Chromosomal rearrangements occurredrepeatedly and independently during species

diversification in Malagasy geckos,genus Paroedura

Gennaro Aprea1, Franco Andreone

2, Domenico Fulgione

1,

Agnese Petraccioli1& Gaetano Odierna

1*

1

Dipartimento di Biologia, Università di Napoli Federico II, Via Cinthia, I-80126, Napoli, Italy

2

Museo Regionale di Scienze Naturali, Via G. Giolitti, 36, I-10123 Torino, Italy.

Received 12 October 2012. Accepted 2 April 2013

We conducted a phylogenetic study through karyological data, by standard staining andAg-NOR banding, and molecular analysis (by 12S and 16S mitochondrial rRNA genes andnuclear gene C-mos) on 11 species of Malagasy geckos, genus Paroedura, and two relatives(Ebenavia inunguis and Uroplatus phantasticus). Ebenavia inunguis and U. phantasticus had2n = 36 telocentric elements, NORs on the first chromosome pair in E. inunguis, and on thethird chromosome pair in U. phantasticus. All examined Paroedura showed NORs on thesmallest chromosome pair; moreover, six of the eleven examined species show a 2n = 36karyotype, with a pair of metacentrics and 17 telocentric pair. The remaining species exhibitedkaryotypes with a diploid chromosome number ranging from 2n = 31 to 2n = 38. We assumethat these karyotype assemblages derived from the 2n = 36 karyotype by cryptic and/or simplerearrangements, such as inversions, fissions and fusions. Furthermore, molecular and/orchromosomal data indicate that Paroedura is a monophyletic genus, in which chromosomerearrangements occurred repeatedly and independently during the specific diversification.Moreover both P. bastardi and P. gracilis in current definitions are paraphyletic assemblages ofseveral related species, since their population proves more closely related to P. ibityensis orP. oviceps than co-specific populations.

Key words: chromosome, Gekkonidae, Madagascar, Paroedura, molecular phylogeny.

INTRODUCTIONParoedura Günther, 1879, is a gekkonid genusendemic to Madagascar and the Comoros (Glaw &Vences 1994, 2007; Nussbaum & Raxworthy 2000).This genus has been the subject of several taxo-nomic and systematic revisions (e.g. Mocquard1909; Angel 1942; Guibé 1956). Dixon & Kroll(1974) described nine Paroedura species on the basisof morphological and osteological characters. Afurther species (P. masobe) was described byNussbaum and Raxworthy (1994). Later, the sameauthors (Nussbaum & Raxworthy 2000) synony-mised P. guibeae with P. bastardi, described fivemore species and partitioned the genus Paroedurainto two major clades, featured by differentnostril–rostril contact: the picta-group (includingP. androyensis, P. bastardi, P. maingoka, P. picta andP. vahiny), with the nostril–rostril not in contactand typical of xeric regions in southern and south-western Madagascar, and the sanctijohannis-group

(including P. sanctijohannis, P. gracilis, P. homalorhina,P. oviceps, P. stumpffi, P. masobe, P. karstophila,P. tanjaka and P. vazimba) with nostril–rostral incontact and distributed in humid habitats of west-central, northern and eastern Madagascar, and onthe Comoro Islands. Glaw et al. (2001) described a15th species (P. lohatsara) as a large gecko withblackish markings on the head and distinctivelyenlarged and spinous tubercles. More recently,Jackman et al. (2008) elevated to species rankP. ibityensis, formerly considered a subspecies ofP. bastardi, and they also recognised two other can-didate species: Paroedura sp.n. I (from Montagnedes Français) and Paroedura sp.n. II (fromBemaraha-Andafiabe). Moreover, Jackman et al.(2008) in their extensive molecular phylogeneticanalysis of mitochondrial (ND2, ND4) and nuclear(RAG1, phosducin) DNA sequences on 16 of 18known Paoredura species did not treat the picta andsanctijohannis species-groups as valid; instead,these authors proposed a biogeographical*Author for correspondence. E-mail: gaetano.odierna@unina.it

African Zoology 48(1): 96–108 (April 2013)

scenario in which eastern species (P. masobe andP. gracilis) are basal and western and southwesternspecies form a monophyletic group. In their analy-sis Jackman et al. (2008) used Ebenavia inunguis asthe outgroup species, because from unpublisheddata concerning a phylogenetic study on a largernumber of Afro-Malagasy genera of geckos,Ebenavia retrieved as the closest relative toParoedura. Raxworthy et al. (2008), in presentingtheir comprehensive phylogeny for Uroplatus, indi-cate that Paroedura is the sister taxon to Uroplatus,and both are closely related to Ebenavia.

So far, karyological data on Paroedura are avail-able for P. picta and an undescribed Paroedura sp.molecularly related to P. bastardi species group(Main et al. 2012). The former species possesses2n = 36 chromosomes, with the elements ofthird pair biarmed and the remainder telocentric;the Paroedura sp. related to P. bastardi possesses2n = 34 chromosomes, with the elements of thefirst and fourth pairs biarmed and the rest telo-centric.

In order to contribute to define karyological rela-tionships among species of the genus Paroedura inMadagascar, here we re-evaluated the karyologicalprofile of 11 species of this genus, performed bystandard staining and Ag-NOR banding by oneof us (Aprea 2006). Since samples attributed onmorphological grounds to P. gracilis were karyo-logically variable, we also extended our study tothe analysis of sequence variation in mitochondrial12S+16S rRNA genes, and nuclear gene C-mos,which were successfully used in this gecko’smolecular phylogenetic studies (Gamble et al.2008; Rocha et al. 2009; Raxworthy et al. 2008).Furthermore, we extended our study to Uroplatusphantasticus and Ebenavia inunguis; both specieswere used as outgroups in our chromosomal andmolecular analyses

MATERIALS & METHODS

Field work and karyotypingThe individuals used in the analysis were

collected during fieldwork in the period 2002–2005.Sampling localities, Genbank accession numbersand sex of Ebenavia inunguis, Uroplatus phantasticusand Paroedura individuals collected for this studyare given in Table 1. After capture, animals wereinjected with a 0.5 mg/ml colchicine solution(0.1 ml/10 g body weight). One hour later theywere euthanased using a 0.5% MS 222 injection.After dissection, intestine, lungs, spleen and gonads

were removed. These organs were incubated for30 min in a solution of sodium citrate (0.5%) andfixed in 3:1 methanol and acetic acid. The fixedmaterial was preserved at 4°C and transferred tothe laboratory. Chromosomes were obtainedusing the air-drying method. They were firststained in 5% Giemsa (at pH7) and subsequentlystained with Ag-NOR banding (Howell & Black1980). For karyotyping we used at least five well-spread metaphase plates, following the chromo-some nomenclature proposed by Levan et al.(1964).

Mitochondrial 12S, 16S rRNA and C-mos gene analysisDNA was extracted from liver, gonads or blood

by the proteinase K+SDS digestion and conven-tional method of phenol-chloroform (Sambrooket al. 1989), or from a tip of the tail by means of theDNeasy Tissue kit (Qiagen). The 12S rRNA,16S rRNA and C-mos genes were amplified andsequenced using pairs of primers given in Table 2.All reactions were conducted in 50 µl of PCR mix(5 µl of Buffer 10x, 1.5 µl of MgCl2 25 mM, 0.5 µlof dNTP-mix 10 mM, 0.5 µM of each primers, 1.5 Uof Taq Polymerase Invitrogen, 100 ng of DNAtemplate and ddH2O). The cycling profile for12S+16S rRNA was: 2 min initial denaturationstep, 35 cycles including denaturation, 92°C for30 s, 55°C for 30 sec and 72°C for 45 sec, final exten-sion for 7 min at 72°C. For the C-mos gene, thecycling profile (Saint et al. 1998) was: 3 min of initialdenaturation step, 35 cycles including denatur-ation, 94°C for 45 sec, 52°C for 45 sec and 72°C for60 sec, final extension for 6 min at 72°C. The sizeand quality of PCR products were visualizedon 1% agarose gels. PCR products were cleanedwith the Qiaquick purification kit (Qiagen) anddirectly sequenced using BigDye™ terminatorcycle sequencing chemistry following the manu-facture protocol (Applied Biosystems, Foster City,U.S.A.). Samples were run on an ABI 377 auto-matic sequencer (Perkin-Elmer, Waltham, U.S.A.).Sequences were aligned using ClustalW (Thomp-son et al. 1994) with homologous fragments ofE. inunguis and U. phantasticus. We first analysedthe three datasets separately noting that theyproduced a similar topology. Then we combinedthe three datasets to optimize the statistical weightof the nodes and higher definition of clades.Pooled sequences were aligned using ClustalW(Thompson et al. 1994) with homologous frag-ments of E. inunguis and U. phantasticus. ThejModelTest program (Posada 2008) was used on

Aprea et al.: Chromosomal rearrangements and species diversification in Malagasy geckos 97

98 African Zoology Vol. 48, No. 1, April 2013Ta

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individual alignments of genes to estimate thebest-fit models of nucleotide substitution (result-ing in Jukes-Cantor model). Phylogenetic relation-ships among the samples were assessed bypartitioned Bayesian analyses conducted usingMrBayes 3.1 (Ronquist & Huelsenbeck 2003) withdefault priors. Analyses were initiated with randomstarting trees and run for 5 000 000 generations;Markov chains were sampled every 1000 genera-tions and a burn-in length of 500 000. Two separateanalyses with two independent chains were per-formed to check for convergence of log-likeli-hoods in stationary points (Huelsenbeck &Bollback 2001). The first 10% of the trees werediscarded as burn-in. A consensus tree from theretained trees was computed with MrBayes.

Patristic genetic distances were measuredamong taxa, to infer about the amount of geneticchanges along the branches.

RESULTS

Chromosome analysis

Samples of E. inunguis and U. phantasticus had2n = 36 elements. In these species all chromosomeswere telocentric (T), decreasing progressively inlength, arm number (A.n.) = 36 (Fig. 1, Table 3);Ag-NOR positive spots were on the peritelomericregions of the first chromosome pair in E. inunguis,or on the apical regions of third chromosome pairin U. phantasticus (Fig. 1). In Paroedura the chromo-some number ranged from 2n = 31 to 2n = 38(Figs 1 & 2, Table 3). Ag-NOR staining invariablymarked chromosomes of the smallest pair in allParoedura species (Figs 1 & 2).

Paroedura sp. n. I ‘Montagne des Français’, P.lohatsara, P. picta, P. stumpffi, P. vazimba and P. andro-yensis had a karytype of 2n = 36 chromosomes,with a pair of metacentric elements (the third) and

telocentric the chromosomes of the remainingpairs (A.n. = 38) (Fig. 1). No chromosomal varia-tion was found between sexes or among localitiesin each of these latter species, as well as in P.masobe, P. oviceps, P bastardi and P. ibityensis. How-ever, the latter four species exhibited a chromo-some formula different from the above sixParoedura species. In fact, P. masobe differed in hav-ing the chromosomes of the first pair (A.n. = 40)submetacentric; P. oviceps in showing chromo-somes of the first two pairs larger than the othersand with the second pair M (A.n. = 38); P. bastardiand P. ibityensis in possessing 2n = 34 chromo-somes, with Ms the first and fourth pairs andremaining pairs, (A.n. = 38) (Fig. 2, Table 3). Eachexamined population of P. gracilis showed a differ-ent chromosome number (Fig. 2): northern sam-ples (Montagne d’Ambre) had both sexes with2n = 38, (all T chromosomes, A.n. = 38), northeast-ern samples, from Ambolokopatrika, had bothsexes with 2n = 36 (the third pair M, the others Ts,A.n. = 38); from the central-eastern samples(Fiherenana) only females had metaphase platessuitable for karyotyping, they comprised 31 chro-mosomes of which pairs 1–4 were metacentric,pairs 5–14 were telocentric, and two pairs weremedium-sized Ts and the last was a a large M(A.n. = 39) (Fig. 2, Table 3).

Sequence analysis of 12S, 16S rRNA and

C-mos genes

Alignments of 22 Paroedura, U. phantasticus andE. inunguis sequence data produced fragments407 bp long for 12S rRNA, 571 bp for 16S rRNA and374 bp for C-mos; these fragments were pooled ina single nucleotide sequence of 1352 bp. Themultialignment was used to estimate nucleotidevariability in the pooled gene (Table 4) and geneticdistance among species (Table 5). The level of

Aprea et al.: Chromosomal rearrangements and species diversification in Malagasy geckos 99

Table 2. Primers used for PCR and sequencing.

Primer name Primer sequence Reference

12S rRNA12Sa 5’-AAACTGGGATTAGATACCCCACTAT-3’ Kocher et al. 198912Sb 5’-GAGGGTGACGGGCGGTGTGT-3’ Kocher et al. 1989

16S rRNA16SA-L 5’-CGCCTGTTTACCAAAAACAT-3’ Palumbi et al. 199116SB-H 5’-CCGGTCTGAAACTCAGATCAGT-3’ Palumbi et al. 1991

C-mos673 Cmos 5’-GCGGTAAAGCAGGTGAAGAAA-3’ Saint et al. 1998674 Cmos 5’-TGCAAAGTCTCCAATAGCATC-3’ Saint et al. 1998

100 African Zoology Vol. 48, No. 1, April 2013Ta

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2.6

±4.

00

(t)

123.

3±

3.5

0(t

)3.

3±

3.5

0(t

)2.

8±

.50

(t)

3.6

±3.

50

(t)

3.7

±3.

90

(t)

3.9

±.5

0(t

)2.

4±

.50

(t)

133.

0±

2.7

0(t

)3.

1±

2.7

0(t

)2.

1±

2.7

0(t

)3.

2±

2.7

0(t

)3.

2±

.00

(t)

3.7

±2.

70

(t)

2.3

±2.

70

(t)

142.

8±

3.0

0(t

)3.

0±

3.0

0(t

)1.

7±

3.0

0(t

)2.

5±

3.0

0(t

)2.

5±

3.0

0(t

)3.

0±

3.0

0(t

)1.

8±

3.0

0(t

)15

2.8

±2.

80

(t)

3.0

±2.

80

(t)

1.7

±2.

80

(t)

2.2

±2.

80

(t)

2.2

±3.

40

(t)

2.8±

2.8

0(t

)1.

6±

2.8

0(t

)16

2.6

±2.

50

(t)

2.6

±2.

50

(t)

1.5

±2.

50

(t)

1.8

±2.

50

(t)

1.8

±2.

90

(t)

2.4

±2.

50

(t)

1.2

±2.

50

(t)*

172.

2±

2.9

0(t

)2.

5±

2.9

0(t

)1.

4±

2.9

0(t

)1.

7±

2.9

0(t

)1.

5±

2.7

0(t

)2.

1±

2.9

0(t

)18

1.7

±2.

50

(t)

1.9

±2.

50

(t)

1.3

±2.

50

(t)*

1.4

±2.

50

(t)*

1.4

±26

0(t

)*1.

8±

2.5

0(t

)19

1.3

±2.

50

(t)*

P.lo

hats

ara

P.st

umpf

fiP.

vazi

mba

P.m

asob

e

Nos

yB

eA

nkar

ana

Mah

ajan

kaM

.des

Fran

çais

Chr

.R

.L.

C.I.

R.L

.C

.I.R

.L.

C.I.

R.L

.C

.I.R

.L.

C.I.

R.L

.C

.I.R

.L.

C.I.

111

.3±

4.8

0(t

)11

.3±

4.8

0(t

)11

.4±

4.8

0(t

)11

.4±

4.8

0(t

)11

.5±

4.8

0(t

)11

.1±

.10

(t)

11.4

±5.

30.

21(s

t)2

10.7

±.9

0(t

)10

.7±

.90

(t)

10.6

±.9

0(t

)10

.7±

3.9

0(t

)10

.5±

.90

(t)

10.7

±4.

00

(t)

10.0

±2.

70

(t)

310

.6±

2.7

0.44

(m)

10.3

±2.

70.

44(m

)10

.4±

2.7

0.45

(m)

10.5

±2.

70.

44(m

)10

.4±

2.7

0.45

(m)

10.5

±.3

0.45

(m)

9.7

±.2

0.46

(m)

47.

8±

3.0

0(t

)7.

9±

3.0

0(t

)8.

0±

3.0

0(t

)7.

9±

3.0

0(t

)8.

1±

3.0

0(t

)7.

8.0

±3.

30

(t)

8.4

±3.

00

(t)

57.

8±

3.1

0(t

)7.

7±

3.1

0(t

)7.

8±

3.1

0(t

)7.

9±

3.1

0(t

)8.

0±

3.1

0(t

)7.

5±

3.0

0(t

)8.

1±2.

90

(t)

67.

1±

2.6

0(t

)7.

1±

2.6

0(t

)6.

9±

2.6

0(t

)7.

0±

2.6

0(t

)7.

1±

2.6

0(t

)7.

1±

2.9

0(t

)7.

8±

2.7

0(t

)7

7.0

±.1

0(t

)6.

9±

3.1

0(t

)6.

8±

.10

(t)

7.0

±.1

0(t

)6.

9±

.10

(t)

6.7

±.7

0(t

)7.

0±

2.5

0(t

)

}12.

4±3

}0.4

0(m

)

Aprea et al.: Chromosomal rearrangements and species diversification in Malagasy geckos 101

86.

2±

.70

(t)

6.2

±3.

70

(t)

6.3

±.7

0(t

)6.

2±

.70

(t)

6.3

±.7

0(t

)6.

5±

.10

(t)

7.0

±.4

0(t

)9

6.0

±2.

80

(t)

5.9

±2.

80

(t)

6.2

±2.

80

(t)

6.0

±2.

80

(t)

6.0±

2.8

0(t

)6.

2±

2.5

0(t

)6.

3±

3.3

0(t

)10

4.9

±3.

40

(t)

4.9

±.4

0(t

)4.

8±

.40

(t)

4.5

±.4

0(t

)4.

5±

.40

(t)

5.0

±.5

0(t

)4.

5±

.80

(t)

114.

2±

3.3

0(t

)4.

4±

3.3

0(t

)4.

4±

3.3

0(t

)4.

3±

3.3

0(t

)4.

3±

3.3

0(t

)4.

5±

3.0

0(t

)3.

9±

4.0

0(t

)12

3.6

±3.

10

(t)

3.5

±3.

10

(t)

3.4

±3.

10

(t)

3.5

±3.

10

(t)

3.3

±3.

10

(t)

3.7

±3.

70

(t)

3.6

±3.

50

(t)

133.

2±

2.9

0(t

)3.

3±

2.9

0(t

)3.

2±

2.9

0(t

)3.

2±

2.9

0(t

)3.

3±

2.9

0(t

)3.

3±

2.4

0(t

)3.

0±

2.7

0(t

)14

2.8

±2.

70

(t)

2.5

±2.

70

(t)

2.6

±2.

70

(t)

2.5

±2.

70

(t)

2.6

±2.

70

(t)

2.6

±2.

50

(t)

2.7

±3.

00

(t)

152.

1±

2.6

0(t

)2.

3±

2.6

0(t

)2.

4±

2.6

0(t

)2.

4±

2.6

0(t

)2.

.5±

2.6

0(t

)2.

0±

2.4

0(t

)2.

0±

2.8

0(t

)16

17±

2.0

0(t

)1.

9±

2.0

0(t

)1.

8±

2.0

0(t

)1.

9±

2.0

0(t

)1.

7±

2.0

0(t

)1.

7±

1.7

0(t

)1.

7±

2.5

0(t

)17

1.6

±2.

70

(t)

1.7

±2.

70

(t)

1.6

±2.

70

(t)

1.6

±2.

70

(t)

1.7

±2.

70

(t)

1.6

±1.

90

(t)

1.6

±2.

90

(t)

181.

4±

2.4

0(t

)*1.

5±

2.4

0(t

)*1.

4±

2.4

0(t

)*1.

5±

2.4

0(t

)*1.

5±

2.4

0(t

)*1.

5±

2.0

0(t

)*1.

3±

2.5

0(t

)*

P.an

droy

ensi

sP.

pict

aP.

ibity

ensi

sP.

bast

ardi

Ilaka

kaM

iand

rivaz

oTo

liara

Chr

.R

.L.

C.I.

R.L

.C

.I.R

.L.

C.I.

R.L

.C

.I.R

.L.

C.I.

R.L

.C

.I.

111

.0±

4.8

0(t

)11

.2±

4.2

0(t

)15

.2±

5.1

0.41

(m)

15.5

±4.

00.

44(m

)16

.0±

5.1

0.43

(m)

15.8

±5.

10.

44(m

)2

10.5

±.9

0(t

)10

.8±

.50

(t)

11.4

±3.

00

(t)

11.0

±3.

00

(t)

11.0

±3.

60

(t)

11.1

±3.

60

(t)

310

.4±

2.7

0.41

(m)

10.5

±.0

0.44

(m)

11.2

±.2

0(t

)10

.4±

4.3

0(t

)10

.8±

4.0

0(t

)10

.6±

4.0

0(t

)4

8.0

±3.

00

(t)

8.1

±3.

20

(t)

10.4

±2.

70.

47(m

)10

.0±

2.3

0.45

(m)

10.4

±2.

90.

44(m

)10

.3±

2.9

0.45

(m)

57.

9±

3.1

0(t

)8.

0±

3.3

0(t

)8.

3±

2.9

0(t

)8.

3±

3.1

0(t

)8.

1±

3.5

0(t

)8.

6±

3.5

0(t

)6

7.1

±2.

60

(t)

7.1

±.2

0(t

)7.

2±

2.7

0(t

)7.

2±

2.4

0(t

)7.

1±

2.9

0(t

)7.

2±

2.9

0(t

)7

6.8

±3.

10

(t)

6.8

±.4

0(t

)7.

2±

2.5

0(t

)6.

8±

2.2

0(t

)7.

0±

2.5

0(t

)7.

1±

2.5

0(t

)8

6.5

±.7

0(t

)6.

2±

.00

(t)

5.8

±.4

0(t

)6.

1±

.00

(t)

5.8

±.0

0(t

)6.

0±

.00

(t)

96.

4±

2.8

0(t

)5.

8±

2.5

0(t

)5.

0±

3.3

0(t

)5.

1±

3.8

0(t

)5.

2±

3.4

0(t

)5.

2±

3.4

0(t

)10

4.7

±.4

0(t

)5.

0±

.10

(t)

3.2

±4.

00

(t)

3.5

±.5

0(t

)3.

4±

.90

(t)

3.2

±.9

0(t

)11

4.5

±3.

30

(t)

4.4

±3.

00

(t)

2.9

±.8

0(t

)3.

0±

3.2

0(t

)3.

0±

.00

(t)

3.0

±.0

0(t

)12

3.7

±3.

10

(t)

3.5

±2.

90

(t)

2.4

±3.

00

(t)

2.4

±3.

50

(t)

2.5

±3.

90

(t)

2.4

±3.

90

(t)

133.

0±

2.9

0(t

)3.

2±

2.7

0(t

)2.

2±

2.7

0(t

)2.

3±

2.7

0(t

)2.

3±

.00

(t)

2.2

±.0

0(t

)14

2.4

±2.

70

(t)

2.5

±2.

80

(t)

2.1

±2.

90

(t)

2.2

±2.

60

(t)

2.1

±2.

90

(t)

2.2

±2.

90

(t)

152.

0±

2.6

0(t

)2.

2±2.

90

(t)

1.9

±.5

0(t

)2.

0±

.00

(t)

2.0

±.6

0(t

)1.

9±

.60

(t)

161.

9±

2.0

0(t

)1.

8±

2.4

0(t

)1.

9±

2.5

0(t

)2.

0±

2.4

0(t

)1.

8±

2.9

0(t

)1.

7±

2.9

0(t

)17

1.7

±2.

70

(t)

1.5

±2.

10

(t)

1.7

±2.

80

(t)*

1.8

±2.

50

(t)*

1.5

±2.

70

(t)*

1.5

±2.

70

(t)*

181.

5±

2.4

0(t

)*1.

4±

2.3

0(t

)*

102 African Zoology Vol. 48, No. 1, April 2013

nucleotide diversity was high in P. bastardi andP. gracilis and low in P. stumffi (Table 4). Parwisegenetic distance ranged from a minimum of 0.127(P. stumffi vs P. lohatsara) to a maximum of 0.450(P. vazimba vs P. gracilis in average with all popula-

tions) (Table 5). In P. bastardi, no significant diver-sification was observed between samples ofMarofandilia and Miandrivazo (genetic distanceof 0.014); in contrast, samples from Toliara andIlakaka were highly differentiated (genetic dis-

Fig. 1. Karyotypes and sampling localities of Ebenavia inunguis, Uroplatus phantasticus and Paroedura speciesshowing a ‘primitive’ like chromosome set. The inserts include the NOR bearing pair and the Ag-NOR banded pair onthe right.The hatched inserts show the NOR-bearing pair in scale (on the left) and the magnified Ag-NOR-banded pair(on the right). Scale bar is the same for all images

tance 0.236). Results of Bayesian phylogeneticanalysis yielded a well-supported relationshippattern (Fig. 3). Relationships among Ebenavia,Uroplatus and Paroedura are unsolved, retrievingthem as a tricotomy. However, the monophyly ofthe genus Paoredura was strongly supported in allanalyses by bootstrap of 99% and a posterior

probability of 1.0. Paroedura species clustered intwo main lineages: the first retrieved the central-northeastern species, with P. masobe as sisterspecies to Paroedura sp. n. I ‘Montagne des Fran-çais’, P. gracilis and P. oviceps. The second lineageincludes two subclades: one with P. lohatsara andP. stumpffi; the other clade with central-south-

Aprea et al.: Chromosomal rearrangements and species diversification in Malagasy geckos 103

Fig. 2. Karyotypes and sampling localities of Paroedura species possessing a ‘derivative’ chromosome set. Thehatched inserts show the NOR-bearing pair in scale (on the left) and the magnified Ag-NOR-banded pair (on theright). Scale bar is the same for all images.

104 African Zoology Vol. 48, No. 1, April 2013

western species (P. androyensis, P. bastardi, P.ibityensis, P. picta and P. vazimba). In the lattergroup, P. vazimba was sister to the clade ((P.androyensis, P. picta) (P. bastardi + P. ibityensis))(Fig. 3). Furthermore, the topology of P. bastardi +P. ibityensis suggests that the former species belongsto a paraphyletic assemblage, because samples ofP. bastardi from Toliara or Ilakaka are more closelyrelated to P. ibityensis than their conspecifics fromMiandrivazo or Marofandilia (Fig. 3).

DISCUSSIONData for Malagasy geckoes other than the twospecies studied by Main et al. (2012) and thoseconsidered here are available only for species andsubspecies of Phelsuma and Uroplatus (Aprea et al.1996; Aprea 2006). The latter studies providedevidence that specific chromosome diversificationin Phelsuma and Uroplatus occurred withoutmodifications either in number and shape of theirchromosome sets (in fact all examined taxashowed 2n = 36 elements, mostly Ts), in contrastto loci of NORs that extensively varied both in thenumber and/or position on the chromosomes.Conversely, species diversification in Paroeduraseems to have been accompanied by extensivevariations of chromosome number, ranging from2n = 31 to 38, and shape, whereas no changeoccurred in the number or position of NORs.However, our data allow us to put forward ahypothesis on the derivation of the karytotypes ofthe studied taxa. The most parsimonious scenario(Fig. 4) is to assume that the common ancestor ofParoedura+Ebenavia (according to Jackman et al.2008) or Paroedura+Uroplatus (Roxworthy et al.2008) possessed a karyotype of 2n = 36, alltelocentric chromosomes with NORs on the small-est pair. One step occurs for the transition to thekaryotype of E. inunguis (NOR translocation onthe first pair) or U. phantasticus (NOR translocationon the third pair); similarly, one step (an inversion,

Table 4. Genetic diversity in the species of Paroedura inwhich we have considered more than one population,namely P. stumpfii, P. bastardi and P. gracilis. Statisticswere based on 1352 sites of 12S and 16S rRNA andC-mos gene sequences; #ps, number of polymorphicsites;Pi, nucleotide diversity;#H, number of haplotypes.

#ps Pi #H

Paroedura stumpffi 86 0.026 6Paroedura bastardi 217 0.105 4Paroedura gracilis 187 0.103 3

Tab

le5.

Pat

ristic

gene

ticdi

stan

ceam

ong

stud

ied

Mal

agas

yge

ckos

.The

inte

nsity

ofth

egr

eyis

muc

hhi

gher

whe

nth

ege

netic

sim

ilarit

yis

elev

ated

.

involving the primitive third pair) occurs forthe transition to a karyotype of 2n = 36, withmetacentric elements of the third pair, that weassume to be the Paroedura primitive karyotype.Six out the 11 studied Paroedura taxa would haveconserved the hypothesized primitive karyotype,namely P. picta (Main et al. 2012 and presentstudy), P. stumpffi, P. androyensis, P. lohatsara,Paroedura sp. I ‘Montagne des Français’, P. gracilisfrom Ambolokopatrika and P. vazimba. Derivativekaryotypes would have from complex (P. oviceps)or simple chromosome rearrangements [P. masobe,Paroedura sp. molecularly related to P. bastardispecies group studied by Main et al. (2012),P. bastardi, P. ibityensis and P. gracilis from Fihere-nana and Montagne d’Ambre] (Fig. 3). An inver-sion involving chromosomes of the first pair mayaccount for the elements of this pair being shapedas subtelocentric in P. masobe, without changing indiploid chromosome number (Fig. 4); a centricfusion, probably occurring between fourth andfifth chromosomes pairs, may justify the reductionof elements from 2n = 36 to 2n = 34 in P. bastardi,and P. ibityensis (Fig. 3); note that this fusionevent also can be applied to the complement of 2n= 34 chromosomes of Paroedura sp. studied byMain et al. (2012); a centric fission of the primitivemetacentric third pair may account for theincrease of the of elements from 2n = 36 to 2n = 38chromosomes, all telocentric in P. gracilis fromMontagne d’Ambre; three centric fusions and oneinversion would occur for derivation of the karyo-type of 2n = 31 with four metacentric pairs andone unpaired metacentric element of P. gracilisfrom Fiherenena.

Our chromosome evolutionary hypothesis alsosuggests that somehow primitive chromosomesof Paroedura ancestor are prone to a certain kind ofrearrangement. The primitive telocentric chromo-somes of the fourth pair were involved in twoseparate events of centric fusions: with the telo-centric chromosomes of the second pair givingplace to the first metacentric pair of P. gracilis fromFiherenena; with the telocentric chromosomes ofthe fifth pair giving place to the first metacentricpair of P. bastardi or P. ibityensis. The primitive telo-centric chromosomes of the first pair were in-volved in two separate events of inversion: oneshaping as metacentric the chromosomes of thefirst pair of P. masobe; the other inversion shaped asmetacentric the chromosomes of the second pairof P. gracilis from Fiherenena.

Molecular relationships of 16 out of 18 known

Paroedura species were previously studied byJackman et al. (2008) by analysis of mitochondrial(ND2, ND4) and nuclear (RAG1, phosducin)DNA sequences. Although based on three differ-ent molecular markers, our analysis also producesa very similar topology for the 11 consideredspecies. In fact, our tree shows two main clades.One includes the central-northeastern species,P. masobe, P. sp.n.I Montagne des Français,P. oviceps, and P. gracilis. According to Jackmanet al. (2008) P. masobe appears as the sister speciesto all other Paroedura species. However, both in thepresent study and that by Jackman et al. (2008), thephylogenetic position of P. masobe was supportedby low values of bootstrap (55 or 57). Our molecu-lar data further support the scenario given byJackman et al. (2008) concerning the close relation-ships between P. picta and P. androyensis and P.lohatsara and P. stumpffi. However, our originalmolecular results concern P. stumpffi, P. gracilisand P. bastardi, evidencing different degrees ofdiversification within their populations, with orwithout differentiation at chromosomal level orchromatic morphological pattern. In these threespecies we studied samples of five populations ofP. stumpffi, four of P. bastardi and three of P. gracilis;in each species the geographic distance betweenlocalities ranged from 50 to 300 km. Samples ofP. stumpffi retrieved as a homogenous monophy-letic assemblage; this uniformity was evident alsoin chromosome and chromatic pattern. In con-trast, samples both of P. bastardi and P. gracilis clus-ter as a paraphyletic assemblage. In the latterspecies, the sample from Fiherenena is moreclosely related to P. oviceps than its conspecific fromAmbolokopatrika or Montagne d’Ambre. TheParoedura bastardi sample from Toliara or Ilakaka ismore closely related to P. ibityensis than theirconspecific from Marofandilia or Miandrivazo.Furthermore, patristic distances suggest that ascurrently defined P. gracilis and P. bastardi areat least two assemblages of closely related taxa. InP. gracilis, populations from Fiherenena, Ambolo-kopatrika and Montagne d’Ambre exhibit a differ-entiation at molecular level and each also shows adistinctive karyotype and morphological chro-matic pattern. Indeed, they have a differentdiploid number, respectively 2n = 31, 36 and 38. Interms of colouration, the individuals fromFiherenana, central-eastern Madagascar, show adorsal pattern of longitudinal blackish stripes,those from Ambolokopatrika, northeastern Mada-gascar, have irregular blackish patterns, and those

Aprea et al.: Chromosomal rearrangements and species diversification in Malagasy geckos 105

from Montagne d’Ambre, extreme northeasternMadagascar, have transverse blotches (Figs 2 & 4).In P. bastardi no significant diversification wasobserved between samples from Marofandilia andMiandrivazo, which, in contrast, are highly differ-entiated from samples from Toliara and Ilakaka.Note that the sequence divergence values foundby Main et al. (2012) for their Paroedura sp. speci-mens which are closely related to P. bastardi aresimilar to values found for P. bastardi specimens inthis study. Unfortunately, the samples studied byMain et al. (2012) were from the pet trade fromunknown localities. However, their specimens

showed a karyotype of 2n = 34 elements very simi-lar to those of P. bastardi and P. ibityensis here stud-ied. This suggests that before a moleculardiversification from their common ancestor, acentric fusion occurred, reducing the chromo-some number from 2n = 36 to 2n = 34.

Our findings with regard to P. bastardi andP. gracilis indicate that, as currently defined, eachis a paraphyletic assemblage of several relatedspecies. However, because the overall distributionpatterns of the three P. gracilis populations, as wellas that of P. bastardi, are still lacking, we refrainfrom advancing taxonomic proposals.

106 African Zoology Vol. 48, No. 1, April 2013

Fig. 3. Bayesian (Monte Carlo-Markov chain) consensus phylogenetic tree of 22 Paroedura mitochondrial andnuclear sequences rooted by Ebenavia inunguis and Uroplatus phantasticus. Posterior probabilities and maximumparsimony bootstrap values of the major nodes are listed below and above the branches, respectively. Colours ofbranches represent general distribution of species:dark blue = eastern and northeastern Madagascar;green = north-western and northern Madagascar; red = central and western-south Madagascar.Karyograms and live image profilesof each of examined taxa are included. In Paroedura, unstained haploid karyograms are considered primitive, while inkaryograms considered derivative the rearranged elements are coloured, as in Fig. 4.

Aprea et al.: Chromosomal rearrangements and species diversification in Malagasy geckos 107

ACKNOWLEDGEMENTS

We are grateful to the Malagasy authorities forpermits. G. Aprea and G. Odierna were supportedby a grant from the University of Naples FedericoII. F. Andreone thanks several organizations, suchas WWF, WCS, CI, and Gondwana Conservationand Research. We thank J.E. Randrianirina, F.Mattioli, F. Glaw and M. Vences for field assistanceand for providing us with tissue of some Paroedurasamples. For the live specimen images of P. ibity-ensis, P. stumpffi, P. vazimba, P. lohatsara, P. gracilis,and P. oviceps, we thank Jon and Stacy Bone,Jorome Gublin, Friedrich Wilhelm Henkel, JesseHosmann and Emmanuel Van Heygen for permis-sion to use their original pictures.

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Responsible Editor: B. Jansen van Vuuren

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