a new species of horned lizard (genus … · a new species of horned lizard (genus phrynosoma) from...

Herpetologica, 70(2), 2014, 241–257! 2014 by The Herpetologists’ League, Inc.

A NEW SPECIES OF HORNED LIZARD (GENUS PHRYNOSOMA) FROMGUERRERO, MEXICO, WITH AN UPDATED MULTILOCUS

PHYLOGENY

ADRIAN NIETO-MONTES DE OCA1, DIEGO ARENAS-MORENO

2, ELIZABETH BELTRAN-SANCHEZ3, AND

ADAM D. LEACHE4,5

1 Museo de Zoologıa and Departamento de Biologıa Evolutiva, Facultad de Ciencias, Universidad Nacional Autonoma deMexico, Ciudad Universitaria, Mexico 04510, Distrito Federal, Mexico

2 Departmento de Zoologıa, Instituto de Biologıa, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria,Mexico 04510, Distrito Federal, Mexico

3 Instituto de Investigacion Cientıfica Area Ciencias Naturales, Universidad Autonoma de Guerrero, Av. LazaroCardenas s/n, Cd. Universitaria, Chilpancingo 39000, Guerrero, Mexico

4 Department of Biology and Burke Museum of Natural History and Culture, University of Washington, Seattle, WA98195-1800, USA

ABSTRACT: We describe a new species of Phrynosoma from central northeastern Guerrero, Mexico;perform a phylogenetic analysis of mitochondrial and nuclear sequence data to estimate its phylogeneticrelationships; and investigate the monophyly of Phrynosoma asio, P. braconnieri, and P. taurus. The newspecies can be distinguished from all of its congeners by the possession of a unique combination ofmorphological characteristics. The molecular genetic data include three fragments of the mitochondrialgenome and six nuclear genes (2419 and 3909 base pairs in total, respectively) for 31 samples belonging to the16 previously recognized species of Phrynosoma and the new species. The new species is strongly supportedin maximum likelihood analyses of both the concatenated mitochondrial and nuclear data as a monophyletic,distinct evolutionary lineage sister to, and moderately divergent from, P. taurus, and highly divergent from allof the other species of Phrynosoma. A Bayesian species tree analysis also strongly supports the monophyly ofthe Brevicauda clade, and a sister relationship between P. taurus and the new species.

RESUMEN: Se describe una especie nueva de Phrynosoma del centro-noreste de Guerrero, Mexico; serealiza un analisis filogenetico de secuencias de ADN mitocondrial y nuclear para estimar sus relacionesfilogeneticas, y se investiga la monofilia de P. asio, P. braconnieri, y P. taurus. La nueva especie puededistinguirse de todos sus congeneres por la posesion de una combinacion unica de caracteres morfologicos.Los datos geneticos moleculares incluyen secuencias de tres fragmentos del genoma mitocondrial y seis genesnucleares (2419 y 3909 pares de bases en total, respectivamente) para 31 muestras de las 16 especies dePhrynosoma previamente reconocidas y la nueva especie. La nueva especie es fuertemente apoyada enanalisis de maxima verosimilitud de los datos concatenados (tanto mitocondriales como nucleares) como unlinaje evolutivo distinto y monofiletico, hermano y moderadamente divergente de P. taurus y altamentedivergente de todas las otras especies de Phrynosoma. Un analisis Bayesiano de arbol de las especies tambienapoya fuertemente la monofilia del clado Brevicauda y la relacion de especies hermanas entre P. taurus y lanueva especie.

Key words: Phrynosoma brevicauda clade; Phrynosomatidae; Species tree; Systematics; Taxonomy

HORNED lizards (genus Phrynosoma) areamong the most distinctive of North andMiddle American reptiles because of theirunusual morphology and life history (Leacheand McGuire, 2006). The genus is composedof 16 currently recognized species distributedfrom Canada to Guatemala: Phrynosoma asio,P. blainvillii, P. braconnieri, P. cerroense, P.cornutum, P. coronatum, P. ditmarsi, P.douglasii, P. goodei, P. hernandesi, P. mcallii,P. modestum, P. orbiculare, P. platyrhinos, P.

solare, and P. taurus (Leache and McGuire,2006; Leache et al., 2009). With the exceptionof P. ditmarsi described in 1906, all of thesespecies were described in the 19th century or,in the case of P. orbiculare (Linnaeus, 1758),even earlier. However, only 12 of them wererecognized before 1997. Since then, thenumber of species within Phrynosoma hasincreased to 16, mainly because of reevalua-tions of species limits within polytypic groups,and the elevation of subspecies (Zamudio etal., 1997; Montanucci, 2004; Leache andMcGuire, 2006; Mulcahy et al., 2006; butsee Bryson et al., 2012).5 CORRESPONDENCE: e-mail, [email protected]

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Of the 16 species of Phrynosoma, only fouroccur south of the Mexican TransvolcanicBelt: Phrynosoma asio, P. braconnieri, P.orbiculare, and P. taurus (Leache andMcGuire, 2006). Phrynosoma braconnieriand P. taurus have consistently been recov-ered as sister species in a number ofphylogenetic studies (e.g., Montanucci, 1987;Zamudio et al., 1997; Reeder and Montanucci,2001; Hodges and Zamudio, 2004; Leache andMcGuire, 2006). These species are viviparous(Montanucci, 1979; Zamudio and Parra-Olea,2000), have extremely short tails (Smith andTaylor, 1950; Reeve, 1952; Leache andMcGuire, 2006), and have been reported tolack blood-squirting capabilities (Sherbrookeet al., 2004; Leache and McGuire, 2006).However, it was recently reported that P.taurus does squirt blood (Garcıa-Vazquez andCanseco-Marquez, 2006). In their phyloge-netic study, Leache and McGuire (2006)provided a phylogenetic taxonomy for thegenus and proposed the name Brevicauda forthe clade composed of P. braconnieri and P.taurus.

Phrynosoma braconnieri and P. taurus haverelatively small distributions in southernMexico (Leache and McGuire, 2006). Phry-nosoma braconnieri is known from Puebla andOaxaca (Montanucci, 1979; Zamudio andParra-Olea, 2000), whereas P. taurus is knownfrom Guerrero, Morelos, Puebla, northernOaxaca, and possibly northwestern Veracruz(Montanucci, 1979; Zamudio and Parra-Olea,2000; Garcıa-Vazquez and Canseco-Marquez,2006; Leache and McGuire, 2006; Canseco-Marquez and Gutierrez-Mayen, 2010). How-ever, Montanucci (1979) reported consider-able ecological diversity in both of thesespecies. Phrynosoma braconnieri has beencollected in pine–oak forests at approximately2438 m in elevation and also in xeric, thorn–scrub vegetation from 1950 to 2255 m inelevation (total range, 1600–2500 m in eleva-tion; Zamudio and Parra-Olea, 2000), whereasP. taurus has been collected in oak–chaparralin Guerrero and also in desert areas in Puebla(locality elevation ranges from 1300 to 1900m; Zamudio and Parra-Olea, 2000). Further-more, Montanucci (1979) reported color andmorphological differences between specimensfrom pine–oak forests and xeric, thorn–scrub

vegetation in P. braconnieri and suggestedthat recognition of two geographic races ofthis species might be warranted should thesedifferences be consistent when more materialis available. Montanucci (1979) also reportedsome color difference between specimensfrom oak–chaparral and desert areas in P.taurus, although too few specimens wereavailable to determine whether this differencewas constant. Although several phylogenies ofPhrynosoma have been published (see above),the monophyly of P. braconnieri and P.taurus, as well as that of P. asio, has not beentested.

In May 2011, herpetologists from theUniversidad Autonoma de Guerrero and theUniversidad Nacional Autonoma de Mexicocollected one specimen of Phrynosoma in thevicinity of Tenexatlajco, in the Sierra Madredel Sur of Guerrero, that appeared torepresent an undescribed species of thisgenus. Thirteen additional specimens collect-ed subsequently in this, and other, localities inthe Sierra Madre del Sur of Guerreroconfirmed this notion. The undescribed spe-cies is small, has an extremely short tail, andmost closely resembles P. braconnieri. Herein,we describe the new species, perform aphylogenetic analysis of Phrynosoma basedon mitochondrial DNA (mtDNA) and nucleargene sequence data to investigate phylogenet-ic relationships and provide some informationabout the ecology and reproductive biology ofthe new species. Also, we investigate for thefirst time the monophyly of P. asio, P.braconnieri, and P. taurus.

MATERIALS AND METHODS

Morphological DataWe examined 14 specimens of the unde-

scribed species of Phrynosoma from severaldifferent localities in the state of Guerrero: 11from the type locality in the municipality ofChilapa de Alvarez and three from twolocalities in the municipality of Olinala. Tissuesamples were taken from eight specimenscollected on 18 June 2012 at the type locality(field numbers ADL 4173–4176, 4178–4181),and the tissues were used for the molecularphylogenetic analyses (see below). Three ofthe specimens from the type locality (ADL

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4175, 4177, 4180) were released upon theirexamination. The remaining eight specimensfrom this locality and the three specimensfrom the municipality of Olinala were fixed in10% buffered formalin, stored in 70% ethanol,and deposited at the Museo de ZoologıaAlfonso L. Herrera of the Facultad deCiencias, Universidad Nacional Autonoma deMexico (MZFC).

The diagnosis is based on the examinedspecimens and the relevant literature (Gentry,1885; Smith and Taylor, 1950; Reeve, 1952;Montanucci, 1987). Because the new speciesexhibited a combination of characters thatreadily distinguishes it from all other speciesof Phrynosoma except those in the Brevicaudaclade (i.e., P. braconnieri and P. taurus), wecompared it directly only to the latter species.A list of the specimens examined is provided

in the Appendix. Institutional abbreviationsfor museums and collections follow Sabaj-Perez (2010), except for the Burke Museum ofthe University of Washington (UWBM) andthe herpetological collection at the Universi-dad de la Sierra Juarez (ICAH).

Nomenclature of scales follows Smith(1946), Reeve (1952), and Montanucci (1987,2004). Scale counts were performed with theaid of a dissecting microscope. Measurementsfollowed Montanucci (2004) and were takenwith calipers (60.1 mm). Color descriptionsand codes (in parentheses) follow Smithe(1975). In the case of characters examinedon both the left and right sides of eachspecimen, the corresponding conditions arereported in this order, separated by a slash.The superciliary, occipital, and temporalspines were measured on the right side ofthe specimens.

DNA SequencingWe collected new DNA sequence data for

all species of Phrynosoma, and a total of 31samples are included in the phylogeneticanalyses. The majority of these specimenswere used in previous studies of Phrynosoma(Reeder and Montanucci, 2001; Hodges andZamudio, 2004; Leache and McGuire, 2006;Leache et al., 2009), and we augmented thesewith new specimens for P. asio, P. braconnieri,P. douglasii, P. orbiculare, and P. taurus(Table 1). Multiple individuals of P. asio (n¼ 3), P. braconnieri (n¼ 4), P. taurus (n¼ 3),and the new species (n¼ 8) were included totest the monophyly of these taxa.

Six nuclear exons were polymerase chainreaction (PCR) amplified and sequenced,including brain-derived neurotrophic factor(BDNF, 529 base pairs [bp]), exophilin 5(EXPH5, 609 bp), NK-tumor recognitionprotein (NKTR, 639 bp), RNA fingerprintprotein 35 (R35, 704 bp), recombinationactivating gene-1 (RAG1, 1054 bp), andsuppressor of cytokine signaling 5 (SOCS5,374 bp). We also sequenced three fragmentsof the mitochondrial genome (Table 2),including the 12S rRNA gene (12S, 769 bp),NADH1 (ND1, 969 bp) and NADH4 (ND4,681 bp) protein coding genes, and severaltRNA genes (histidine, serine, and leucine).Standard methods of DNA extraction and

TABLE 1.—Samples of Phrynosoma included in thephylogenetic analyses. UWBM ¼ University of Washing-ton Burke Museum. ANMO ¼ Adrian Nieto-Montes deOca, CDR ¼ Christopher D. Rivera, JAC ¼ Jonathan A.Campbell, RRM ¼ Richard R. Montanucci.

Species Samples Vouchers

P. asio 3 MVZ 161505, CDR 279,UWBM:HERP:7280

P. blainvillii 1 MVZ 150066P. braconnieri 4 UWBM:HERP:7282,

UWBM:HERP:7283, ANMO2167, ANMO 2161

P. cerroense 1 MVZ 161188P. cornutum 1 MVZ 238582P. coronatum 1 MVZ 137778P. ditmarsi 1 RRM 2459P. douglasii 1 UWBM:HERP:7227P. goodei 1 CAS 229922P. hernandesi 1 MVZ 245875P. mcallii 1 CAS 229923P. modestum 1 MVZ 238583P. orbiculare 1 UWBM:HERP:7284P. platyrhinos 1 MVZ 161495P. solare 1 MVZ 241510P. taurus 3 UWBM:HERP:7295,

UWBM:HERP:7296, JAC 22525P. sp. nov. 8 UWBM:HERP:7286, (MZFC

28101), UWBM:HERP:7287,(MZFC 27898),UWBM:HERP:7288,UWBM:HERP:7289, (MZFC27899), UWBM:HERP:7291,(MZFC 27900),UWBM:HERP:7292, (MZFC27896), UWBM:HERP:7293,UWBM:HERP:7294, (MZFC27897)

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PCR amplification were used (see Leache andMcGuire, 2006; Portik et al., 2012), andpurified PCR products were sequenced usingan ABI 3730 automated sequencer. All newsequences are deposited in GenBank (acces-sions KJ123951–KJ124157) and alignmentsare provided in the Dryad Digital Repository,http://datadryad.org (DOI:10.5061/dryad.n7h53).

Sequences were edited using Sequencherversion 4.9, and multiple sequence alignmentswere generated using Muscle version 3.8.31(Edgar, 2004). Open reading frames for theprotein-coding genes were identified usingMacClade version 4.08 (Maddison and Mad-dison, 2005). The 12S alignment was guidedusing previous alignments generated for 12Sin Phrynosoma guided by secondary structuremodels (Reeder and Montanucci, 2001). Forthe nuclear genes, heterozygous sites werecoded using ambiguity codes.

Phylogenetic AnalysesPhylogenetic relationships were inferred

using maximum likelihood (ML) and Bayesianspecies tree estimation. ML gene trees wereestimated for each mtDNA and nuclear geneseparately, the combined mtDNA data, andthe combined nuclear data. We did notconduct analyses of combined mtDNA þnuclear data because previous phylogeneticstudies of Phrynosoma have identified strongconflict between mtDNA and nuclear genesresulting from hybridization and mtDNAintrogression (Leache and McGuire, 2006).ML analyses were conducted with RAxML

version 7.0.4 (Stamatakis, 2006) with theGTRþGAMMA model of nucleotide substitu-tion. All ML analyses executed 100 rapidbootstrap replicates followed by a thoroughML search under the specified model.

We used *BEAST version 1.7.4 (Heled andDrummond, 2010) to obtain a species tree forPhrynosoma that accommodates incompletelineage sorting of the nuclear genes. Thespecies tree analysis contained 31 samplesbelonging to 17 species. Species tree methodsproduce rooted phylogenetic trees; however,we conducted species tree analyses both with,and without, an outgroup to test the sensitivityof root inference at the base of the Phryno-soma phylogeny. We used the closely relatedsand lizard species Holbrookia maculata as anoutgroup for this purpose (Wiens et al., 2013).The site models, clock models, and partitiontrees were unlinked, and we applied the HKYmodel of nucleotide substitution to each gene.We assumed a Yule tree prior and uncorre-lated lognormal model for branch variation.Two replicate runs were conducted withrandom starting seeds and chain lengths of100 million generations, with parameterssampled every 10,000 steps. Long chains werenecessary for achieving high effective samplesizes (ESSs) for parameters, and ESS values #200 were used as a proxy for convergence ofparameters. Species trees were summarizedafter discarding the first 25% of trees as burn-in. The post burn-in samples were analyzed inTreeAnnotator to produce a maximum cladecredibility tree.

TABLE 2.—Variation in the molecular sequences collected for Phrynosoma and source of primer sequences. Gene treemonophyly values are bootstrap percentages obtained from 100 replicates using maximum likelihood.

Gene Source Length (bp) Variable sitesP. taurus

monophylyP. braconnieri

monophylyP. sp. nov.monophyly

12s Wiens et al., 1999 769 218 0 99 100ND4 Arevalo et al., 1994 681 284 93 100 94ND1 Leache and Reeder, 2002 969 352 99 100 100mtDNA combined 2419 854 89 100 100BDNF Leache and McGuire, 2006 529 29 0 96 0EXPH5 Portik et al., 2012 609 49 0 0 100NKTR Townsend et al., 2011 639 48 0 99 0R35 Leache, 2009 704 69 97 99 92RAG1 Leache and McGuire, 2006 1054 111 69 77 99SOCS5 Alfoldi et al., 2011 374 14 0 91 0Nuclear combined 3909 320 0 100 99


RESULTS

The combined nuclear data matrix contains3909 base pairs and 320 variable sites,whereas the mtDNA data include 2419 basepairs and 854 variable sites (Table 2). Thegene trees provide strong support (bootstrap# 70%) for the monophyly of P. braconnierimore often than they do for P. taurus or thenew species (Table 2). mtDNA genes thatsupport each of these three species asmonophyletic include ND1 and ND4; the12S gene does not support the monophyly ofP. taurus. The nuclear genes contain lessvariation and, as a result, they support themonophyly of these species less frequentlythan mtDNA. For example, only two of thenuclear genes, R35 and RAG-1, support themonophyly of P. taurus, and these are the onlynuclear genes that support the monophyly ofall three species.

The ML analysis of the concatenatedmtDNA data (Fig. 1A) is highly congruentwith previous mtDNA-based estimates ofPhrynosoma relationships. The new Phryno-soma samples from Guerrero form a clade thatis sister to P. taurus (bootstrap¼ 100%) withinthe Brevicauda clade. The concatenatednuclear data also support the monophyly ofthe new samples from Guerrero and placethem sister to P. taurus as well (Fig. 1B). Incontrast, other aspects of the nuclear andmtDNA trees indicate opposing relationshipsthat receive strong support in the separateanalyses. For example, the mtDNA tree placesP. modestum as sister to the remaining short-horned species (P. ditmarsi, P. douglasii, P.hernandesi, and P. orbiculare), whereas thenuclear data places P. modestum as sister to aclade containing P. goodei and P. platyrhinos(Fig. 1). In addition, the mtDNA and nucleardata disagree on the placement of the cladecontaining the short-tailed species (P. bracon-nieri, P. taurus, and P. sp. nov.); this cladediverges early at the base of the mtDNA genetree but has a more recent divergence in thenuclear tree (Fig. 1).

The species tree analysis of the six nuclearloci (Fig. 2) provides strong support (posteriorprobability #0.95) for relationships at shallowlevels in the tree, but the initial divergences inthe group have low posterior probabilitysupport ($0.5). Phrynosoma cornutum is

FIG. 1.—Phylogenetic relationships of Phrynosomabased on a maximum likelihood analysis of (A) concate-nated mtDNA data and (B) concatenated nuclear data.Bootstrap values #50% are indicated on nodes. The treesare rooted with Holbrookia maculata. Phylogenetictaxonomy is noted where applicable.


sister to the remaining species; however, thesupport for this relationship is low (posteriorprobability¼0.5). Including H. maculata as anoutgroup does not change this placement of P.cornutum at the base of the tree. Theremaining species are arranged as follows: P.asio is placed as sister to P. solare and the P.coronatum complex; P. goodei and P. platy-rhinos form a clade sister to a P. mcallii and P.modestum clade that is sister to viviparous andshort-horned species in the Tapaja clade.

The relative divergence times between thenew species and P. taurus are comparable tothose between the pairings of P. goodei and P.platyrhinos, P. hernandesi and P. douglasii,and P. cerroense and P. blainvillii (Fig. 2).

Phrynosoma sherbrookei sp. nov.(Figs. 3–5)

Holotype.—MZFC 28101 (ADL 4173), amale from Tenexatlajco, municipality of Chi-lapa de Alvarez, Guerrero, Mexico,

17.554378N, 99.269738W (datum ¼ WGS84for all localities), 1997 m in elevation,collected on 18 June 2012 by D. Arenas-Moreno, M.A. Dıas-Ojendis, M. Garcıa-Par-eja, P.P. Gonzalez-Alvarado, J. Grummer, A.Hernandez-Rıos, R. Lara-Resendiz, A.D.Leache, A. Nieto-Montes de Oca, and W.C.Sherbrooke.

Paratypes.—Ten specimens, all from Guer-rero, Mexico: three males (MZFC 27894,27896, 27897) and four females (MZFC27893, 27898–27900) from the same localityas the holotype (MZFC 27893–27894 collect-ed on 2011 or 2012 by unrecorded collectors;MZFC 27896–27900 collected on 18 June2012 by the same collectors as the holotype),and three specimens from the municipality ofOlinala: one female (MZFC 27895) from nearLa Encinera (Los Terrenos), 17.852027788N,98.751258W, 1794 m elevation, collected on19 July 2012 by W. Gramajo, and twospecimens, one female and one juvenile ofundetermined sex (MZFC 28303 and 28304,respectively) from 1 km NW Xixila,17.945648N, 98.859968W, 1677 m elevation,collected on 1 October 2011 by E. Rosendo,V.H. Jimenez-Arcos, and S. Santa Cruz-Padilla.

Diagnosis.—Phrynosoma sherbrookei maybe distinguished from P. blainvillii, P. cer-roense, P. coronatum, P. douglasii, P. goodei,P. hernandesi, P. mcallii, P. modestum, P.orbiculare, P. platyrhinos, and P. solare byhaving keeled ventral scales (vs. ventral scalessmooth in the other species).

Among the species with keeled ventralscales, Phrynosoma sherbrookei differs fromP. asio and P. cornutum by having a smalleradult body size (snout–vent length [SVL] ,63.0 mm vs. SVL . 140.0 mm in the otherspecies; Smith and Taylor, 1950), a shorter tail(tail length , 23% of SVL vs. tail length #50% of SVL in the other species; Smith andTaylor, 1950), and one lateral abdominal rowof fringe scales (vs. two lateral abdominal rowsof fringe scales in the other species; Smith andTaylor, 1950; Montanucci, 1987). Phrynosomasherbrookei can be distinguished from P.ditmarsi by having well-developed occipitaland temporal spines (vs. occipital and tempo-ral spines absent, replaced by low, roundedprotuberances in P. ditmarsi; Smith and

FIG. 2.—Species tree analysis of Phrynosoma showingthe phylogenetic position of Phrynosoma sp. nov. in theBrevicauda clade of viviparous and short-tailed speciesendemic to Mexico. Nodes supported by posteriorprobability values # 0.50 are indicated. The outgroup,Holbrookia maculata, is not shown. Phylogenetic taxono-my is noted where applicable.


Taylor, 1950; Reeve, 1952) and one or twoshort rows of sublabials (vs. five or six rows ofsublabials in P. ditmarsi; Reeve, 1952).

Phrynosoma sherbrookei is most closelyrelated to the two species in the Brevicaudaclade (P. braconnieri and P. taurus). The newspecies can be distinguished from P. bracon-nieri by having the outer temporal part of theskull prolonged posteriodorsally into twotemporal spines (vs. the outer temporal partof the skull prolonged posteriodorsally intothree temporal spines in P. braconnieri),posterior chinshields larger than the postla-bials (vs. posterior chinshields smaller than atleast the posterior-most postlabial in P.braconnieri), and more numerous sublabials(10–18, X ¼ 14.7, n ¼ 9) usually arranged intwo rows (vs. 1–7, X¼ 4.8, n¼ 9, sublabials inone row in P. braconnieri).

Phrynosoma sherbrookei differs from P.taurus by having a smaller adult body size(,54 and ,63 mm SVL in males and females,respectively, vs. 55.5–77.0 and 65.0–90.0 mmSVL in P. taurus males and females, respec-tively; Zamudio and Parra-Olea, 2000); byhaving outer temporal spines only slightlylonger than the occipital spines (length ratio1.1–1.6, X ¼ 1.4, n ¼ 12) and not or barelyexceeding the occipital spines in posteriorextension (vs. outer temporal spines muchlonger than the occipital spines [length ratio2.1–3.0, X¼ 2.5, n¼ 6] and far exceeding theoccipital spines in posterior extension in P.taurus), and in lacking enlarged postcloacalscales (vs. the presence of enlarged postcloa-cal scales in P. taurus).

Description of holotype.—Adult male, SVL53.3 mm; head length 13.1 mm (24.6% ofSVL), head width 13.5 mm; shank length 13.7mm (25.6% of SVL); tail length 8.9 mm(67.9% of head length, 16.7% of SVL; Figs. 4and 5).

Dorsal surface of head with polygonal,juxtaposed scales arranged in no discerniblepattern; their surface texture granular. Post-rostral scales small, flat, usually keeled.Frontal and supraocular scales small to mid-sized except for two somewhat enlargedazygous scales; usually with one central,slightly elevated granule and occasionallyone or more keels converging on granule.Parietal scales mid-sized, conical, with nu-

merous, conspicuous granules and keels.Abruptly differentiated transverse series ofpreoccipital tubercles absent. Rostral small,slightly more than twice as wide as high (width1.3 mm, height 0.5 mm). External nares

FIG. 3.—Phrynosoma sherbrookei sp. nov. Head scalesof holotype (MZFC 28101) in dorsal (top), lateral(middle), and ventral view (bottom). Horizontal barsrepresent 5 mm.


situated within canthal lines, dorsally pierced.Eleven scales along midline between rostraland interparietal. Interparietal to rostraldistance 9.1 mm (69.2% of head length);interparietal length 2.0 mm; parietal shelflength 4.3 mm (32.7% of head length). Twocomparatively small occipital spines (maxi-mum width at base 2.12 mm, length 3.1 mm,or 23.6% of head length), projecting poster-odorsally at angle of about 608 to longitudinalaxis, widely separated from each other (dis-tance between bases of spines 2.8 mm, or21.6% of head length) and from temporalspines (distance between bases of occipitaland inner temporal spines 2.0 mm/1.8 mm).Interoccipital and parietotemporal spines ab-sent.

Lateral surface of head with polygonal,juxtaposed scales arranged in no discerniblepattern except on temporal region; theirsurface texture granular. Canthals and super-ciliaries intermediate in size, keeled or striate;superciliary ridge terminating in a well-developed superciliary spine (maximum width

at base 2.16 mm, length 2.2 mm, or 16.8% ofhead length) projecting posterodorsally atangle of about 608 to longitudinal axis. Lorealssmall, usually keeled. Three scale rowsbetween orbit and supralabials; rows immedi-ately below orbit and above supralabialscomposed of small, keeled scales; middlerow composed of increasingly larger, keeledscales extending posteriorly from loreal re-gion, merging with temporal scales. Temporalscales large, each with one slightly elevatedgranule near its posterior border and one ormore keels converging on granule; scalesarranged in oblique, parallel rows, graduallybecoming larger posteriorly; scales in posteri-or-most two rows gradually becoming largerdorsally; those at their dorsal end abruptlydifferentiated into two adjacent, moderatelylarge temporal spines; outer temporal rowcomposed of three large scales followeddorsally by outer and inner temporal spines.Inner temporal spine smaller than outer one(maximum width at base and height ¼ 1.84and 2.36 mm, and 3.0 and 4.1 mm, respec-

FIG. 4.—Holotype of Phrynosoma sherbrookei sp. nov. (MZFC 28101). Photograph was taken at the type locality inGuerrero, Mexico.


tively; length of outer spine 31.2% of headlength) or about as large as, and distinctlylarger than, occipital spines, respectively;temporal spines projecting both posterolater-ally and posterodorsally at angle of about 458to longitudinal axis; projecting slightly fartherposteriorly (about 1.2 mm) than occipitalspines. Supralabials small, keeled, verticallyoriented, not flared; some slightly scallopedalong buccal margin of row. Tympanumexposed; skin fold above tympanum absent.

Mental scale small, shallow. Scales ininfralabial-postlabial row moderately large,keeled; gradually becoming convex, triangularposteriorly in row from about its middle, withlaterally projecting keeled edge; infralabial-postlabial row separated from chinshield rowby 10/14 small sublabials arranged in tworows. Chinshields 8þ (row partially mis-

shaped)/10, left and right rows abutting eachother below mental-infralabials row; scales ineach row gradually becoming larger, convex,triangular, keeled posteriorly in series, withkeeled edge directed slightly lateroventrally,forming a serrate series barely visible fromdorsal view. Posterior chinshields markedlylarger than postlabials. Approximately 41longitudinal gular scale rows between lastchinshields. Pregular fold weakly developed,composed of a single transverse row ofapproximately five moderately enlarged, con-ical, keeled, mucronate scales on each side ofventral surface of neck. Gular fold welldeveloped. One row of slightly enlarged gularson each side of throat extending posteriorly tolevel of posterior end of chinshield row. Tworows of enlarged, keeled, mucronate scales oneach side of neck: one row extending poste-

FIG. 5.—Holotype of Phrynosoma sherbrookei sp. nov. (MZFC 28101) in dorsal (left) and ventral (right) view.


riorly from ear to gular fold, then curvingdorsally bordering dorsal end of fold, and onerow of increasingly larger scales extendingposterodorsally from posterolateral corner ofthroat, at level of posterior end of chinshieldrow, to first scale row on neck at its junctionwith margin of gular fold.

Dorsal body scales heterogeneous, com-posed of enlarged scales interspersed amongirregular, juxtaposed, smaller scales arrangedin no discernible pattern, often keeled onmiddorsum; enlarged scales slightly oval, theirposterior margin slightly rounded, arrangedin: (1) two short paravertebral rows, eachcomposed of three juxtaposed scales increas-ingly larger, keeled, and mucronate posterior-ly between levels of anterior and posteriorinsertions of forelimbs; (2) one enlarged scaleon each side of paravertebral rows at level ofanterior insertion of forelimb, with severalstrong converging keels and strong mucroneprojecting dorsally from center; (3) two poorlydefined paravertebral rows of slightly en-larged, keeled, not or slightly mucronatescales extending between levels of axilla andcloaca; scales of each row separated fromnearest scales in row by distance usually equalto, or shorter than, their own diameter; and,(4) six poorly defined longitudinal scale rowsextending between levels of axilla and cloaca(four medial-most rows) or between levels ofaxilla and groin (two lateral-most rows); scalesin each row strongly keeled; one median keeland two short lateral keels converging nearposterior margin; strongly mucronate; mu-crone originating at posterior end of mediankeel, near posterior edge of scale, projectingdorsally; medial-most rows straight, composedof largest scales on dorsum; remaining rowsincreasingly curved, paralleling body contour,and composed of gradually smaller scaleslaterally; scales of each row separated fromnearest scales in row by distance usually equalto, or greater than, their own diameter.

Dorsal scales of arm and forearm imbricate,keeled, mucronate; those of thigh and shankheterogeneous, composed of enlarged scalesinterspersed among much smaller, irregular,juxtaposed, usually keeled scales arranged inno discernible pattern; enlarged scales round-ed, their posterior margin slightly rounded,strongly keeled; one median keel and two

short lateral keels converging near posteriormargin; strongly mucronate; mucrone origi-nating at posterior end of median keel, nearposterior edge of scale, projecting dorsally.

Ventral body scales keeled, juxtaposed toslightly imbricate, not mucronate or withexceedingly small mucrone directed posteri-orly. One lateral abdominal fringe scale rowextending from level between anterior andposterior insertions of arm to nearly groin oneach side; first two scales in row separatedfrom rest of row by short gap at level of axilla.Approximately 40 transverse scale rows alongmidline between levels of axilla and groin;about 71 longitudinal scale rows betweenlateral abdominal fringe scale rows at level ofmidbody. Approximately 27/26 scales in lateralabdominal fringe scale row. Lateral abdominalfringe scale length at level of midbody »1.6mm. Enlarged postcloacal scales absent. Onesingle row of 12/12 femoral pores; poressurrounded anteriorly by single scale; bor-dered posteriorly by two to three small scales.Approximately 16 precloacal scales betweenfemoral pore rows. Ventral scales of arms andlegs imbricate, keeled, slightly mucronate.Subdigital lamellae under fourth toe 14/14.Tail short, subcylindrical, slightly wider thandeep.

Coloration of holotype in life.—Dorsalsurface of head, except for temporal areasand temporal spines, smoke gray (45); dorsaland lateral surfaces of temporal areas, tempo-ral spines, and remaining lateral surfaces ofhead pale horn (92); frontal, supraocular, andespecially parietal scales and superciliary andoccipital spines darkened by black granulesand keels; parietal and temporal scales andtemporal spines moderately stippled withPeach Red (94). Ventral surface of headwhite, with irregular, scattered, indistinct grayspots (Fig. 4).

Dorsal background color of body glaucus(80). Sulfur yellow (157) middorsal stripeextending from posterior end of head to levelof axilla (i.e., on paravertebral rows ofenlarged, juxtaposed scales between levels ofanterior and posterior insertions of arm),bordered on each side by large, bright jetblack (89) blotch extending laterally to lateralabdominal scale row, between posterior endof head and level of axilla along its medial


margin, and between posterior end of headand level of about one-fifth distance fromaxilla to groin along its lateral margin. Sixindistinct, middle neutral gray (84) crossbandson rest of body and tail: three on body (onebetween anterior jet black blotches and levelof midbody and two between levels of mid-body and groin), one above level of cloaca,and two on tail. Crossbands on body wide,with ill-defined, concave anterior and posteri-or margins, broadly connected to each otheralong midline; that above level of cloaca wide,broadly U-shaped; those on tail narrow.Irregular, poorly defined, medium gray spotsand bars on arms, legs, and digits. Ventralsurface white; chest and venter with irregular,scattered, indistinct gray spots and bars. Aphotograph of the preserved specimen isprovided in Fig. 5.

Variation.—Except as noted, description ofvariation in scalation and morphometric ratiosis based on all paratypes, except the juvenilespecimen (MZFC 28304) and the threereleased specimens (n ¼ 12; nine females,SVL 41.5–62.4 mm, and three males, SVL46.7–53.1 mm). Morphometric ratios aregiven in Table 3. Variation in color pattern(Fig. 6) is based on the eight specimenscollected at the type locality, in addition to theholotype.

Scales between the rostral and interpari-etal scales 11–13 (X ¼ 12.4). Superciliaryand occipital spines approximately equal insize in all of the specimens; both spinesfrom slightly smaller to slightly larger thanthe inner temporal spine in all of thespecimens except about twice as large aslatter spine in one specimen (MZFC 27893);from about two-thirds to nearly as large asthe outer temporal spine. Superciliary spinesprojecting posterodorsally at angle of about608 to longitudinal axis in six of thespecimens; at angle of about 458 in five.Occipital and temporal spines projectingposterodorsally at angle of about 308–408,and about 408 to longitudinal axis, respec-tively, in all of the paratypes included (n ¼9). Outer temporal spines slightly exceedingthe occipital spines in posterior extension in10 specimens; occipital spines slightly ex-ceeding the outer temporal spines in poste-rior extension in two. Chinshields (right

side) 10–11 (X ¼ 10.7). Sublabials (rightside) 10–18 (X ¼ 14.7, n ¼ 9). Longitudinalrows of gular scales between last chinshields37–43 (X ¼ 41.2, n ¼ 9). Transverse rows ofventral scales along midline between levelsof axilla and groin 35–44 (X ¼ 39.1); ventralscales between lateral fringe scale rows atlevel of midbody 66–78 (X ¼ 71.3). Scales inlateral abdominal fringe scale row (rightside) 25–32 (X ¼ 28.5). Femoral pores (rightside) 11–15 (X ¼ 12.0). Precloacal scalesbetween femoral pore rows 11–18 (X ¼15.3). Subdigital lamellae under fourth toe13–16 (X ¼ 14.8).

In general, all specimens showed a dorsalcolor pattern similar to that of the holotype.However, the dorsal background color was adifferent tone of pale horn (92), vinaceous (3),and glaucus (80) in each of four, two, and twospecimens, respectively (Fig. 6). All speci-mens had the parietal area and superciliaryand occipital spines darkened to some degreeby black granules and keels, and the temporalareas and temporal spines sparsely to denselyspeckled with red peach (94). All of thespecimens also exhibited a pale middorsalstripe (in one of several tones of pale horn[92]) on the paravertebral rows of enlarged,juxtaposed scales; a black blotch on each sideof the latter stripe, and several, sometimesindistinct, dark crossbands (in one of severaltones of glaucus [80]) on the body and tail. Inall of the specimens, the dark crossbands hadpoorly defined, concave anterior and posteriormargins, and were broadly connected witheach other along the midline. However, thedark crossbands on the body varied indistinctness (from moderately to clearly dis-tinct in seven specimens; barely distinguish-able in one), number (three, in six specimens;two indistinct ones, in two), and width (widein five specimens; narrow in three). In five ofthe specimens, the flanks were heavily speck-led with red peach (94). In all specimens therewas a wide, often indistinct, dark crossband onthe level of the cloaca, and two narrowcrossbands on the short tail. The specimensalso had irregular, dark marks and bars on thelimbs and digits, and a white throat, chest, andbelly with irregular, scattered, sometimesindistinct gray spots occasionally interconnect-ed forming short bars.


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Etymology.—The specific epithet is a pat-ronym for Wade C. Sherbrooke in recognitionof his many and significant contributions tothe knowledge of horned lizards.

Distribution and ecology.—Phrynosomasherbrookei is currently known from threelocalities at intermediate elevations in centralnortheastern Guerrero: the type locality, at1800–2040 m in elevation in the vicinity ofTenexatlajco, municipality of Chilapa deAlvarez, near central Guerrero, and twolocalities, near La Encinera and near Xixila,between 60 and 65 km to the northeast, at1677–1794 m in elevation in the municipalityof Olinala in northeastern Guerrero (Fig. 7). Itseems likely that the species has a widerdistribution in eastern Guerrero and adjacentPuebla.

The climate at the type-locality is temperateand subhumid with precipitation in thesummer, and the substratum is composed ofclays and sedimentary rocks (INEGI, 2009).The vegetation is oak forest with patches ofgrassland and scrub with Agave spp. and otherherbaceous plants (Rzedowski, 1978). Thearea is heavily impacted by farming and soilerosion (INEGI, 2009). The type series wascollected during the day in a small patch ofgrassland hillside with heavy erosion (Fig. 8).

Life history.—Field observations of oneindividual and fecal analysis of five speci-mens indicate that P. sherbrookei feeds onants, as is true for most Phrynosoma species(Sherbrooke, 2003). The mean body tem-perature of 15 specimens was 30.25 61.618C. The species is viviparous. Matingoccurs in spring and parturition in fall.

FIG. 6.—Color pattern variation in the paratype series of Phrynosoma sherbrookei sp. nov. from the type locality, bothlateral and dorsal views. Females (A–F) and males (G–H) are arranged in descending snout-to-vent length as follows: (A)62.4 mm (ADL 4174), (B) 61.9 mm (ADL 4178), (C) 61.2 mm (ADL 4176), (D) 52.3 mm (ADL 4180), (E) 46.8 mm(ADL 4175), (F) 41.5 mm (ADL 4177), (G) 53.1 mm (ADL 4179), and (H) 46.7 mm (ADL 4181).


Antipredator blood squirting has not beenobserved in P. sherbrookei in the field orinduced in the lab.

DISCUSSION

The distinctness of P. sherbrookei issupported by both morphological and mo-lecular data. The possession of keeled ventralscales separates P. sherbrookei from all butfive species in the genus, and the combina-tion of several other characters (e.g., smallbody size, short tail, distinctive horns) clearlydistinguishes it from those remaining fivespecies. Also, P. sherbrookei was stronglysupported in both the mitochondrial andnuclear trees as a distinct, monophyleticevolutionary lineage, moderately divergentfrom its sister species (P. taurus) and highlydivergent from all of the other species ofPhrynosoma (Figs. 1 and 2).

The phylogenetic placement of P. sherbroo-kei within the Brevicauda clade is notsurprising based on external morphology andgeographic distribution. The species in thisclade (P. braconnieri, P. taurus, and P.sherbrookei) have a short tail and twoparavertebral rows of enlarged, keeled, juxta-posed scales on the dorsal area of the neck(Montanucci, 1987). Also, the three speciesare viviparous and occur in southern Mexicosouth of the Mexican Transvolcanic Belt. Theknown geographic distribution of P. sherbroo-kei (central northeastern Guerrero) is geo-graphically intermediate between the recordsof P. taurus in central Guerrero (»26 km tothe west) and those in southern Puebla (Fig.7). Phrynosoma sherbrookei was stronglysupported as sister taxon to P. taurus ratherthan to P. braconnieri despite it being moresimilar to the latter species in body size,

FIG. 7.—Geographic distribution of Phrynosoma sherbrookei sp. nov. within Mexico (inset). Distributions of P.braconnieri and P. taurus are based on representative localities from Zamudio and Parra-Olea (2000) and Canseco-Marquez and Gutierrez-Mayen (2010) in addition of those from this study. Closed symbols represent type localities.


morphology of the occipital and temporalspines, and overall scalation.

The phylogenetic taxonomy established forPhrynosoma by Leache and McGuire (2006)is supported by ML analysis of the concate-nated nuclear genes, but it receives mixedsupport using mtDNA (Fig. 1). All analysesprovide strong support for the monophyly ofBrevicauda (containing P. braconnieri, P.sherbrookei, and P. taurus) and Tapaja(containing P. douglasii, P. ditmarsi, P.hernandesi, and P. orbiculare), and thisincludes the species tree analysis of thenuclear data. The Anota clade, which containsP. solare, P. mcallii, and the P. coronatumcomplex, is not supported by the mtDNA;instead, P. mcallii is placed sister to P. goodeiand P. platyrhinos (Fig. 1). This conflictingresult is expected because it is hypothesizedthat introgressive hybridization has replacedauthentic P. platyrhinos and P. goodeimtDNA haplotypes with P. mcallii copies(Leache and McGuire, 2006). This aberrantplacement of P. mcallii also disrupts themonophyly of the Doliosaurus clade, whichcontains P. goodei, P. modestum, and P.

platyrhinos (Fig. 1). The multilocus speciestree analysis conducted using *BEAST alsofails to support the monophyly of Anota andDoliosaurus, both of which are disrupted bythe placement of P. mcallii sister to P.modestum (Fig. 2). Whereas the relationshipsthat conflict with the phylogenetic taxonomyare strongly supported by the mtDNA data(i.e., 100% bootstrap support), the speciestree analysis only provides weak support (i.e.,$0.9 posterior probability) for such conflicts.Phylogenetic support is typically lower forcoalescent-based analyses versus concatena-tion (Leache, 2010), but the factor(s) causingP. mcallii to change phylogenetic positionsunder the coalescent model requires furtherinvestigation. Such studies will benefit fromphylogenetic analyses that combine largenumbers of loci with multiple samples foreach species.

The monophyly of P. braconnieri and P. asiowas strongly supported in the analyses of boththe concatenated mitochondrial and nucleardata, whereas the monophyly of P. taurus wassupported by the concatenated mitochondrialdata (bootstrap ¼ 91%; Fig. 1) and not

FIG. 8.—Habitat of Phrynosoma sherbrookei sp. nov. at the type locality, Guerrero, Mexico.


supported in the concatenated nuclear tree(Fig. 1), which supports P. taurus as para-phyletic with respect to P. sherbrookei.Nuclear genes contain less variation; thus,they support the monophyly of species lessfrequently than mitochondrial genes; they alsotake longer to sort to monophyly (Edwardsand Beerli, 2000). Further research is neededto investigate the potential existence ofmultiple lineages within P. asio, P. bracon-nieri, and P. taurus.

Newly described species are usually notconsidered in Red Lists of threatened speciesor similar lists. Phrynosoma braconnieri and P.taurus are listed by the Mexican governmentas ‘‘threatened’’ and ‘‘under special protec-tion,’’ respectively (SEMARNAT, 2010). Giv-en that the biology of P. sherbrookei seemssimilar to that of the former species, and itsgeographic distribution is much smaller, wesuggest that P. sherbrookei also deservesconservation attention and therefore recom-mend that it be added to the above-mentionedlist.

Acknowledgments.—We thank M.A. Dıas-Ojendis, M.Garcıa-Pareja, P.P. Gonzalez-Alvarado, J. Grummer, A.Hernandez-Rıos, R. Lara-Resendiz, and W.C. Sherbrookefor help in collecting specimens of the new species at thetype locality; E. Rosendo, V.H. Jimenez-Arcos, and S.Santa Cruz-Padilla for collecting two of the paratypes ofthe new species; A. Hernandez-Rıos and W.C. Sher-brooke for providing some specimens of Phyrnosoma forcomparative purposes; V. Aguirre-Hidalgo for the loan ofthe specimens at the ICAH; A. Hernandez Gomez and J.Grummer for taking the photographs of the holotype andsome of the paratypes, respectively; E. Perez-Ramos forcataloging type material at the MZFC; R. Santos-Bibianofor providing data on the type locality; T. Gill forcollecting molecular genetic data; C. Linkem, L. Grismer,and an anonymous reviewer for comments on themanuscript; and J.C. Arenas-Monroy for preparing Fig.7. Support for this project was provided by grants fromthe National Science Foundation (DBI-1144630) to ADLand from Conacyt (154093) to A. Nieto-Montes de Oca.

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Accepted: 3 January 2014Associate Editor: Christopher Austin

APPENDIX

Specimens Examined

ANMO is an abbreviation for a field number of an uncataloged

specimen at the MZFC.

Phrynosoma braconnieri.—PUEBLA: Tecali de Herrera (AN-

MO 2161); Tehuacan (MZFC 174); OAXACA: Ixtlan (ICAH 60,

111); Miahuatlan (ANMO 3278–3280); Santiago Yolomecatl

(ANMO 847, 2167).

Phrynosoma taurus.—GUERRERO: Zumpango del Rio (AN-

MO 3059); Municipality of Eduardo Neri, Zumpango de Neri,

Cerro del Tepetlayo (MZFC uncatalogued, 4 specimens);

PUEBLA: Zapotitlan de las Salinas (ANMO 3066).

DATE OF PUBLICATION

Herpetologica, Vol. 70, No. 2 was mailed on 23 May 2014.


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