Evolutionary relationships amongst polymorphic ...

Systematics and Biodiversity (2014), 12(1): 1–22

Research Article Evolutionary relationships amongst polymorphic direct-developing frogs in the Craugastor rhodopis Species Group (Anura: Craugastoridae)

2 JEFFREY W. STREICHER1, URI O. GARCIA-VAZQUEZ , PAULINO PONCE-CAMPOS3, 2 OSCAR FLORES-VILLELA , JONATHAN A. CAMPBELL1 & ERIC N. SMITH1

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Amphibian and Reptile Diversity Research Center, Department of Biology, The University of Texas at Arlington, Arlington TX 76019, USA 2 Museo de Zoologıa, Facultad de Ciencias, UNAM, A.P. 70-399, Mexico, D.F., 04510, Mexico 3 Bosque Tropical Investigacion para la Conservacion de la Naturaleza, Misi on San Antonio, Torre 4a-2. Colonia Plaza Guadalupe, Zapopan, Jalisco 45030, Mexico (Received 23 October 2013; accepted 7 January 2014) The Craugastor rhodopis Species Group includes two leaf-litter frog species (C. loki and C. rhodopis). These directdeveloping frogs inhabit tropical regions of Mexico and northern Central America. Characterizing diversity within the group has been difficult due to high levels of phenotypic polymorphism within and between species. Because of these polymorphisms, each taxon has junior synonyms. Using a fragment of mitochondrial DNA (mtDNA), we investigated genetic diversity in the C. rhodopis Species Group. We then examined type specimens (including types of junior synonyms) to match nomenclature to geographically circumscribed genetic clusters. Our molecular analyses revealed four major lineages within the C. rhodopis Species Group: (1) a widely distributed clade in western Mexico, (2) a highland clade in eastern Mexico, (3) a widely distributed lowland clade occurring in eastern Mexico, Guatemala and El Salvador, and (4) a haplotype from Volcan San Martın in Veracruz, Mexico. We identified the first clade as C. occidentalis, a taxon currently placed in the ecologically similar but phylogenetically distant C. mexicanus Species Series. In light of this we place C. occidentalis in the C. rhodopis Species Group and designate a lectotype and paralectotype for the species. The second and third clades inhabiting eastern Mexico and northern Central America correspond to C. rhodopis and C. loki, respectively. Additionally, we examined the taxonomic distribution of certain colour pattern traits and compensatory mutations in Domain III of the mtDNA 12S ribosomal RNA gene. Our recovery of the divergent Veracruz haplotype and extensive mtDNA structure within species indicates that additional taxonomic revision will be necessary. Key words: amphibian, colour pattern polymorphism, Eleutherodactylus, Mesoamerica, phylogeography, Terrarana

Introduction Direct-developing frogs of the genus Craugastor Cope 1862 are distributed from northern South America northward to the southwestern United States. Until recently, all 115 species of Craugastor were contained within the massive New World genus Eleutherodactylus Dumeril & Bibron 1841. A series of contemporary studies partitioned Eleutherodactylus into several families and genera (Crawford & Smith, 2005; Heinicke et al., 2007; Hedges et al., 2008; Heinicke et al., 2009; Schmid et al., 2010). One of these partitions is the family Craugastoridae which Correspondence to: Jeffrey W. Streicher. E-mail: streicher@ email.arizona.edu. Current Address: Department of Ecology and Evolutionary Biology, University of Arizona, Tucson AZ 85721, USA. ISSN 1477-2000 print / 1478-0933 online Ó The Trustees of the Natural History Museum, London 2014. All Rights Reserved. http://dx.doi.org/10.1080/14772000.2014.882428

contains two extant genera, Craugastor and Haddadus Hedges et al. 2008. While the discussion of what frog groups should be included in Craugastoridae is ongoing (see Pyron & Wiens, 2011), the monophyly of Craugastor and content of three subgenera, Campbellius Hedges et al. 2008, Craugastor and Hylactophryne Lynch 1968, is well established (Crawford & Smith, 2005; Hedges et al., 2008). Within the subgenus Craugastor, some recent molecular studies have further investigated relationships by using population-level sampling (Crawford et al., 2007). One group that has been exceptionally well characterized in this way is the C. rhodopis Species Series (sensu Hedges et al., 2008) where molecular methods have been useful in delimiting diversity because of the colour pattern polymorphism that occurs in most species (Lynch, 1966a; Savage & Emerson, 1970; Savage, 2002).


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Jeffrey W. Streicher et al.

Molecular studies of C. rhodopis Species Series taxa have addressed evolutionary relationships using karyotypes and nucleotide data derived from several species (Miyamoto, 1983; Crawford, 2003a, 2003b; Chen, 2005; Streicher et al., 2009). However, these investigations have been phylogenetically biased towards the lower Central American segment of the C. rhodopis Species Series, the C. podiciferus Species Group. The C. podiciferus Species Group contains eight species (C. bransfordii Cope 1886, C. jota Lynch 1980, C. lauraster Savage et al. 1996, C. persimilis Barbour 1926, C. podiciferus Cope 1875, C. polyptychus Cope 1886, C. stejnegerianus Cope 1893 and C. underwoodi Boulenger 1896) that are distributed from eastern Honduras to western Panama. In contrast, the northern segment of the C. rhodopis Species Series, the C. rhodopis Species Group, contains only two species (C. loki Shannon & Werler 1955 and C. rhodopis Cope 1867) and is distributed from Mexico southeastward through parts of Guatemala, Belize, El Salvador and Honduras (Fig. 1; Lynch, 2000; Hedges et al., 2008). Based on preliminary molecular diagnoses, the C. rhodopis Species Group is known to contain substantially more diversity than current taxonomy would suggest (see Crawford & Smith, 2005; Hedges et al., 2008). In addition to the phenotypic polymorphism observed in C. loki and C. rhodopis, the syntopic occurrence of two ecologically and

morphologically similar Craugastor lineages in Mexico (the C. mexicanus Species Series) and Central America (the C. laticeps Species Series), has contributed to the description of several presumably invalid species (Table 1; Lynch, 2000). In this study we investigated diversity levels within the C. rhodopis Species Group using a 479 bp fragment of mitochondrial DNA (mtDNA) from frogs sampled in Mexico, Guatemala and El Salvador. We use these data to address: (1) taxonomy within the C. rhodopis group, (2) the distribution of colour pattern polymorphism across matrilineal clades, and (3) compensatory mutation in Domain III of the mtDNA 12S ribosomal RNA subunit gene. Based on these findings and an examination of type material we provide a systematic revision with taxonomic accounts for all members of the C. rhodopis Species Group.

Materials and methods Geographic and taxonomic sampling Tissue samples were obtained from C. rhodopis Species Group frogs from Mexico, Guatemala and El Salvador. Collectively, we included 43 frogs from localities in (1) the Mexican states of Hidalgo, Veracruz, Puebla,

Fig. 1. Geographic distribution of Craugastor rhodopis Species Group samples used for the molecular portion of this study. Symbols and geographical groups (1.1–3.3) correspond to mitochondrial clustering patterns depicted in Fig. 2.

Molecular systematics of the Craugastor rhodopis Species Group

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Table 1. Nomenclature associated with the Craugastor rhodopis Species Group. An asterisk indicates those taxa that are currently in the C. mexicanus Species Series but should be placed in the C. rhodopis Species Group based on relationships inferred from mitochondrial DNA. Epithet

Synonym

Type locality

Type material

rhodopis

N/A

Veracruz, MX

USNM 16558

sallaei

rhodopis

Veracruz, MX

BMNH 1857.7.31.27

venustus

rhodopis

Veracruz, MX

BMNH 1901.12.19.37

plicatus

rhodopis

Veracruz, MX

BMNH 1901.12.19.38

beatae

rhodopis

Veracruz, MX

BMNH 1903.9.30.236–237

mystaceus dunnii dorsoconcolor loki

rhodopis rhodopis rhodopis N/A

Veracruz, MX Veracruz, MX Veracruz, MX Veracruz, MX

MCZ 8241 MCZ 8242 USNM 110619 UIMNH 67057

sanmartinensis

loki

Veracruz, MX

UIMNH 67058

occidentalis

N/A

Zacatecas, MX

BMNH 1947.2.16.65–66

calcitrans

occidentalis

Jalisco, MX

BMNH 1901.12.19.35–36

Description Cope, 1867 ‘1866’, Proc. Acad. Nat. Sci. Philadelphia, 19:323. G€ unther, 1869 ‘1868’, Proc. Zool. Soc. London, 1868: 487. G€ unther, 1900, Biol. Centr. Amer. Rep. Batr., Part 164: 234. G€ unther, 1900, Biol. Centr. Amer. Rep. Batr., Part 164: 228. Boulenger, 1903, Ann. Mag. Nat. Hist. Ser. 7, 12:552. Barbour, 1922, Proc. Biol. Soc. Washington, 35:112. Barbour, 1922, Proc. Biol. Soc. Washington, 35:111. Taylor, 1941, Univ. Kansas. Sci. Bull., 27: 152 Shannon and Werler, 1955, Trans. Kansas Acad. Sci., 58: 370. Shannon and Werler, 1955, Trans. Kansas Acad. Sci., 58: 375. Boulenger, 1898, Proc. Zool. Soc. London, 1891: 477 (Secondary homonym of C. mexicanus) Taylor, 1941, Proc. Biol. Soc. Washington, 54:91. (Replacement name for C. mexicanus) G€ unther, 1900, Biol. Centr. Amer. Rep. Batr., Part 164: 230

MX ¼ Mexico; BMNH ¼ The Natural History Museum, London UK; MCZ ¼ Museum of Comparative Zoology, Cambridge, MA, USA; UIMNH ¼ University of Illinois Museum of Natural History, Urbana, IL, USA; USNM ¼ National Museum of Natural History, Smithsonian Institution, Washington, DC, USA.

Guerrero, Oaxaca and Chiapas, (2) the Guatemalan departments of Quetzaltenango, Peten, San Marcos and Suchitepequez, and (3) the Salvadorian department of Santa Ana. For comparative purposes we included 34 individuals that belong to the C. podiciferus Species Group (C. aff. podiciferus and C. underwoodi) and the C. mexicanus Species Series (C. aff. mexicanus Brocchi 1877, C. aff. pygmaeus Taylor 1937 and C. occidentalis Taylor 1941). Craugastor podiciferus Species Group taxa were collected from (1) the Costa Rican provinces of Puntarenas, Heredia and Alajuela, and (2) the Panamanian province of Chiriquı. Craugastor mexicanus Species Series taxa were collected from the Mexican states of Sinaloa, Jalisco, Colima and Mexico. Scientific collecting permits were issued by the Secretaria de Medio Ambiente y Recursos Naturales (SEMARNAT, permits FAUT#0015 and FAUT#0243) in Mexico, the Consejo Nacional de Areas Protegidas (CONAP; permit#030/ 2010) in Guatemala, and the Ministerio de Medio Ambiente y Recursos Naturales in El Salvador (MARN). Costa Rican and Panamanian samples were collected under previously referenced permits (see Streicher et al., 2009). Tissue samples were stored in 100% ethanol or an SDSbased lysis buffer. All animals were handled and euthanized according to UTA IACUC protocols. Individual

voucher information and locality data are listed Table 2 (also see Fig. 1).

DNA extraction, PCR amplification and sequencing Genomic DNA was isolated from either muscle or liver tissue using a standard protocol from the QiagenÒ DNeasy (Valencia, CA, USA) blood and tissue kit. We used a fragment of the mitochondrial 12S ribosomal subunit gene (12S) that was PCR amplified with the forward primer 12SF (50 AAA CTG GGA TTA GAT ACC CCA CTA T 30 ) and the reverse primer 12SR (50 ACA CAC CGC CCG TCA CCC TC 30 ). We used a standard thermal cycling profile that included 40 cycles of 95 C denaturation, 50 C annealing, and 72 C elongation plus an additional 5 s extension after each elongation. PCR reactions were cleaned using either AMPure magnetic beads (AgencourtÒ , Bioscience, Beverly, MA, USA) or ExoSap-IT (USB/Affymetrix, Santa Clara, CA, USA). Cycle sequencing reactions (in both primer directions) and Sanger sequencing were performed using standard protocols associated with BigDyeÒ terminator chemistry (Applied Biosystems, Foster City, CA, USA) at either

4


Table 2. Voucher specimen information including GenBank accession numbers for Craugastor used in the mitochondrial DNA analysis of this study.


Taxon

Locality

rhodopis Species Group Craugastor loki El Salvador: Santa Ana Craugastor loki El Salvador: Santa Ana Craugastor loki Guatemala: Peten: Municipio La Libertad: Parque Nacional Sierra de Lacandon: Distrito Yaxchilan Craugastor loki Guatemala: Peten: Municipio La Libertad: Parque Nacional Sierra de Lacandon: Sitio Arqueologico La Pasadita Craugastor loki Guatemala: Quetzaltenango: Zunil: Volcan Santa Marıa: Santa Marıa de Jesus Zunil Craugastor loki Guatemala: San Marcos: San Rafael Pie de la Cuesta: Finca America El Vergel Craugastor loki Guatemala: Suchitepequez: Eco Lodge Los Tarrales Craugastor loki Guatemala: Suchitepequez: Eco Lodge Los Tarrales Craugastor loki Guatemala: Suchitepequez: Eco Lodge Los Tarrales Craugastor loki Guatemala: Suchitepequez: Eco Lodge Los Tarrales Craugastor loki Guatemala: Suchitepequez: Eco Lodge Los Tarrales Craugastor loki Guatemala: Suchitepequez: Eco Lodge Los Tarrales Craugastor loki Mexico: Chiapas: Carretera Canton Villa Flor to Rancherıa Los Placeres Craugastor loki Mexico: Chiapas: Carretera Canton Villa Flor to Rancherıa Los Placeres Craugastor loki Mexico: Chiapas: Carretera Tonala-Costa Rica: La Sepultura Craugastor loki Mexico: Chiapas: Carretera Tonala-Costa Rica: La Sepultura Craugastor loki Mexico: Oaxaca: Camino Niltepec-El Palmar Craugastor loki Mexico: Oaxaca: Carretera a El Progreso desde interseccion con MEX 185 Craugastor loki Mexico: Oaxaca: Carretera Santa Marıa Chimalapa-Lazaro Cardenas Craugastor loki Mexico: Oaxaca: Carretera Santa Marıa Guienagati to Santiago Lachiguiri Craugastor loki Mexico: Oaxaca: Municipio Santa Marıa Chilchotla: El Mirador Craugastor loki Mexico: Oaxaca: Road between San Juan Mazatlan and La Mixtequita Craugastor loki Mexico: Oaxaca: Road from Santo Domingo Petapa to Loma Santa Cruz

Elevation (m)

GPS N

GPS W

Voucher ID

12S

800 800 162

13.9894 13.9894 17.0000

89.5803 89.5803 91.0000

EBG 192 EBG 194 UTA A-55245

JX002066 JX002054 JX002071

N/A

N/A

N/A

UTA A-62383

JX002098

1600

14.7507

91.5505

UTA A-61044

JX002078

1590

14.9167

91.8914

UTA A-52268

JX002086

760

14.5267

91.1441

UTA A-60427

JX002079

760

14.5267

91.1441

UTA A-60428

JX002074

760

14.5267

91.1441

UTA A-60429

JX002085

760

14.5267

91.1441

UTA A-60430

JX002092

760

14.5267

91.1441

UTA A-60431

JX002099

760

14.5267

91.1441

UTA A-60432

JX002038

45

15.1129

92.4047

UTA A-56524

JX002069

45

15.1129

92.4047

UTA A-56525

JX002044

197

16.1496

93.6489

UTA A-56532

JX002091

197

16.1496

93.6489

UTA A-56533

JX002090

340

16.6808

94.5707

UTA A-56536

JX002097

62

17.1626

95.0603

UTA A-56594

JX002084

250

16.8237

94.7795

UTA A-56561

JX001974

1170

16.7594

95.4617

UTA A-56562

JX002065

1058

18.25700

96.76700

MZFC-16333

JX002053

633

17.1365

95.4172

UTA A-62325

JX002073

560

16.9119

95.2210

UTA A-62320

JX002043

(continued)


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Table 2. (Continued ). Taxon Craugastor loki Craugastor loki Craugastor loki


Craugastor loki Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis Craugastor occidentalis

Locality

Elevation (m)

GPS N

GPS W

Voucher ID

12S

Mexico: Oaxaca: Vicinity slightly N of Palomares Mexico: Veracruz: Sierra de Las Tuxtlas: Municipio Catemaco: Victoria, La Compuerta Mexico: Veracruz: Sierra de Las Tuxtlas: Volcan San Martın: Martires de Chicago Mexico: Veracruz: Sierra de Las Tuxtlas: Volcan San Martın: Rancho Primero de Mayo Mexico: Colima: Road from HWY 54 to Ixtlahuacan Mexico: Colima: Road from HWY 54 to Ixtlahuacan Mexico: Guerrero: HWY 134 from Ixtapa to Cd. Altamirano Mexico: Jalisco: Bosque Primavera Mexico: Jalisco: Bosque Primavera Mexico: Jalisco: Bosque Primavera Mexico: Jalisco: Bosque Primavera Mexico: Jalisco: Bosque Primavera Mexico: Jalisco: Bosque Primavera Mexico: Jalisco: Bosque Primavera Mexico: Jalisco: Carretera entre Las Cruces y Atenguillo, Mex 90 Mexico: Jalisco: Carretera La Estancia-La Mascota, a El Real Alto Mexico: Jalisco: Carretera La Estancia-La Mascota, a El Real Alto Mexico: Jalisco: Carretera Las Palmas-La Estancia Mexico: Jalisco: Coquio

70

17.16260

95.06040

UTA A-62318

JX002093

398

18.35900

95.1018

UTA A-54820

JX002100

N/A

N/A

N/A

UTA A-56590

JX002055

725

18.59100

95.23417

UTA A-54821

JX002101

221

19.0522

103.7841

UTA A-62340

JX002039

221

19.0522

103.7841

UTA A-62341

JX002040

473

17.8470

101.3806

UTA A-62343

JX002088

1840

20.6410

103.5561

PPC 8

JX002052

1840

20.6410

103.5561

PPC 10

JX002047

1840

20.6410

103.5561

PPC 11

JX002062

1840

20.6410

103.5561

PPC 12

JX002051

1840

20.6410

103.5561

PPC 13

JX002041

1840

20.6410

103.5561

PPC 14

JX002068

1840

20.6410

103.5561

PPC 15

JX002045

1936

20.3721

104.5881

UTA A-60767

JX002058

1957

20.6990

104.8714

UTA A-59511

JX002046

1957

20.6990

104.8714

UTA A-60772

JX002089

557

20.8187

104.9718

UTA A-60773

JX002087

1800

20.9368

103.0274

L 197 397

JX002050

2238

19.9663

103.7256

UTA A-62298

JX002048

1119

19.6909

104.4228

UTA A-62294

JX002061

1412

19.6955

104.3904

UTA A-60779

JX002060

1684

19.6885

104.3954

UTA A-60781

JX002064

1687

19.6884

104.3955

UTA A-62295

JX002049

Mexico: Jalisco: Roads between Sayula and Zacoalco de Torres, and Tapalpa Mexico: Jalisco: Sierra de Manantlan: cerca de Puerto Las Mazos Mexico: Jalisco: Sierra de Manantlan: Puerto Las Mazos: Rancho Los Mojos Mexico: Jalisco: Sierra de Manantlan: Puerto Las Mazos: Rancho Los Mojos Mexico: Jalisco: Sierra de Manantlan: Puerto Las Mazos: Rancho Los Mojos

(continued)

6


Table 2. (Continued ). Taxon Craugastor occidentalis Craugastor rhodopis Craugastor rhodopis Craugastor rhodopis


Craugastor rhodopis Craugastor rhodopis Craugastor rhodopis Craugastor rhodopis Craugastor rhodopis Craugastor rhodopis Craugastor rhodopis Craugastor rhodopis Craugastor rhodopis Craugastor rhodopis Craugastor rhodopis Craugastor rhodopis Craugastor sp. 1

podiciferus Species Group Craugastor aff. podiciferus Craugastor aff. podiciferus Craugastor aff. podiciferus Craugastor aff. podiciferus Craugastor aff. podiciferus Craugastor aff. podiciferus Craugastor aff. podiciferus Craugastor aff. podiciferus

Locality

Elevation (m)

GPS N

GPS W

Voucher ID

12S

Mexico: Sinaloa: HWY 40 between Villa Union and Concordia Mexico: Hidalgo: Municipio Tlanchinol: Carretera Tlanchinol a Tierra Colorada Mexico: Hidalgo: Municipio Tlanchinol: Carretera Tlanchinol a Tierra Colorada Mexico: Hidalgo: Municipio Tlanchinol: Carretera Tlanchinol a Tierra Colorada Mexico: Hidalgo: Municipio Tlanchinol: Carretera Tlanchinol a Tierra Colorada Mexico: Hidalgo: Municipio Tlanchinol: Comedor Estrella, La Monta~na Mexico: Hidalgo: Municipio Tlanchinol: Comedor Estrella, La Monta~na Mexico: Hidalgo: Municipio Tlanchinol: La Caba~na Mexico: Hidalgo: Municipio Tlanchinol: La Caba~na Mexico: Hidalgo: Mx Hwy 105 Mexico: Hidalgo: Mx Hwy 105, Tlanchinol-San Salvador Mexico: Veracruz: HWY 140 N of Banderilla Mexico: Veracruz: HWY 140 N of Banderilla Mexico: Veracruz: HWY 140 N of Banderilla Mexico: Veracruz: Municipio La Perla: Metlac Mexico: Veracruz: Road between Totutla and Huatusco Mexico: Veracruz: Ejido Xagapan: Volcan San Martın: Los Tuxtlas

162

23.2673

106.1167

UTA A-62342

JX002070

1472

20.9824

98.6378

UTA A-62258

JX002080

1472

20.9824

98.6378

UTA A-62259

JX002096

1474

20.9893

98.6243

UTA A-62351

JX002082

1474

20.9893

98.6243

UTA A-62352

JX002083

1531

21.0171

98.6492

UTA A-60310

JX002081

1531

21.0171

98.6492

UTA A-62261

JX002094

1538

21.0362

98.6460

UTA A-62260

JX002076

1538

21.0362

98.6460

UTA A-62262

KF739348

1486 1486

21.0279 21.0215

98.6428 98.6124

UTA A-62264 UTA A-62263

JX002095 JX002037

1644

19.5872

96.9710

UTA A-62299

JX002072

1644

19.5872

96.9710

UTA A-62300

JX002042

1686

19.5821

96.9682

UTA A-62301

JX002067

1911

18.9767

97.1431

UTA A-56584

JX002059

1258

19.1836

96.9596

UTA A-62317

JX002075

N/A

18.5500

95.2000

UTA A-62344

JX002057

2100

10.0830

84.0660

MVZ 164825

EF562303

1900

10.1680

84.0990

UCR 18062

EF562302

2000

10.1502

84.1490

UCR 17439

EF562298

2000

10.1502

84.1490

UCR 17441

EF562299

1410

8.7800

82.9752

FMNH 256657

EF562295

1410

8.7800

82.9752

FMNH 257755

EF562290

1410

8.7800

82.9752

FMNH 257756

EF562293

1500

10.3020

84.7790

FMNH 257653

EF562311

Costa Rica: Heredia: near Cerro Dantas Costa Rica: Heredia: near San Rafael Costa Rica: Heredia: near Vara Blanca Costa Rica: Heredia: near Vara Blanca Costa Rica: Puntarenas: near Las Cruces Costa Rica: Puntarenas: near Las Cruces Costa Rica: Puntarenas: near Las Cruces Costa Rica: Puntarenas: near Monteverde

(continued)


7

Table 2. (Continued ). Taxon Craugastor aff. podiciferus Craugastor sp. A Craugastor underwoodi mexicanus Species Series Craugastor aff. mexicanus


Craugastor aff. pygmaeus

Locality

Elevation (m)

GPS N

GPS W

Voucher ID

12S

1520

10.3020

84.7923

UTA A-52449

EF562312

1663 800

8.8020 10.4010

82.3980 84.0480

AJC 892 USNM 561403

EF562282 EF562323

2264

19.1221

100.1397

UTA A-62348

JX001952

1771

20.3771

105.0479

UTA A-62347

JX001984

Costa Rica: Puntarenas: near Monteverde Panama: Chiriquı Costa Rica: Heredia: near La Selva

Mexico: Mexico: Avandaro: Road from Avandaro to El Manzano, E of Cerro Gordo Mexico: Jalisco: Road between El Cuale and Talpa de Allende

FMNH ¼ Field Museum, Chicago, IL, USA; MVZ ¼ Museum of Vertebrate Zoology, University of California, Berkeley, CA, USA; MZFC ¼ Museo de Zoologıa, Universidad Nacional Autonoma de Mexico, Mexico City, MX; UCR ¼ Universidad de Costa Rica, San Jose, CR; UTA A ¼ University of Texas at Arlington Amphibian Collection; AJC ¼ Andrew J. Crawford personal field series; EBG ¼ Eli B. Greenbaum personal field series; PPC & L series ¼ Paulino Ponce-Campos personal field series.

SeqWright (Houston, TX, USA; www.seqwright.com) or the UTA genomics core facility (Arlington, TX, USA; gcf.uta.edu).

Sequence alignment and phylogenetic analyses Sequences were assembled and cleaned using the program Sequencher 4.1 (Gene Codes Corporation, Ann Arbor, MI, USA). We also used this program to perform multiple sequence alignments. We used maximum likelihood and Bayesian criteria to reconstruct plausible evolutionary scenarios for the diversification of 12S in C. rhodopis Species Group frogs. We tested nucleotide substitution models and conducted maximum likelihood analyses (using a nearest-neighbour-interchange search heuristic) in the program MEGA 5 (Tamura et al., 2011). Support for branching patterns was assessed using 2000 bootstrap pseudoreplicates (Hedges, 1992). Paired Bayesian analyses were conducted in MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003) using default settings with sampling occurring every 1000 generations for 10 million iterations. Convergence was assessed by observing the standard deviation of split frequencies between runs. Phylogenetic trees were visualized using the program FigTree 1.5 (http://tree.bio.ed.ac.uk/software/figtree).

Colour pattern, morphology and advertisement call diagnoses We examined colour pattern variation in 216 museum specimens referable to C. rhodopis (n ¼ 97; 6 populations) and C. loki (n ¼ 119; 9 populations) to better

understand the distribution of phenotypic polymorphism in these species. Specimens were examined at the University of Texas at Arlington (UTA; Arlington, TX, USA), the Field Museum of Natural History (FMNH; Chicago, IL, USA), the Illinois Natural History Survey (INHS; Champaign-Urbana, IL, USA), and the University of Kansas (KU; Lawrence, KA, USA). A list of specimens examined for colour pattern can be found in Appendix 1 (see online supplemental material, which is available from the article’s Taylor & Francis Online page at http:// dx.doi.org/10.1080/14772000.2014.882428). Many colour pattern characteristics in C. rhodopis Species Group frogs are associated with bright colours (i.e. reds, oranges etc.). Unfortunately these characteristics become faded or disappear in preserved specimens, thus we only used dark characters (i.e. melanin defined) which regardless of preservation methodology remain visible. We used presence/absence data from ten characters: (1) canthal masque, (2) supratympanic masque, (3) dorsal lateral stripes, (4) mid-dorsal stripe, (5) cloacal blotch, (6) dorsal flecking, (7) interocular blotch, (8) knee blotch, (9) mid-dorsal spot and (10) lip barring. We used the program SYSTAT 11 (Systat Software, Inc., Chicago IL, USA) to generate histograms based on these observations. To describe chromatotypes and glandular ridging patterns observed in living frogs we used the terminology of Savage (2002) for ‘Eleutherodactylus’. To link morphological variation to genetic data, we measured several characters on adult specimens with matched DNAs in our molecular analysis. We used a digital calliper to the nearest 0.01 mm or from high-quality photographs using the program Image J version 1.47 (Schneider et al., 2012) to measure characters that are variable between other Craugastor species (Savage, 2002): snout–vent length (SVL), tympanum


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width, shank length, head length and head width. These measurements were also taken on all type specimens that we examined. We determined sex by the presence of a subgular vocal sac in males and by observing eggs through the ventral surface of females. We recorded vocalizations of C. occidentalis from a population in Colima, Mexico during June and July 2009 (Fig. 8). Specifically, we made recordings of two individuals (UTA A-62340–41) along an access road of Mexican Highway 110 between Colima and Tecoman (N19.05220 W 103.78407, 342 m; WGS84). Advertisement calls were recorded using the video function on a Kodak EasyShare Z710 digital camera and audio files were extracted using Bink Video software (RAD Game Tools, Kirkland, WA, USA). We generated waveforms from these recordings using the program Raven Lite 1.0 (Bioacoustics Research Program, Ithaca, NY, USA).

Inference of ribosomal secondary structure In animals, the most frequently sequenced region of the 12S ribosomal RNA subunit gene is Domain III, one of four domains in the 12S gene. This region is comprised of several conserved fragments (typically corresponding to stem regions in the secondary structure of transcribed RNA molecules) that are interspersed amongst ‘hypervariable’ regions (typically corresponding to loop regions of the RNA secondary structure). Given the utility and frequent use of this Domain III fragment in biodiversity studies, several structural comparisons have been performed (Hickson et al., 1996; Kjer, 1997; Carapelli et al., 2004). To infer secondary structure in our 12S dataset we used the model described for amphibians by Kjer (1997) along with an annotated 12S sequence for Bos taurus Linnaeus 1758 from GenBank (V00654). We focused our examination on stems 32 and 33, because these areas are part of the backbone of Domain III making them critical for the stability of the subunit domain. Furthermore, the nucleotides that are transcribed to make the Watson-Crick base pairings of these stems are separated by several hundred nucleotides (c. 300 bp) making them valuable for the investigation of compensatory mutation rates (see Springer et al., 1995).

Results Phylogenetic analysis of mitochondrial DNA Based on AIC scores, the most appropriate model for our 12S dataset was the GTRþIþG model (lnL -2861.69). Thus, we used this model in our Bayesian and Maximum Likelihood analyses. Our phylogenetic analyses were largely concordant and revealed several well-supported clades (Fig. 2). We found that Craugastor mexicanus Species Series taxa (C. occidentalis, C. aff. mexicanus and C.

aff. pygmaeus) were paraphyletic relative to the ingroup. Frogs referable to C. occidentalis were nested within the C. rhodopis Species Group in all topological reconstructions including additional analyses (not shown here) that featured a more extensive sampling of the C. mexicanus Species Series. Given these results, we remove C. occidentalis from the C. mexicanus Species Series and place it in the C. rhodopis Species Group. Similar to previous studies (Crawford & Smith, 2005; Hedges et al., 2008), we recovered the C. podiciferus Species Group as monophyletic and as the sister lineage of the C. rhodopis Species Group. Within the C. rhodopis Species Group there are three major haplogroups with geographically circumscribed distributions: (1) a western Mexico clade, (2) an upland clade found in the Sierra Madre Oriental of Mexico and (3) a clade distributed in the lowlands of eastern Mexico and northern Central America. We also identified a distinctive haplotype from Volcan San Martın in the Mexican state of Veracruz. The earliest branching 12S lineage is the western Mexico clade and the divergent Volcan San Martın haplotype was recovered as the sister lineage to a clade containing the reciprocally monophyletic Sierra Madre Oriental and eastern lowlands clades. By georeferencing type localities of all 12 names referrable to the C. rhodopis Species Group (Table 1, Fig. 3), we found that the western mitochondrial clade contains samples that correspond to the name C. occidentalis and that the eastern highland and lowland mitochondrial clades correspond to the names C. rhodopis and C. loki, respectively. Based on levels of divergence, we refer to the Volcan San Martın haplotype as Craugastor sp. 1 (see discussion).

Colour pattern polymorphism analysis and morphometrics All of the colour pattern characters that we scored were present in C. rhodopis and C. loki, but at different frequencies. We found that supratympanic masque was a monomorphic character across the individuals we sampled, so we excluded it from our frequency histogram. Based on this examination, none of the dark colour pattern traits that we examined seem to be taxonomically correlated (Fig. 4). Although they are parapatrically distributed (Fig. 1; Lynch, 2000), C. loki and C. rhodopis are similar in morphological gestalt. Using adults from the molecular analysis and type specimens, we identified a putative morphological difference between these taxa. Male and female specimens of C. loki (clades 3.2, 3.3 [Fig. 2] and the holotype [N ¼ 7]) and C. rhodopis (clades 2.1. 2.2 [Fig. 2] and all types known from Veracruz [N ¼ 7]) revealed that relative shank length differentiates these taxa (Table 3). Based on our sampling, C. loki has a shank



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Fig. 2. Bayesian phylogram depicting relationships in the Craugastor rhodopis Species Series. Support values appear below branches and correspond to Bayesian posterior probabilities and maximum likelihood bootstrap support, respectively. Symbol and clade designations (1.1–3.3) correspond to geographically circumscribed groups (see Fig. 1). See online edition for the colour version of this figure.

that ranges from 46–54% SVL whereas C. rhodopis has a longer shank that ranges from 56–65% SVL.

lineages that according to our phylogenetic analysis are not sister taxa (Craugastor sp. 1 and C. podiciferus; Fig. 5).

Secondary structure analysis Amongst the nucleotides that comprise stems 32 and 33, we found evidence that several compensatory mutations have occurred in members of the C. rhodopis Species Series. Interestingly, a stem 32 G-C Watson-Crick pairing that is invariant in most anurans (Kjer, 1997) has shifted to a U-A pairing in two C. rhodopis Species Series

Taxonomic accounts Below we provide taxonomic accounts based on our revised understanding of the C. rhodopis Species Group. We provide some diagnostic morphological traits for differentiating taxa which when used in combination with collection locality information seem to be reliable.


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Fig. 3. Lateral view of type specimens (and some junior synonyms) of the Craugastor rhodopis species group: (A) Craugastor rhodopis, lectotype, USNM 16558; (B) C. loki, holotype, UIMNH 67057; (C) C. occidentalis, lectotype, BMNH 1947.2.16.65; (D) Hylodes venustus, holotype; BMNH 1901.12.19.37: (E) H. sallaei, holotype, BMNH 1857.7.31.27; (F) Eleutherodactylus sanmartinensis, holotype, UIMNH 67058; (G) H. calcitrans, syntype, BMNH 1901.12.19.35; and (H) H. plicatus, holotype, BMNH 1901.12.19.38. Map indicates the geographic distribution of type localities. Shaded/coloured regions correspond to the approximate distribution of mitochondrial clades from Fig. 2. See online edition for the colour version of this figure.

However, we encourage readers to interpret these diagnoses cautiously given the extreme levels of morphological polymorphism in the group.

Craugastor rhodopis Species Group Members of this group range in adult body size from c. 16 mm SVL (adult male C. loki) to c. 45 mm SVL (adult female C. rhodopis). Notable geographic variation in maximum adult body size has been doumented in C. loki and C. rhodopis (Lynch, 2000). All species appear to have sexual dimorphism in adult body size with females being larger. The width of the tympanum is sexually dimorphic (at least in C. occidentalis and C. rhodopis) with females possessing smaller tympana than males. The head is narrow (c. 30% SVL, C. rhodopis, lectotype) to moderately wide (c. 43% SVL, C. loki; Eleutherodactylus sanmartinensis, holotype). The following character descriptions are modified from Lynch (2000) and Hedges et al. (2008): The dorsum is either rugose or tuberculate, the venter is smooth, and the snout is acuminate to subacuminate in lateral view. Vocal slits are absent, nuptial pads present in all species with the exception of C. occidentalis. Finger I is slightly longer than Finger II with all digits bearing small disks. The toes are not webbed and the plantar tubercles are mostly inconspicuous. A subtle to large inner

metatarsal tubercle or fold is present in all taxa including C. occidentalis. All species have a small outer metatarsal tubercle. All species have well-developed vomers (described elsewhere as ‘large and prominent’; Hedges et al., 2008). Dorsal colour patterns are complex and polymorphic within populations. The venter is cream white with varying degrees of dark flecking (some specimens of C. loki and C. rhodopis from the Carribean slopes of the Sierra Madre Oriental have uniformly white throats [Lynch, 2000], while the holotype of C. loki has substantial dark flecking on the throat). Content: The Species Group includes three species: Craugastor loki, C. occidentalis and C. rhodopis. A fourth taxon may occur in Veracruz (Craugastor sp. 1, see Discussion). Distribution: The Species Group is distributed throughout mainland Mexico (south of the tropic of Cancer) southeastward to Guatemala, Belize, El Salvador and possibly Honduras (see C. loki account below). Comments: Historically, two members (C. loki and C. rhodopis) were contained within the Eleutherodactylus rhodopis cluster/group (Smith & Taylor, 1948; Shannon & Werler, 1955; Lynch, 2000; Savage, 2002; Crawford & Smith, 2005). Craugastor occidentalis was considered to be a member of the subgenus Hylactophryne (Lynch,



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Fig. 4. Bar plots depicting polymorphism of certain colour characteristics in Craugastor rhodopis and C. loki. See Appendix 1 for locality information and specimen voucher information (see supplemental material online). See online edition for the colour version of this figure.

1976) and more recently the Craugastor mexicanus Species Series (Hedges et al., 2008). Craugastor loki Shannon & Werler 1955 Common Leaf-litter Frog (Fig. 6) Eleutherodactylus loki Shannon & Werler (1955). Holotype: Illinois Natural History Survey (UIMNH) 67057 (original field number Frederick A. Shannon [FAS] 4748). Type locality: ‘Volcan San Martın, Veracruz’, Mexico. Eleutherodactylus sanmartinensis – Shannon & Werler (1955). Eleutherodactylus rhodopis – Duellman (1961) Eleutherodactylus rhodopis – Lee (1996) Eleutherodactylus rhodopis – Campbell (1998) Eleutherodactylus rhodopis – Lee (2000) Eleutherodactylus loki – Lynch (2000) Eleutherodactylus rhodopis – part. Lynch (2000) Eleutherodactylus loki – McCranie & Wilson (2002)

Eleutherodactylus rhodopis – K€ohler et al. (2005 “2006”) Craugastor loki – Crawford & Smith (2005) Craugastor loki – Frost et al. (2006) Craugastor rhodopis – McCranie (2007) Craugastor loki – Hedges et al. (2008) Craugastor loki – Garcıa-Vazquez et al. (2009) Craugastor loki – Pyron & Wiens (2011) Description. Adults used in the molecular dataset ranged in SVL from 21.09–29.14 mm (males; N ¼ 8) and from 30.23–39.67 mm (females; N ¼ 3). Craugastor loki can typically be differentiated from other members of the C. rhodopis Species Group as follows (character condition in parentheses): Unlike C. occidentalis, C. loki has a small supratympanic fold that is often subtended by two tubercles that are occasionally fused (‘Y-shaped’ supratympanic fold that extends onto the lateral surfaces [flanks]). Unlike C. rhodopis, C. loki has a relatively short shank rarely exceeding 54% SVL in length (relatively long shank up to 65% SVL in length). In general, this

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Table 3. Character ratios used to differentiate Craugastor loki and C. rhodopis. Clade designations refer to groupings from Figs 1, 2 and ‘Type’ indicates that the specimen is a holotype or name-bearing junior synonym of the C. rhodopis Species Group. Individual (Sex)

Taxon (Category)

Character Ratio (SHANK/SVL)

UIMNH 67057 (Female) JAC 30823 (Female) JAC 30821 (Male) JAC 30822 (Female) UTA A-60430 (Female) UTA A-60431 (Male) UTA A-60432 (Male) BMNH 1901.12.19.38 (Male) BMNH 1947.2.15.79 (Male) BMNH 1901.12.19.37 (Female) MCZ A-8241 (Male) USNM 16558 (Female) JAC 29875 (Female) UTA A-62301 (Male) UTA A-62260–64 (Both sexes) UTA A-62260–64 (Both sexes) UTA A-62260–64 (Both sexes) UTA A-62260–64 (Both sexes)

loki (Type) loki (clade 3.2) loki (clade 3.2) loki (clade 3.2) loki (clade 3.3) loki (clade 3.3) loki (clade 3.3) rhodopis (Type) rhodopis (Type) rhodopis (Type) rhodopis (Type) rhodopis (Type) rhodopis (clade 2.1) rhodopis (clade 2.1) rhodopis (clade 2.2) rhodopis (clade 2.2) rhodopis (clade 2.2) rhodopis (clade 2.2)

0.46 0.50 0.51 0.52 0.52 0.54 0.54 0.65 0.57 0.60 0.56 0.56 0.59 0.57 0.62 0.57 0.62 0.59

Jonathan A. Campbell field series, uncatalogued at UTA. Individuals from Hidalgo, Mexico. Photographs were used to obtain measurements and were not labelled with individual voucher numbers, so we were unable to match specimens to measurements within the series.

species possesses a more uniformly coloured dorsum than most C. rhodopis Species Group taxa; however, we have observed some individuals from Guatemala that possess mottled dorsa. Variation. Most individuals of C. loki possess distinctive black canthal and supratympanic masks. Dorsal ground colour can be orange, tan, brown or almost cream white. The dorsum is covered by many small glands (substantially smaller than those observed in C. occidentalis) and many individuals have faint linear ridging that runs the length of the dorsum. Some individuals have a thin middorsal pinstripe. Some individuals from populations in southern Veracruz, Mexico (and the holotype, a female) have a white subocular patch.

Fig. 5. Evidence for convergent compensatory mutations in a structurally integral portion of stem region 32 of the 12S rRNA domain III in Craugastor rhodopis Species Series taxa. See online edition for the colour version of this figure.

Distribution. This species was described as endemic to the Volcan San Martın in the Los Tuxlas region of southern Veracruz, Mexico. Lynch (2000) extended its distribution to include lower elevation sites (sea level to 2100 m) from San Luis Potosı southward across the Isthmus of Tehuantepec to Belize, El Salvador and northwestern Honduras. Our recovery of a widespread lowland clade that occurs from Central Mexico southward into Guatemala and El Salvador is largely consistent with the hypotheses presented by Lynch (2000) for C. loki (Figs 1, 2). Most specimens of C. loki have been collected in tropical wet forests from near sea level to elevations of c. 1600 m.



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Fig. 6. Population level colour pattern variation in Craugastor loki from near Matıas Romero, Oaxaca, Mexico (left of dotted line), and from near El Chupadero, Suchitepequez, Guatemala (right of dotted line). See online edition for the colour version of this figure.

Comments. The original description of C. loki (named after the Teutonic god of strife and fire) used two colour pattern characteristics to distinguish it from congeners (Shannon & Werler, 1955); however, these traits seem to be regionally restricted to southern Veracruz. Following its description, C. loki was synonymized with C. rhodopis by Duellman (1961). Lynch (2000) resurrected this taxon and described it as a widespread species distributed throughout much of lowland Mexico and northern Central America. A small group of specimens was reported from the northern department of Cortes in Honduras, but the actual occurrence of C. loki in Honduras is questionable given the abundance of the morphologically similar C. chac, C. gollmeri and C. laticeps, in this region and the historic precedent of misidentification between C. rhodopis Species Group and C. laticeps Species Group taxa (see Lynch, 1965; Lynch & Fugler, 1965; Savage, 1987 for discussion of this phenomenon). Unfortunately, contemporary verification of the Honduran records is doubtful given that local agricultural practices have heavily modified the only known locality (McCranie & Wilson, 2002). McCranie (2007), however, listed this taxon (using the name C. rhodopis) as known from Cortes. The common name of this species was listed as the ‘Volcan San Martın Robber Frog’ by Liner & CasasAndreu (2008) since it was thought to be endemic to this region of Veracruz. However, given the extensive geographic distribution of C. loki, we suggest using the common name proposed by Campbell (1998) for northern Guatemalan populations of C. rhodopis (herein referred to as C. loki) of ‘Common Leaf-litter Frog’. This name is an allusion to the relatively high abundance of this species at many localities in Mexico and northern Central America. Craugastor occidentalis Taylor 1941a Western Leaf-litter Frog (Figs 7, 8)

Eleutherodactylus occidentalis Taylor 1941a. Replacement name for Borborocoetes mexicanus Boulenger 1898. Lectotype: British Museum of Natural History (BMNH) 1947.2.16.65 (see formal designation below). Type locality: ‘Hacienda El Florencio, Zacatecas, Mexico’ (Boulenger, 1898). Here restricted to mountains north of Florencia de Benito Juarez, Zacatecas, Mexico (see comments). Hylodes calcitrans – part. G€unther (1900) Eleutherodactylus calcitrans Stejneger (1904) Hylactophryne mexicanus – part. Lynch (1976) Hylactophryne occidentalis – Lynch (1976) Eleutherodactylus occidentalis – Garcıa & Ceballos (1994) Eleutherodactylus occidentalis – Lynch (1996) Craugastor occidentalis – Crawford & Smith (2005) Craugastor occidentalis – Frost et al. (2006) Craugastor occidentalis – Hedges et al. (2008) Craugastor occidentalis – Arenas-Monroy et al. (2012) Description. Adults used in the molecular dataset ranged in SVL from 27.79–32.24 mm (males; N ¼ 4) and from 31.40–40.44 mm (females; N ¼ 6). Males often had dark throats and proportionally larger tympana than females. Craugastor occidentalis can typically be differentiated from all other members of the C. rhodopis Species Group by a distinctive ‘Y-shaped’ supratympanic fold that extends onto the lateral surfaces of the dorsum. This fold is usually unbroken, but we have examined some individuals from Colima and Jalisco where this fold is broken. The dorsum is highly glandular, sometimes tubercles form a short longitudinal series of warts on the posterior half of the dorsum, and there are often dark blotches associated with individual glands. Most adults have open hourglass glandular ridging on the dorsum in addition to short linear ridging.


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Fig. 7. Colour pattern variation in Craugastor occidentalis from the states of Jalisco, Colima, Guerrero and Sinaloa in western Mexico. Note red thigh colouration (bottom right panel) observed in an individual from Guerrero, Mexico (UTA A-62343). See online edition for the colour version of this figure.

Variation. Individuals of this species typically possess a supratympanic dark stripe or patch. Dorsal ground colour occurs in varying shades of grey, tan or brown. Extensive lip barring is common. Some individuals have a thin middorsal stripe, white arms and/or a distinctive white upper labium. We observed bright red colouration on the axial and postaxial thigh surfaces of an individual from Guerrero (UTA A-62343; Fig. 7). Advertisement call: The call consists a series of five high frequency bird-like chirps (Fig 8; A) followed by 1–3 quieter single note peeps (Fig. 8; B and C). The duration of the call is between 5 and 6 seconds. We observed males calling from positions at forest floor level to c. 3.5 m into the understorey. This call is so conspicuous that on most evenings during the wet season it dominates the anuran soundscape at this locality, which in addition to C. occidentalis is comprised of Eleutherodactylus (¼ Syrrhophus Cope 1878) spp., Smilisca baudinii Dumeril & Bibron 1841, Smilisca fodiens Boulenger 1882, Rhinella marina Linnaeus 1758, Incilius marmoreus Wiegmann 1833, Agalychnis dacnicolor

Cope 1864, and Diaglena spatulata G€unther 1882. Our description of this vocalization differs remarkably from others where it is described as a quiet mono-pulse (Hedges et al., 2008) which may indicate that at least one cryptic species occurs within the concept of C. occidentalis we present here. Distribution. This species is distributed in western Mexico across a wide range of elevations (15–2500 m). It occurs primarily in lowland tropical dry forests and high elevation pine-oak forests and may be distributed as far north as the Mexican state of Sonora. Comments. We designate the syntype specimen (BMNH 1947.2.16.65; Fig. 3C) collected by A.C. Buller at ‘Hacienda el Florencio’ in Zacatecas as the lectotype. The type locality is described as being directly north of San Cristobal on the eastern slope of the Sierra de Florencio. We restrict this locality to the mountains east of Florencia, Zacatecas; this region is located directly north of San Cristobal. A recent report also describes C. occidentalis in



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Fig. 8. Advertisement calling behaviours of Craugastor occidentalis from along Mexican Highway 110 between Colima and Tecoman in Colima, Mexico (UTA A-62340–41). Pictured are perch locations on the forest floor and in trees ranging in height from c. 6 cm to 3.5 m (top) and the waveform of a typical advertisement call (bottom). See text for description of vocalization characteristics (A–C).

Zacatecas (Arenas-Monroy et al., 2012). The remaining syntype (BMNH 1947.2.16.66) is designated a paralectotype. Amongst field workers, populations of C. occidentalis occurring along the Pacific coast south of the TransMexican Volcanic Cordilleras are often referred to as C. hobartsmithi. We examined the type series of C. hobartsmithi (holotype, FMNH 100114; paratypes FMNH 100579, 100899) and all three frogs are small (SVL < 2.5 cm) and referable to the C. mexicanus Species Series as previously suggested in Hedges et al. (2008). We found that the type series of Hylodes calcitrans (currently a junior synonym of C. omiltemanus) is a composite taxon that includes C. omiltemanus from Guerrero and C. occidentalis from Jalisco, a finding that was originally noted by Kellogg (1932), who through correspondence with H. W. Parker and E.R. Dunn also linked these specimens to Borborocoetes mexicanus. As such we place the H. calcitrans Jalisco individuals (BMNH 1901.12.19.35–36) in the synonymy of C. occidentalis. Liner (1994) recommended the use of ‘Taylor’s Barking Frog’ as a Standard English name for C. occidentalis. This was primarily based upon an earlier hypothesized relationship with barking frogs of the C. augusti complex (Lynch, 1976). Since our phylogenetic results place C. occidentalis within the C. rhodopis group, we recommend the standard name ‘Western Leaf-litter Frog’ in reference to the geographic distribution of this species.

Museum of Natural History (USNM) 16558. Type locality: ‘Vicinity of Orizaba’ (Smith & Taylor, 1948) subsequently modified to ‘Orizaba, Veracruz, Mexico’ (Smith & Taylor, 1950). Hylodes sallaei – G€unther (1869 “1868”) Hylodes plicatus – G€unther (1900) Hylodes venustus – G€unther (1900) Hylodes beatae – Boulenger (1903) Eleutherodactylus dunnii – Barbour (1922) Syrrhophus mystaceus – Barbour (1922) Eleutherodactylus dorsoconcolor – Taylor (1941b) Eleutherodactylus rhodopis – Lynch (2000) Craugastor rhodopis – Crawford & Smith (2005) Craugastor rhodopis – Frost et al. (2006) Craugastor rhodopis – Hedges et al. (2008) Craugastor rhodopis – Pyron & Wiens (2011)

Craugastor rhodopis Cope 1867 “1866” Polymorphic Leaf-litter Frog (Fig. 9)

Description. Adults used in the molecular analysis ranged in SVL from 23.82–30.43 mm (males; N ¼ 2) and from 36.77–42.33 mm (females; N ¼ 6). Males sometimes had proportionally larger tympana than females. Craugastor rhodopis can typically be differentiated from other members of the C. rhodopis Species Group as follows (character condition in parentheses): Unlike C. loki, C. rhodopis has a relatively long shank typically more than 56% SVL in length (typically less than 54% SVL). Unlike C. occidentalis, C. rhodopis has a small supratympanic fold that is typically subtended by two tubercles ( ‘Y-shaped’ supratympanic fold).

Lithodytes rhodopis Cope (1867 “1866”) Lectotype (Kellogg, 1932): Smithsonian Institution National

Variation. Individuals of this species typically possess a masque extending in some individuals from the canthus to


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Fig. 9. Population level colour pattern variation in Craugastor rhodopis from near Banderilla, Veracruz, Mexico (above dotted line), and from near Tlanchinol, Hidalgo, Mexico (below dotted line). See online edition for the colour version of this figure.

the posterior side of the tympanum and in others being restricted to the posterior side of the tympanum. Dorsal ground colour occurs in varying shades of grey, orange, tan or brown. The dorsum is highly glandular and typically there are no dark blotches associated with individual glands, as in C. occidentalis. Dorsal ridges are present and more prominent than in C. loki, but not as prominent as in C. occidentalis. Some individuals have a mid-dorsal stripe varying from thin to thick, or tan dorsolateral stripes. A solid tapered dorsum colouration is sometimes observed in specimens of C. rhodopis (Chromatotype of Eleutherodactylus dorsoconcolor). As observed in C. occidentalis, specimens of C. rhodopis can have white arms and/or a distinctive white upper labium (chromatotype of Syrrhophus mystaceus). Distribution. This highland species occurs in the Sierra Madre Oriental range of eastern Mexico. Populations are known from pine-oak forest habitat in the states of San

Luis Potosı, Hidalgo, Veracruz and Puebla (1258– 1862 m). Based on large adult body sizes, Lynch (2000) assigned two populations from Chiapas to C. rhodopis (his population systems 11 and 12); however, based on phylogeography (Fig. 1) we suspect these populations will be referred to C. loki once molecular data can be used to test their phylogenetic affinities. Comments. Craugastor rhodopis may have one of the more complex taxonomic histories for a Mexican amphibian. The original description of Craugastor rhodopis is attributed to Cope, who based his descriptions on four syntypes USNM 16557–60. Kellogg (1932) designated USNM 16558 as the lectotype (Fig. 3, A). The original description places the type locality at Orizava (¼ Orizaba) and Cordova (¼ Cordoba), Veracruz, but this was later restricted to Orizaba by Smith & Taylor (1950). Two species were described from the vicinity of Jalapa (¼ Xalapa), Veracruz, Hylodes plicatus and H. venustus.


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Fig. 10. Lateral, dorsal, and ventral surfaces of a recently hatched Craugastor sp. 1 (UTA A-62344) from Volcan San Martın, Veracruz, Mexico. The specimen’s left leg was taken as a tissue sample for genetic analysis. See online edition for the colour version of this figure.

Barbour (1922) described two additional forms from just west of Xalapa (specifically a locality he referred to as Cerro de los Estropajos), Syrrhophus mystaceus and Eleutherodactylus dunnii (spelled dunni by several subsequent authorities [e.g. Shannon & Werler, 1955; Frost, 2013]) both later considered junior synonyms of C. rhodopis by Kellogg (1932) and Lynch (1966a), respectively. Eleutherodactylus dorsoconcolor was described by Taylor (1941b) from Tequeyutepec, Veracruz about 7 miles west of Xalapa, which Duellman (1960) placed in the synonymy of C. rhodopis. In terms of dorsal colour pattern, it appears that C. rhodopis is the most polymorphic species in the C. rhodopis Species Series (Fig. 9), rivalled only by some populations of C. podiciferus in central Costa Rica (JWS, pers. obs.). In light of this, it is not surprising that several authors have recommended the common name ‘Polymorphic Robber Frog’ for C. rhodopis (e.g. Liner, 1994; Lee, 2000). The use of the term ‘Robber’ relates to the dark canthal masque that many Craugastor species possess. This ‘masque’ reminded some workers of the black domino-style masque often worn by stererotypical bank robbers and burglers. However, since not all C. rhodopis possess a full canthal masque (see Fig. 9) but all are ecologically associated with leaf-litter, we propose a more inclusive common name: ‘Polymorphic Leaf-litter Frog’.

Discussion Taxonomic implications We used evidence from our mtDNA analyses to hypothesize that there are at least three extant species in the Craugastor rhodopis Species Group: C. loki, C. occidentalis and C. rhodopis. Given that intraspecific levels of 12S mtDNA divergence are similar to those observed between some Craugastor species (Streicher, 2012), all three

species may require additional taxonomic subdivision in the future. Volcan San Martın in the Tuxtlas region of Veracruz is home to several endemic species (Firschein & Smith, 1956; Perez-Higareda & Navarro, 1980). Therefore we believe it is reasonable to suspect that the divergent haplotype we described as belonging to Craugastor sp. 1 represents an independent evolutionary lineage. However, given that we also sequenced several mtDNA haplotypes of C. loki from the region (Figs 1, 2), at present it is impossible to confirm this speculation given that incomplete lineage sorting in C. loki could also result in the observed patterns of sequence variation. The specimen that the mtDNA sequence of Craugastor sp. 1 originated from is a small metamorph (UTA A-62344, SVL ¼ 8.95 mm; Fig. 10). It is similar in gestalt to a metamorph paratype specimen of E. sanmartinensis, described as possessing a startling grey dorsal pattern, deeply contrasting black bands, paramedial bands extending between the eyes to the tip of the snout, and distinct bands on the upper labium, arms and legs (Shannon & Werler, 1955) and it also possesses a white subocular patch (like the holotype of C. loki). If future reconnaissance links this specimen to the type series of C. loki and/or E. sanmartinensis, the geographically widespread concept of C. loki we present herein will require revision. Streicher (2012) discussed a divergent mitochondrial haplotype from the C. rhodopis Species Group that originated from the Cerro de Los Flores region of Tabasco (Field ID: UOGV 385, uncatalogued). We further investigated this haplotype (which was derived from a small recently hatched frog), and discovered that it is closer in per cent mtDNA sequence identity to members of the C. laticeps Species Series (sensu Hedges et al., 2008) than the C. rhodopis Species Series. Cluster analyses performed using a broad sampling of Craugastor presented in Streicher (2012) also placed this haplotype with the C.

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laticeps Species Series. Thus, it is likely that this small frog was a misidentified C. laticeps Species Series taxon and that the divergent C. rhodopis Species Series lineage from Tabasco discussed in Streicher (2012) is invalid.


Comparative phylogeographic patterns Amongst the individuals of C. occidentalis that we sampled, we recovered two strongly supported clades (1.1 and 1.2; Fig. 2): (1.1) a group occurring in the northern portion of the species range (Jalisco and Sinaloa) and (1.2) a group primarily occurring in the southern portion of the species range from Jalisco southward through Colima and Guerrero. Recently a series of studies using DNA from amphibians and reptiles have identified several deep phylogeographic breaks along the western coast of Mexico (e.g. Devitt, 2006; Cox et al., 2012a, 2012b). Our analysis of the Western Leaf-litter Frog (C. occidentalis) resulted in consistent patterns with these studies in that the phylogeographic break between the two clades of C. occidentalis occurs more or less at the terminus of the Trans-Mexican Volcanic Belt (TMVB) in western Jalisco. As such, the TMVB may have been a dispersal filter barrier for C. occidentalis, a finding that is consistent with patterns observed in other amphibian species inhabiting both sides of this putative boundary (e.g. Smilisca fodiens; Cox et al., 2012a). Phylogeographic structure in the highlands of Mexico has been demonstrated to be extensive in many reptile and amphibian species (Mulcahy & Mendelson, 2000; Bryson et al., 2010; Bryson & Riddle, 2011; Bryson et al. 2011a, 2011b, 2011c). Furthermore, pine-oak and cloud forest habitats have revealed particularly complex genetic structuring (Castoe et al., 2009; Ornelas et al., 2013). Within C. rhodopis, a taxon presumably restricted to pine-oak and cloud forests of the Sierra Madre Oriental, we recovered two distinct clades (2.1 and 2.2; Figs 1, 2): (2.1) individuals inhabiting the highlands of western Veracruz (1200þ m), and (2.2) individuals distributed in the Tlanchinol region of Hidalgo, Mexico (1400þ m). We suspect that more regionally restricted haplogroups will be discovered once C. rhodopis is sampled from additional localities in the Sierra Madre Oriental. Within C. loki, we recovered substantial phylogeographic structure in the form of three geographically circumscribed clades (3.1–3.3; Figs 1, 2). The geographic distribution of clades 3.2 and 3.3 across the Isthmus of Tehuantepec is similar to phylogeographic patterns reported for a small salamander species, Bolitoglossa rufescens Cope 1869 (Rovito et al., 2012). We obtained two samples of C. loki from the northern Guatemalan department of Peten. One of these (UTA A-55245) was found to be closely related to individuals from the Los Tuxtlas region of Veracruz, Mexico, while the other (UTA A-62383) was closely related to individuals from El Salvador (the southernmost region of the distribution

of C. loki). We suspect that the latter individual is a mislabelled sample from southern Guatemala, but were unable to confirm this assumption.

Distribution of colour pattern polymorphism The phenomenon of intraspecific colour pattern polymorphism (or polychromatism; Savage, 2002) is observed throughout the vertebrate tree of life (e.g. Hoffman & Blouin, 2000; Galeotti et al., 2003; Chapple et al., 2008). The selective pressures that maintain colour pattern polymorphism in vertebrates are complex and often decoupled from genetic diversity (Cox & DavisRabosky, 2013). By mapping simple colour pattern traits in two C. rhodopis Species Group lineages inferred from our mitochondrial phylogeny (Fig. 4), we found that discrete colour pattern phenotypes occurring at the population level are not taxonomically correlated. This finding is consistent with previous findings in the C. podiciferus Species Group (see Savage & Emerson, 1970; Savage, 2002; Crawford, 2003a). What selective pressures might maintain such high levels of polychromatism? Visual predator-driven balancing selection may be a strong candidate for explaining population level polychromatism in Craugastor given that (1) several neotropical bird species are known to prey on terraranans (Poulin et al., 2001) and (2) the frequency of colour morphs is known to shift temporally and spatially in other polychromatic terraranans (Eleutherodactylus coqui Thomas 1966; Woolbright & Stewart, 2008). However, future investigations explicitly testing the determinants of colour pattern polymorphism will be necessary to reveal why recurrent colour pattern variation is observed in distantly related Craugastor lineages.

Compensatory mutations and ribosomal secondary structure The phenomenon of compensatory mutations that correspond to secondary structure stem regions of ribosomal RNA is well documented (Hancock et al., 1988; Dixon & Hillis, 1993). The frequency and fixation rate of these mutations is likely related to the strength of selection against deleterious intermediate states and thus strongly influenced by population size and mutation rate (Nasrallah & Huelsenbeck, 2013). Given that C. rhodopis Species Series frogs are thought to have large population sizes (Crawford, 2003a), they may represent a unique opportunity to explore the evolution of compensatory mutation in vertebrates. While a formal analysis of this phenemenon is beyond the scope of our study, the patterns we report in stem region 32 (Fig. 5) are noteworthy. The lineages that possess U-A modifications in stem 32 (C. podiciferus and Craugastor sp. 1) are not sister taxa according to our

Molecular systematics of the Craugastor rhodopis Species Group phylogenetic hypothesis. This may indicate that convergent compensatory mutations occurred during the Miocene to present diversification of the group (Crawford & Smith, 2005; Heinicke et al., 2007).


Future directions Lynch (2000) discusses a putative zone of sympatry between C. rhodopis and C. loki at intermediate elevations in Puebla, Veracruz, and central Chiapas. In particular, Lynch (2000) speculated that C. rhodopis and C. loki occur synoptically in the Huatusco and Xalapa regions of Veracruz. One of the populations sampled in our mtDNA analysis is from Banderilla (c. 6.6 km north of Xalapa) and we only observed haplotypes of C. rhodopis in the individuals we sampled. While we did not recover overlap in the geographic distribution of mtDNA haplotypes between C. loki and C. rhodopis, sampling nuclear loci and additional specimens will be necessary to test for hybridization and elucidate the distributional limits of these sister taxa. Both Lynch (2000) and Savage (2002) provided revealing summaries on the issue of phenotypic polymorphism in certain Craugastor lineages (also see [Lynch, 1966b]). These qualities produce an almost insurmountable challenge for identifying diagnostic characters that may aid in field identification. Since many distinct lineages within the C. rhodopis Species Series have been shown to possess overlapping variation in colour pattern, glandular ridging, and tubercle quality/density, how can field biologists reliably identify these frogs for ecological and conservation studies? We feel that the answer to this issue will be dependent on information obtained from dense geographic sampling and multilocus coalescent-based methods of species delimitation (e.g. Leache & Fujita, 2010). Although it is not a stand-alone diagnostic character (see Bauer et al., 2011) geographic distribution may ultimately be the most reliable way to differentiate some C. rhodopis Species Series taxa. Although external morphology may show low levels of phylogenetic signal in C. rhodopis Species Series frogs, following objective species delimitation, a multivariate analysis of morphology would provide valuable insights into the partitioning of morphological variation amongst species. Furthermore, skeletal, muscular and visceral morphologies may also prove useful for differentiating taxa in natural history collections. Lynch (2000) provided some descriptions of skull morphology in C. rhodopis Species Series taxa (C. bransfordii, C. rhodopis and C. stejnegerianus) and used these examinations to differentiate these taxa from members of the morphologically similar Craugastor mexicanus Species Series (C. mexicanus, C. pygmaeus and C. sartori Lynch 1965). Lynch (1993) also identified some unique conditions of jaw musculature in C. rhodopis relative to other Craugastor. Thus, similar

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studies that include C. loki and C. occidentalis may yield hitherto unidentified autapomorphies for these hypervariable species. When commenting on putative instances of hybridization amongst C. rhodopis Species Group frogs at Volcan San Martın, Shannon & Werler (1955) wrote ‘Persistent collecting on the mountain by a genetically-minded person should produce fruitful study material’. Based on the mtDNA survey we conducted in this study, we agree with these authors and encourage future molecular exploration to elucidate the yet undocumented diversity that almost certainly exists in these highly polymorphic tropical frogs.

Acknowledgements We thank the following individuals (and their respective institutions) for allowing access to specimens in their care: A. Resetar, T. F. Lian and R. Inger (FMNH), A. Wynn, R. Heyer and R. Wilson (USNM), B. Clarke, D. Gower and M. Wilkinson (BMNH), C. Phillips and M. Dreslik (INMH), R. Brown and A. Campbell (KU). M. Ingrasci kindly photographed museum specimens at FMNH. We are indebted to many individuals who assisted us with field and laboratory work during the course of this investigation. E. Greenbaum greatly aided this study by his donation of material from El Salvador. J. C. Arenas and E. Wostl provided helpful comments on early drafts of the manuscript. The following individuals helped by collecting leaf-litter frogs for this study: L. CansecoMarquez, C. Vasquez-Almazan, C. Cox, C. Sheehy III, T. Eimermacher, C. Franklin, M. Vaughn, R. Tovar, M. Ingrasci, J. Reyes-Velasco, G. Weatherman, T. Devitt, R. Garcia-Anleu, A. Carbajal Saucedo, F. R. Mendoza Paz, M. E. Acevedo, J. L. Camarillo Rangel and J. Meik. We thank B. Barker for discussions on Eleutherodactylus colour pattern polymorphism.

Funding Funding was provided by NSF grants to JAC [DEB0613802 & 0102383] and a Bioclon grant to ENS, CONACYT [no. 47590-Q], and DGAPA, UNAM [PAPIIT no. IN 224009] to A. Nieto-Montes de Oca. We thank two anonymous reviewers for comments that greatly enhanced the quality of the manuscript.

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