molecular and karyologic evidence of the taxonomic status of coendou and sphiggurus (rodentia:...
TRANSCRIPT
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: [email protected]
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
TA
BL
E1.
—P
hylo
gene
tic
dist
ance
betw
een
spec
imen
sas
indi
cate
dby
P-d
ista
nces
for
spec
imen
sof
Ere
thiz
onti
dae
(spe
cim
ens
1–8)
,C
teno
myi
dae
(9,
10),
Ech
imyi
dae
(11,
12),
Cav
iida
e(1
3,14
),O
ctod
onti
dae
(15,
16),
and
Mur
idae
(17)
.In
terg
ener
icdi
stan
ces
betw
een
mem
bers
ofth
esa
me
fam
ily
are
show
nin
bold
face
.
Spe
cim
ens
12
34
56
78
910
1112
1314
1516
1.Sp
higg
urus
vill
osus
2.S.
vill
osus
3.S.
mel
anur
a4.
Coe
ndou
preh
ensi
lis
5.C
.pr
ehen
sili
s
0.03
00.
105
0.12
10.
121
0.09
00.
099
0.09
90.
104
0.10
40.
000
6.C
.pr
ehen
sili
s7.
C.
bico
lor
8.C
.bi
colo
r9.
Cte
nom
ysbo
livi
ensi
s
0.11
90.
126
0.12
60.
287
0.09
80.
105
0.10
50.
272
0.10
20.
109
0.10
90.
282
0.00
10.
004
0.00
40.
261
0.00
10.
004
0.00
40.
261
0.00
60.
006
0.26
30.
000
0.26
50.
265
10.
C.
haig
i11
.D
acty
lom
ysda
ctyl
inus
12.
Mes
omys
hisp
idus
0.28
20.
253
0.26
7
0.28
00.
246
0.25
4
0.28
40.
239
0.26
0
0.26
90.
236
0.26
8
0.26
90.
236
0.26
8
0.26
90.
238
0.26
6
0.27
40.
240
0.27
3
0.27
40.
240
0.27
3
0.06
30.
225
0.21
80.
214
0.21
10.
179
13.
Cav
iapo
rcel
lus
14.
C.
porc
ellu
s15
.Sp
alac
opus
cyan
us16
.O
ctod
onde
gus
17.
Mus
mus
culu
s
0.26
80.
268
0.32
70.
302
0.28
3
0.26
70.
267
0.30
50.
286
0.26
5
0.28
70.
287
0.28
40.
294
0.25
4
0.25
10.
251
0.27
30.
253
0.25
6
0.25
10.
251
0.27
30.
253
0.25
6
0.25
10.
251
0.27
60.
251
0.25
8
0.25
10.
251
0.27
60.
253
0.25
6
0.25
10.
251
0.27
60.
253
0.25
6
0.27
50.
275
0.22
00.
209
0.25
5
0.25
80.
258
0.21
60.
200
0.24
8
0.26
20.
262
0.22
20.
216
0.23
4
0.27
30.
273
0.23
10.
221
0.26
1
0.00
00.
244
0.24
90.
273
0.24
40.
249
0.27
30.
123
0.24
40.
237
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.
LITERATURE CITED
BENIRSCHKE, K. 1968. The chromosome complementand meiosis of the North American porcupine. Joun-al of Heredity 59:71–76.
BONVICINO, C. R., F. C. ALMEIDA, AND R. CERQUEIRA.2000. The karyotype of Sphiggurus villosus (Roden-tia: Erethizontidae). Studies on Neotropical Faunaand Environment 35:81–83.
CABANIS, J. 1848. Saeugethiere. Pp. 766–786 in Reisenin Britisch-Guiana in den Jahren 1840–1844, Ver-such einer Fauna und Flora von Britisch-Guiana (R.Schomburgk, ed.). Vol. 3. J. J. Weber, Leipzig, Ger-many (not seen, cited in Voss and Angermann1997).
CABRERA, A. 1961. Catalogo de los mamıferos deAmerica del Sur. Revista del Museo Argentino deCiencias Naturales ‘‘Bernardino Rivadavia’’ 4:309–732.
CONCEPCION, J. L., AND J. MOLINARI. 1991. Sphiggurusvestitus pruinosus (Rodentia, Erethizontidae): the
karyotype and its phylogenetic implications, de-scriptive notes. Studies on Neotropical Fauna andEnvironment 26:237–241.
FELSENSTEIN, J. 1995. PHYLIP (Phylogeny inferencepackage). Version 3.5c. University of Washington,Seattle.
GEORGE, W., AND B. J. WEIR. 1974. Hystricomorphchromosomes. Symposia of the Zoological Societyof London 34:79–108.
HANDLEY, C. O., JR., AND R. H. PINE. 1992. A newspecies of prehensile-tailed porcupine, genus Coen-dou Lacepede, from Brazil. Mammalia 56:237–243.
HUSSON, A. M. 1978. The mammals of Suriname(Zoologische Monographieen Van Het RijksmuseumVan Natuurlijke Historie). E. J. Brill, Leiden, TheNetherlands 2:1–569.
KUMAR, S., K. TAMURA, I. JAKOBSEN, AND M. NEI.2000. MEGA: molecular evolutionary genetics anal-ysis. Version 2. Pennsylvania State University, Uni-versity Park.
LIMA, F. S. 1994. Cariotipos em especies de Dasyproc-tidae e Erethizontidae, com discussao da evolucaocromossomica (Rodentia, Caviomorpha). BrazilianJournal of Genetics 17, supplement:135.
SEABRIGHT, M. 1971. A rapid banding technique forhuman chromosomes. Lancet 2:971–972.
SMITH, L. J., R. C. BRAYLAN, J. E. NUTKIS, K. B. ED-MUNDSON, J. R. DOWNING, AND E. K. WAKELAND.1987. Extraction of cellular DNA from human cellsand tissues fixed with ethanol. Analytical Biochem-istry 160:135–138.
SMITH, M. F., AND J. L. PATTON. 1993. The diversifi-cation of South American murid rodents: evidencefrom mitochondrial DNA sequence data for the ako-dontine tribe. Biological Journal of the Linnean So-ciety 50:149–177.
SWOFFORD, D. L. 1993. PAUP: phylogenetic analysisusing parsimony. Version 3.1.1. Smithsonian Insti-tute, Washington, D.C.
VOSS, R. S., AND R. AGERMANN. 1997. Revision noteson neotropical porcupines (Rodentia: Erethizonti-dae). 1. Type material described by Olfers (1818)and Kuhl (1820) in the Berlin Zoological Museum.American Museum Novitates 3214:1–42.
WOODS, C. A. 1993. Suborder Hystricognathi. Pp.771–806 in Mammal species of the world: taxonom-ic and geographic reference (D. E. Wilson and D.M. Reeder, eds.). 2nd ed. Smithsonian InstitutionPress, Washington, D.C.
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).