, our morphological examination of the specimens included in this work showed that dorsal colour pattern of males also appear to be informative at species level. Morphological delimitation of Sphenarium species was therefore based on six male adult features, including dorsal colour pattern and five features recorded from male genitalia. Male internal genitalia were dissected and cleared with KOH, following the terminology employed in previous works (Kevan and Akbar, 1964). Characters of male genitalia were recorded from a total of 72 specimens. These features were examined for three specimens from each sampled locality, except for those with specimens assigned to S. p. purpurascens, where only six specimens from two localities were examined. Male dorsal colour pattern was recorded from live individuals collected by the authors. Digital photographs of male genitalia were taken and edited with a LeicaÒ Z16 APO-A stereoscopic microscope, a LeicaÒ DFC295/DFC290 HD camera, and the Leica Application SuiteÒ program. A list with the six morphological features selected and their recorded variation is shown in Supplementary material 3. Photographs of male dorsal pattern and genitalia structures (endopallus and epiphallus) of some of the species delimited in this study are shown in Fig. 1 and Supplementary material 4, respectively. Morphospecies were delimited by the presence of at least one exclusive feature and/or by the presence of a particular combination of characters. The delimited morphospecies were compared with the species boundaries obtained by the sequence-based approaches, and an integrative taxonomic approach was followed based on a congruence criterion (Padial et al., 2010) in order to establish the number of species in Sphenarium.

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Phylogenetic relationships among representative specimens of the species delimited using the above integrative taxonomic approach were reconstructed with MrBayes and RAxML using the parameters aforementioned. A Bayesian relaxed molecular clock analysis was performed with BEAST using a concatenated COI + cyt b + 28S data set. Four independent runs of 20 million generations each were performed to ensure convergence in estimates, sampling every 2000 generations and using the above partitions and evolutionary models (or their nearest available options). Mean node ages and their 95% highest posterior densities (HPDs) were estimated using a Yule tree prior and an uncorrelated relaxed lognormal clock rate. Burn-in was determined to occur after 2 million generations with the program Tracer version 1.4 (Rambaut and Drummond, 2007). A range of previously published mutation rates obtained for several insect groups, including orthopterans, was implemented for COI and cyt b to estimate absolute node ages (percentage of changes per million years). These mutation rates varied from 1.15% to 1.7% (Brower and DeSalle, 1998; Papadopoulou et al., 2010) and from 0.35% to 1.15% (Brower and DeSalle, 1998; Gray et al., 2006) for COI and cyt b, respectively. The mutation rate for 28S was of 0.06% ± 0.03% (Papadopoulou et al., 2010).

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A total of 1685 bp were obtained for the three gene fragments examined. A summary of the features of each partition is shown in Supplementary material 2. The number of sequences included for the separate COI, cyt b and 28S analyses was of 115, 71 and 105, respectively. The most variable marker was cyt b, followed by COI and 28S (28.2%, 24.4% and 4.1% of parsimony informative sites, respectively). The 28S topology was largely unresolved, though this and the mt topologies were mostly concordant in their recovered relationships (Fig. 3). The few incongruent relationships observed between the 28S and mt topologies mainly involved specimens of two populations assigned to S. variabile (Oaxaca 1 and 3), both located near the Tehuantepec Isthmus, Oaxaca. Various specimens of these populations shared the same 28S genotype in the 28S tree, though four of them were nested separately due to the presence of two exclusive nucleotide substitutions. In contrast, in the cyt b topology members of the above two populations were intermingled within a major clade exclusively composed of specimens from coastal populations. Other incongruence between the mt and nuclear phylogenies involved specimens assigned to one of the samples of S. p. purpurascens from Oaxaca (Oax. 5), which were nested in a single clade together with all members assigned to this subspecies in the mt analyses, but appeared nested separately in the 28S tree due to the presence of three exclusive nucleotide substitutions.

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3.2. Species boundaries

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All analyses excluded the seven specimens assigned to S. variabile (Oax. 1, Oax. 3) and three specimens of S. p. purpurascens (Oax.5) due to their conflicting relationships in the nuclear and mt trees. Moreover, the specimens of S. variabile were also intermingled with S. m. mexicanum in the cyt b topology despite being morphologically distinct (Supplementary material 3). Together this suggests that there has been gene introgression or/and incomplete lineage sorting into the latter three taxa. Pairwise genetic distances for the COI locus and the number of species delimited with the GMYC method are shown in Supplementary materials 5 and 6, respectively. The three GMYC analyses performed were congruent in their species boundaries, and therefore only the results derived from the analysis with the combined mt markers were employed for further comparisons. Ten of the species delimited by the 2%D and GMYC approaches and by morphology were congruent in their specimen composition (Fig. 4). Morphospecies discrimination supported the recognition of 16 species (Supplementary material 3), 13 of which were congruent with the GMYC analysis and one belonged to S. variabile. Among the cases of incongruence observed between the above three species delimitations, two sample combinations, S. p. purpurascens + Gro. 4 + Gro. 1, 8 and S. rugosum + Gro. 4, each formed a single BIN, but were divided into five (two S. p. purpurascens species, S. rugosum, Gro. 4, Gro. 1 + 8) and three (S. p. purpurascens, S. rugosum + Gro. 4, Gro. 1, 8) putative species by the GMYC approach and morphology, respectively. The species delimited by the BSD analyses using the four tested models are shown in Fig. 5. The results show that different prior combinations for l and s0 considerably affect the posterior probabilities for nodes representing speciation events. In particular, the priors assuming small ancestral population sizes and shallow divergences among species recovered most of the speciation events with strong posterior probabilities in the four tested models. We therefore only mention those results obtained with the latter prior combination.

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Fig. 3. Phylograms derived from the Bayesian analyses performed for the separate mt (COI and cyt b) and nuclear (28S) data sets. Numbers below nodes are Bayesian posterior probabilities P 95%. Numbers above nodes are bootstrap values P 75% recovered from the ML analyses. Taxon colours are those assigned in the phylogram derived from the Bayesian concatenated analysis showed in Fig. 4.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Seven taxa were strongly supported as distinct putative species by the BSD analyses carried out with the four tested models: S. p. minimum, S. m. mexicanum, S. macrophallicum, S. sp. Gro. 2, S. sp. Gro. 3, S. sp. Gro. 7 and S. borrei. Sphenarium p. purpurascens also was strongly supported with the three models that regarded all its populations as a single species. Moreover, the Oax. 9 population was strongly supported as a separate species from S. sp. Gro. 6 + S. sp. Gro. 9 by two of the models. The two tested species hypotheses that regarded S. rugosum, Gro. 1 + 8 and Gro. 4 each as a separate species recovered them with strong support.

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3.3. Phylogenetic relationships and divergence time estimates

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A subset of 37 specimens comprising all the putative species recovered by the different approaches was employed for the concatenated Bayesian and ML analyses. The topologies obtained with the phylogenetic methods (Fig. 4 and 6) were congruent with the individual gene genealogies, with all their strongly supported relationships being congruent among them. Sphenarium was significantly supported as monophyletic. Sphenarium borrei, the species with the northwesternmost distribution of of the genus, appears as sister to the remaining species of the genus in the ML and BEAST phylograms, though with weak support. Two significantly supported major clades were consistently recovered, one exclusively

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composed by coastal species and the other one by species from montane (above 800 m) regions. Phylogenetic relationships among species within these two clades were geographically congruent. The chronogram reconstructed with BEAST is shown in Fig. 6. According to this, the origin of Sphenarium and separation of the coastal and montane occurred during the late Miocene, 6.25–10.8 and 5.54–9.27 MY ago, respectively. The most recent common ancestors of the latter two clades on the other hand were estimated to diverge during the mid Pliocene to Pleistocene and late Miocene to mid Pliocene, 2.26–4.12 and 3.93–6.91 MY ago, respectively. All the cladogenesis events that appear within the coastal and montane clades appeared to have occurred during the Pleistocene.

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4. Discussion

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4.1. Performance of DNA sequence-based species delimitation approaches

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The results obtained with the three DNA sequence-based approaches performed differ in some of the delimited species for the examined populations of Sphenarium. This incongruence in species composition corresponds to pairs of lineages with low genetic divergence, where each of the approaches used employs a different criterion for distinguishing between evolving entities.

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Fig. 3 (continued)

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However, despite these differences, the three methods allowed us to unambiguously delimit seven species within Sphenarium, including three undescribed taxa (see below). The Barcoding gap of 2% implemented in the BIN system represents a rapid identification tool that is especially useful for highly diverse invertebrate taxa (Ratnasingham and Hebert, 2013). This criterion, however, has limited accuracy in recently diverged species, which often have considerably low COI divergence due to incomplete lineage sorting or large effective population sizes and/or low mutation rates (van Velzen et al., 2012). The GMYC method on the other hand shows considerable stability for delimiting species under different conditions (tree-building method employed, high presence of singletons, number of included species; (Ceccarelli et al., 2012; Fujisawa and Barraclough, 2013;

Talavera et al., 2013)) though it tends to overestimate species in the presence of pronounced population structure (Hamilton et al., 2011; Satler et al., 2013). The differences observed in our species delimitation analyses are concordant with the above criticisms when it comes to the two latter approaches, with the 2%D approach recognising fewer and the GMYC more putative species. The BSD is a multilocus-based method that has as its main advantage the validation of species hypotheses, while accommodating lineage sorting and uncertainties that could occur in separate gene trees (Zhang et al., 2011). However, as observed with our sequence data, the performance of this method still needs to be further assessed since it is highly sensitive to the use of different priors combination (McKay et al., 2013).

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Fig. 4. Bayesian phylogram derived from the concatenated COI + cyt b + 28S analysis for the putative species of Sphenarium. Posterior probabilities of nodes are showed above branches; bootstrap values of nodes obtained from the ML analysis are showed below branches. Coloured bars at the right of the topology show the species boundaries recovered by the 2%D and GMYC approaches and by morphology. ⁄ = specimens of S. variabile were not included in the tree (see results), they only were delimited based on morphological evidence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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4.2. Species boundaries in Sphenarium

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Following an integrative taxonomic approach based on congruence among our examined morphological/molecular data, as well as on concordance among the DNA sequence-based methods performed, we can unambiguously delimit seven species. Moreover, between five and ten additional species were recovered depending the data/method analysed. The seven species can be confidently delimited within Sphenarium are those assigned in this work to: (1) S. p. minimum, (2) S. borrei, (3) S. m. mexicanum (Fig. 1B), (4) S. macrophallicum, (5) S. sp. Gro. 2, (6) S. sp. Gro. 3 and (7) S. sp. Gro. 7 (Fig. 1E). We therefore propose that S. p. minimum and S. m. mexicanum should be elevated to species level. Of these seven species, the latter three represent previously overlooked and undescribed taxa (S. spp. Gro. 2, Gro. 3 and Gro. 7). Moreover, at least one additional undescribed spe-

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cies is represented by the Gro. 6, Gro. 9 and Oax. 9 populations, though the GMYC and morphological approaches indicate that each one represents a separate evolutionary unit. Additional morphological and molecular information is thus required to clarify the status of the latter populations. For the currently recognised S. p. purpurascens and S. rugosum, both from high altitude regions, as well as for the coastal S. m. histrio, we propose that they represent valid species according to their populations examined in this work until further studies assess their taxonomic status and composition in more detail. Specifically, additional work will reveal whether S. p. purpurascens represents a taxon composed of more than one species. We also propose to maintain the specific status of S. variabile (Fig. 1F) based on its morphological distinctiveness, though the presumable occurrence of mitochondrial introgression or incomplete lineage sorting needs to be assessed with respect to the populations assigned here to S.

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0.275 0.347 1.0

0.134 0.030 0.943

0.119 0.166 1.0

0.169 0.129 0.993

0.050 0.075 0.999

0.999 1.0 1.0

0.962 0.839 1.0 1.0 1.0 1.0

1.0 1.0 1.0

0.159 0.016 0.959

0.009 0.002 0.853

0.044 0.020 0.999

0.015 0.001 0.916

0.267 0.319 1.0

S. rugosum

Gro4 Gro4

S. rugosum

0.019 0.050 0.996

0.049 0.046 0.990

0.113 0.229 1.0 1.0 1.0 1.0

0.948 0.720 1.0 1.0 1.0 1.0

1.0 1.0 1.0

1.0 1.0 1.0

1.0 1.0 1.0

1.0 1.0 1.0

1.0 1.0 1.0 1.0 1.0 1.0

Gro1 + Gro8

S. macrophalicum

Gro3

S. p. minimum

Gro7

S. m. histro

Oax2

S. m. mexicanum

Gro6

Gro9

Oax9

MODEL 4 (Morphology + S. sp. Gro4, 16 species)

S. borrei

Sphenacris crassicornis

Gro1 + Gro8

S. rugosum + Gro4

S. p. purpurascens

D

Gro2

S. macrophalicum

0.299 0.338 1.0

1.0 1.0 1.0

S. p. purpurascens 2

1.0 1.0 1.0

1.0 1.0

Gro3

S. p. minimum

Gro7

S. m. histro

Oax2

S. m. mexicanum

Gro6

Gro9

Oax9

S. borrei

Sphenacris crassicornis

1.0 1.0 1.0

1.0 1.0 1.0

0.008 0.022 0.911

1.0 1.0 1.0 1.0 1.0 1.0

1.0 1.0 1.0

MODEL 3 (Morphology, 15 species)

0.007 0.004 0.833

Gro1 + Gro8 1.0 1.0 1.0

1.0 1.0 1.0

C

0.014 0.030 0.998

0.381 0.539 0.377 0.134 0.176 1.0

1.0 1.0 1.0

1.0 1.0 1.0

S. p. purpurascens 1

Gro2

Gro3

S. macrophalicum

S. m. histro

Gro7

Oax2

S. m. mexicanum

S. p. minimum

0.309 0.269 0.999

0.062 0.048 0.990

0.806 0.941 1.0

1.0 1.0 1.0

1.0 1.0 1.0

0.004 0.011 0.908 0.043 0.071 1.0

0.795 0.003 0.795

0.004 0.032 0.921

0.795 0.799 1.0

Gro6

Gro9

S. borrei

1.0 1.0 1.0

Oax9

Sphenacris crassicornis

S. p. purpurascens

Gro2 0.056 0.072 0.991

S. p. purpurascens

0.093 0.112 1.0

S. macrophalicum

Gro3

S. p. minimum

Gro7

S. m. histro 0.005 0.011 0.912

Model 2 (GMYC, 17 species)

Gro2

0.036 0.004 0.953

Oax2

S. m. mexicanum

Gro6

Gro9

Oax9

S. borrei

Sphenacris crassicornis

MODEL 1 (2% D, 14 species)

0.002 0.000 0.851

B

S. rugosum + Gro4+Gro1+Gro8

A

1.0 1.0 1.0

1.0 1.0 1.0

Fig. 5. Results from the species delimitation analyses using the BSD method and testing four different hypotheses of species boundaries in Sphenarium. The following values near nodes refer to posterior probabilities of speciation under distinct Gamma parameter distributions of h and s0: above, large ancestral population sizes h  G (1, 10) and deep divergences among species s0  G (1, 10); middle, large ancestral population sizes h  G (1, 10) and shallow divergences among species s0  G (1, 1000); bellow, small ancestral population sizes h  G (1, 1000) and shallow divergences among species s0  G (1, 1000). Species delimitation models: (a) putative species recovered by the 2% distance criterion (2%D); (b) putative species recovered bt the GMYC method; (c) delimited morphospecies; (d) delimited morphospecies, except by dividing S. rugosum and S. Gro. 4 into two separate species.

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Fig. 6. Chronogram reconstructed with BEAST and the concatenated data set. 95% HPD intervals (in MY) are shown for selected nodes. Black circles above branches are Bayesian posterior probabilities P 95%.

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m. mexicanum. Further studies will also help to establish whether the populations belonging to Gro. 1 + 8, Gro. 4 and Oax. 2 each actually represent additional undescribed species.

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4.3. Gene tree incongruence

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Analyses based on mt markers often provide robust phylogenetic and phylogeographic estimates. However, under some circumstances, it is known that mt trees might reflect evolutionary processes other than phylogenetic relationships (Doyle, 1997; Nichols, 2001; Pamilo and Nei, 1988), including introgressive hybridization and incomplete lineage sorting (Carr et al., 1986; Funk and Omland, 2003; Patton and Smith, 1994). Therefore, far from being problematic, mt markers can provide valuable insights into evolutionary phenomena that otherwise would remain undetected only using nuclear markers (Bryson et al., 2014; McGuire et al., 2007). The complex geological and environmental history of the Mexican territory has shaped a mosaic of regions in which numerous contact zones between lineages have been described for different animal groups, making mt introgression a recurrent phenomenon (McGuire et al., 2007). We suggest that the lack of coalescence of the sequences belonging to S. variabile could represent a case of mt introgression by hybridization with S. mexicanum, which is sup-

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ported by the consistent morphological distinctiveness of these two taxa and their apparent parapatric distribution.

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4.4. Evolution and historical biogeography of Sphenarium

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According to the phylogenetic relationships recovered for Sphenarium, most of its species diversification occurred within two major clades with exclusive geographic distributions, one composed of coastal and the other one of montane taxa (Fig. 5). Our results also indicate that the origin and early diversification in the genus probably occurred during the late Miocene, whereas a series of Pleistocene events could have originated the current species diversity within the above two geographically structured clades. Late Miocene–Pliocene divergences between major lineages have also been estimated for other montane (e.g. jays: (McCormack et al., 2008); rattlesnakes: (Bryson et al., 2011b)) and coastal (e.g. toads: (Mulcahy and Mendelson III, 2000) cichlid and characid fish: (Hulsey et al., 2004)) taxa with similar distribution to Sphenarium. The divergence ages proposed for these groups appear to be congruent with the formation of the TMVB. The emergence of this mountain range could therefore have influenced the evolutionary history of Sphenarium, both by segregating its high and low altitude populations, as well as by creating a complex

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physiography that promoted subsequent cladogenesis through Pleistocene climatic oscillations. Significant Pleistocene changes in the isometric sea levels influenced by glacial climatic cycles (Ferrari et al., 2000) could have originated speciation in Mexican coastal taxa, including Sphenarium (Fig. 6). These cladogenetic events probably occurred when the sea level was significantly high, splitting coastal populations situated between mountain ranges, such as the TMVB and Sierra Madre del Sur. Drastic sea level changes have also been proposed as the major force for cladogenesis in other coastal groups distributed along Mesoamerica, including cichlid fish (Haq et al., 1987) and frogs (Rˇícˇan et al., 2013). Similar to some North American montane grasshopper taxa (Knowles, 2001; Lacey Knowles and Alvarado-Serrano, 2010; Zaldivar-Riveron et al., 2004), Pleistocene climatic oscillations could also have promoted speciation in high altitude populations of Sphenarium through repeated expansion/contraction of their distribution ranges. Species of Sphenarium belonging to the montane clade are mainly distributed along the TMVB and Sierra Madre del Sur at altitudes above 800 m, including the widespread S. p. purpurascens, whose populations are generally found at altitudes above 1400 m. This supports the existence of a close biogeographic relationship of montane fauna between these two morphotectonic provinces (Knowles and Alvarado-Serrano, 2010).

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Acknowledgments

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We thank Valeria Salinas, Gabriela Aguilar Velasco, Sara Ceccarelli, Marysol Trujano, Uri García, Marilyn Mendoza, Patricia Ornelas, Mario García, Oscar Franke, Sabina Zaldívar and David Ortíz for collecting some of the specimens included in this study; Donald L.J. Quicke for his helpful comments to this work; Andrea Jiménez for her help in the laboratory; and Cristina Mayorga, Enrique Mariño, Eduardo Nuple and Gabriela Aguilar for mounting and labelling the specimens. This work was in part funded by a grant given by CONACyT (Red Temática del Código de Barras de la Vida) to AZR, and by a postdoctoral fellowship given by UNAM (DGAPA) to CPL.

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Appendix A. Supplementary material

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2015.01. 001.

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Please cite this article in press as: Pedraza-Lara, C., et al. Montane and coastal species diversification in the economically important Mexican grasshopper

Q1 genus Sphenarium (Orthoptera: Pyrgomorphidae). Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.01.001