1071

Journal of Mammalogy, 83(4):1071–1076, 2002

MOLECULAR AND KARYOLOGIC EVIDENCE OF THETAXONOMIC STATUS OF COENDOU AND SPHIGGURUS

(RODENTIA: HYSTRICOGNATHI)

C. R. BONVICINO,* V. PENNA-FIRME, AND E. BRAGGIO

Genetics Division, Instituto Nacional de Cancer, Rio de Janeiro, RJ, Brazil (CRB, EB)Laboratorio de Biologia e Controle da Esquistosomose, Medicina Tropical Dept., IOC-FIOCRUZ,

Rio de Janeiro, Brazil (CRB, VP-F)

Mitochondrial cytochrome-b DNA and karyologic data were used to clarify taxonomicstatus of genera Coendou and Sphiggurus within the family Erethizontidae (Rodentia: Hys-tricognathi). Molecular and karyologic data were concordant in demonstrating that Coendouand Sphiggurus represent 2 evolutionary lineages. Comparative analyses showed that spe-cies of Coendou are karyotypically conservative, sharing the same diploid and fundamentalnumber, whereas those of the genus Sphiggurus diverge in diploid number despite sharingthe same fundamental number. We herein describe the karyotype of Sphiggurus melanura,a species considered a junior synonym of S. insidiosus by some authors. Our karyologicdata (2n 5 72, fundamental number 5 76), however, clearly contradict this hypothesis andsuggest that S. melanura is a valid taxon.

Key words: Coendou, cytochrome b, Erethizontidae, karyotype, Sphiggurus melanura, taxonomy

Voss and Angermann (1997) recentlypublished a review of neotropical porcu-pines which served to clarify the taxonomyof some erethizontids. But this family stillis poorly understood with respect to the tax-onomic status of several taxa and their phy-logenetic relationships. Erethizon andEchinoprocta are monotypic and recog-nized genera, whereas other species of er-ethizontids are either included in the generaCoendou and Sphiggurus (Woods 1993) orin a single genus (Coendou—Handley andPine 1992; Voss and Angermann 1997).Sphiggurus melanura also was consideredeither a junior synonym of S. insidiosus(Woods 1993) or a valid species of the ge-nus Coendou (Voss and Angermann 1997).

The karyotypes of 6 erethizontid taxa at-tributed to the genera Erethizon, Coendou,and Sphiggurus have been reported (Benir-schke 1968; Bonvicino et al. 2000; Con-cepcion and Molinari 1991; George and

* Correspondent: cibelerb@inca.gov.br

Weir 1974; Lima 1994), and these karyo-logic data have been helpful in clarifyingtheir taxonomic status.

In this paper, molecular analyses of se-quence data for mitochondrial cytochrome-bDNA for Coendou and Sphiggurus werecarried out to assess phylogenetic relation-ships, taxonomic status, and intragenericvariation. The results of our molecular anal-ysis were compared with karyologic data.The karyotype of S. melanura has also beendescribed.

MATERIALS AND METHODS

DNA of Coendou and Sphiggurus specimenscaptured in Brazil (Appendix I) was isolatedfrom livers preserved in ethanol following Smithet al. (1987). Mitochondrial cytochrome-b DNAwas amplified with primers MVZ05, MVZ14,and MVZ16 and sequenced with an automaticDNA sequencer (ABI Prismy 377, Applied Bio-systems, Foster City, California) followingSmith and Patton (1993). Homologous sequenc-es of 2 specimens of Coendou bicolor (access

1072 Vol. 83, No. 4JOURNAL OF MAMMALOGY

FIG. 1.—Consensus parsimony tree showingphylogenetic relationships of specimens ofCoendou and Sphiggurus with specimens of thefamilies Caviidae, Ctenomyidae, Echimyidae,and Octodontidae as out-groups. Numbers belownodes represent bootstrap values estimated byheuristic search with 1,000 replicates with a 1:1ratio of transitions to transversions.

numbers U34852 and U34851) were obtainedfrom GenBank and used in the phylogeneticanalyses of Coendou species. We also used se-quences of 7 other species of Hystricognathi,Cavia porcelus (GenBank accession numbersAJ222767, NC000884), Ctenomys boliviensis(AF155869), Ctenomys haigi (AF007063), Spa-lacopus cyanus (AF007061), Octodon degus(AF007059), Dactylomys dactylinus (L23335),and Mesomys hispidus (L23395), and 1 murine,Mus musculus (ABO42809) as out-groups. Dis-tances were estimated with Kimura’s 2-parame-ter method with 1:1 transition to transversion ra-tio, and the tree was constructed by neighborjoining using MEGA (version 2—Kumar et al.2000). Bootstrap values were estimated with1,000 replicates. Maximum-parsimony analyseswere carried out considering a 1:1 transition totransversion ratio using PAUP (Swofford 1993).Bootstrap values were estimated with 1,000 rep-licates. Maximum likelihood trees were obtainedusing the DNAML program with a 2:1 transitionto transversion ratio using PHYLIP 3.5c (Fel-senstein 1995).

We karyotyped 1 male S. melanura. Chro-mosome preparations were obtained from bonemarrow cultures in Roswell Park Memorial In-stitute (RPMI)–1640 tissue-culture medium(Sigma Chemical Company, St. Louis, Missou-ri), 20% fetal calf serum (Sigma), ethidium bro-mide (5 mg/ml; Sigma), and colchicine 1026 M(Sigma) for 2 h or from primary cultures of kid-ney epithelium in Dulbecco’s Minimal EssentialMedium (Sigma) with 10% fetal calf serum after5 h of colchicine. G-band preparations weredone following Seabright (1971). The funda-mental number (FN) refers only to the autosomalcomplement.

RESULTS

Mitochondrial cytochrome-b DNA se-quences of 2 specimens of Sphiggurus vil-losus, 3 of Coendou prehensilis (about1,140 base pairs), and 1 of S. melanura(786 base pairs) were deposited in Gen-Bank (Appendix I). Phylogenetic analyseswith neighbor joining, maximum parsimo-ny, and maximum likelihood showed thesame topology comprising 2 major clades(Fig. 1). One of these clades grouped spec-imens belonging to the families Erethizon-tidae and Caviidae (with 70% and 66%

bootstrap values in neighbor-joining andmaximum-parsimony analyses, respective-ly), and another clade grouped specimensbelonging to the Echimyidae, Octodontidae,and Ctenomyidae (76% and 87% bootstrapvalues in neighbor-joining and maximum-parsimony analyses, respectively). These 5families appeared to be monophyletic andwere supported by bootstrap values above65% (Fig. 1). Within the erethizontid clade(100% bootstrap values in neighbor joiningand maximum parsimony), 2 groups wereevident, 1 formed by Coendou haplotypesand the other by Sphiggurus haplotypes; the1st was supported by 100% bootstrap inboth neighbor joining and maximum par-simony, and the 2nd was supported by 93%and 86% bootstrap in neighbor joining andmaximum parsimony, respectively. Also, inthe maximum likelihood tree, the monophy-ly of C. prehensilis was evident, differingfrom the arrangement observed in neighbor-joining and maximum-parsimony topolo-gies.

Within the Erethizontidae, a comparisonof sequence data showed low divergenceestimates between interspecific and inter-individual haplotypes (Table 1). Interindi-vidual variation was observed in C. prehen-silis from Mato Grosso, Brazil, and S. vil-losus from Rio de Janeiro, Brazil. Distance(D) estimates were lower than 0.1% be-

BONVICINO ET AL.—TAXONOMY OF COENDOU AND SPHIGGURUSNovember 2002 1073

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1074 Vol. 83, No. 4JOURNAL OF MAMMALOGY

FIG. 2.—G-band karyotype of a male Sphig-gurus melanura (UFPB 3001) from Sao Joao daBaliza, State of Roraima, Brazil.

tween sympatric specimens and lower than3% between allopatric specimens (Table 1).D estimates (X 5 0.11) between Coendouand Sphiggurus were similar to intergenericestimates in other Hystrictognathi families(D 5 0.12 between Spalacopus and Octo-don within the Octodontidae; Table 1) butlower than those between Echimyidae gen-era.

One specimen of S. melanura had 2n 572 and FN 5 76 karyotype (Fig. 2). Theautosomal complement consisted of 3 pairsof biarmed chromosomes (1 large metacen-tric pair and 2 medium-sized submetacen-tric pairs) and 32 acrocentric pairs varyingin size from medium to small. The X chro-mosome was a medium-sized submetacen-tric, and the Y chromosome was a small-sized acrocentric.

DISCUSSION

Distance estimates, D, between Coendouand Sphiggurus were similar to those foundbetween genera of other Hystricognathifamilies (e.g., Octodontidae; Table 1), in-

dicating that Coendou and Sphiggurus arevalid genera. These analyses show that theCoendou–Sphiggurus clade was monophy-letic, as are both the Sphiggurus and theCoendou clades (Fig. 1). Neighbor-joiningand maximum-parsimony analyses show 2well-defined branches, 1 with C. bicolorand C. prehensilis and the other with S. vil-losus and S. melanura. The 1st lineage cor-responds to the karyotypically conservativegenus Coendou, with species sharing thesame diploid (2n 5 74) and fundamentalnumber (FN 5 82), as is the case of C.prehensilis (Lima 1994) and C. rothschildi(George and Weir 1974). The 2nd lineagecorresponds to the karyotypically diversegenus Sphiggurus, with S. melanura (2n 572, FN 5 76), S. vestitus pruinosus (2n 542, FN 5 76—Concepcion and Molinari1991), S. villosus (2n 5 42, FN 5 76—Bonvicino et al. 2000), and S. insidiosus(2n 5 62, FN 5 76—Lima 1994). The factthat Sphiggurus species share the same FNsuggests that these karyotypes can be de-rived from one another by Robertsonian re-arrangements. Our data do not support thehypothesis of Voss and Angermann (1997)that Coendou and Sphiggurus are conge-neric.

The karyotype of S. melanura (2n 5 70,FN 5 76) does not support the hypothesisof Woods (1993), who considered S. me-lanura as a junior synonym of S. insidiosus(2n 5 62, FN 5 76–Lima, 1994). Con-versely, this finding reinforces the claimthat S. melanura and S. insidiosus are validspecies as previously proposed by Voss andAngermann (1997). These authors com-mented (1997:39) that ‘‘C. melanura wasfirst misidentified as C. insidiosus by Ca-banis (1848), an error subsequently propa-gated by Cabrera (1961) and Husson(1978).’’ Our studies strongly suggest thatSphiggurus deserves a generic status andthat S. melanura is a valid species of thisgenus.

RESUMO

Dados de ADN do gene mitocondrial ci-tocromo b e cariotıpicos foram utilizados

BONVICINO ET AL.—TAXONOMY OF COENDOU AND SPHIGGURUSNovember 2002 1075

para clarificar o status taxonomico dos ge-neros Coendou e Sphiggurus dentro da fam-ılia Erethizontidae (Rodentia: Hystricogna-thi). Os dados moleculares e cariotıpicoscoincidiram em mostrar que Coendou eSphiggurus representam 2 linhagens evo-lutivas diferentes. Analise comparativamostrou que as especies do genero Coen-dou sao conservadas citogeneticamente,compartilhando o mesmo numero diploidee fundamental, enquanto aquelas do generoSphiggurus diferem no numero diploideapesar de compartilharem o mesmo numerofundamental. Aqui descrevemos o cariotipode S. melanura, uma especie consideradasinonimo junior de S. insidiosus por algunsautores. Nossos dados cariotıpicos (2n 572, FN 5 76), entretanto, claramente con-tradizem esta hipotese e sugerem que S. me-lanura e um taxon valido.

ACKNOWLEDGMENTS

We are grateful to R. Cerqueira for laboratoryfacilities and C. Vieira da Silva for helping uswith karyotypic analyses. We are also gratefulto B. Lemos for collecting blood of Coendou,Furnas Centrais Eletricas and C. Alho for allow-ing us to collect samples in Manso reservoir, andH. N. Seuanez for reviewing this manuscript.This work was supported by Programa de ex-celencia (PRONEX)/100-Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico, Fur-nas Centrais Eletricas, and Zoologico do Rio deJaneiro.

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Submitted 29 August 2001. Accepted 1 May 2002.

Associate Editor was Willian L. Gannon.

APPENDIX I

The 6 Brazilian specimens sequenced werefrom the following museums: Museu Nacional,Rio de Janeiro (MN), mammal collection ofUniversidade Federal da Paraıba, Joao Pessoa(UFPB), mammal collection of the hydroelectricdam of Manso, Cuiaba (Manso). GenBank ac-cess number (AF) follows museum specimennumber.

1076 Vol. 83, No. 4JOURNAL OF MAMMALOGY

Sphiggurus villosus.—State of Rio de Janeiro,Rio das Ostras, 228319370S, 418569420W, 4 m el-evation (MN 46938, AF407277); Sumidouro,228029590S, 428409290W, 355 m elevation (MN46937, AF411580).

Sphiggurus melanura.—State of Roraima, Sao

Joao da Baliza, 08579020S, 598549410W, 255 melevation (UFPB 3001, AF411583).

Coendou prehensilis.—State of Mato Grosso,Cuiaba, 158359460S, 568059480W, 176 m eleva-tion, UHE Manso (Manso 849, AF411584; Man-so 212, AF411581; Manso 138, AF411582).