Herpetologica. 59(2), 2003, 203-218© 2003 by The Herpetologists' League, Inc.

CHROMOSOME BANDING OF SIX DENDROBATID FROGS(COLOSTETHUS, MANNOPHRYNE)

HINRICH KAISER1,3,4, CLAUS STEINLEIN2, WOLFGANG FEICHTINGER2, ANDMICHAEL SCHMID2

IDepartment of Biology, La Sierra University, Riverside, CA 92515, USA2Institut fiir Humangenetik, Universitat Wiirz"burg, Biozentrum, Am Huhland, 97074 Wiirz"burg, Germany

ABSTRACT: We conducted a chromosome banding analysis (heterochromatin, nucleolus organiz­er regions, DAPI fluorescence, distamycin A/mithramycin fluorescence) of six phylogenetically basaldendrobatid frog species (Colostethus chalcopis, C. leopardalis, Mannophryne henninae, M. nehlina,M. olmonae, M. trinitatis). With the exception of C. chalcopis (2n = 22), all examined species hada chromosome complement of 2n = 24 chromosomes. The C- and Q-band analyses showed thatconstitutive heterochromatin is present at the centromeres of all species, with Q--regions occurringat the positions of the nucleolus organizer regions (NORs) of C. leopardalis, M. olmonae, and M.trinitatis. The C-band polymorphisms were detected in M. henninae and M. nehlina on chromosomeNo.6 and in M. henninae on chromosome No.7. Silver-staining and distamycin A/mithramycinfluorescence resolved a Single pair of NORs in each species. The DAPI fluorescence revealedpericentromeric bands on chromosome No.1 and 5 in M. trinitatis and on chromosome No.1 and4 in C. chalcopis. Chromosome data clearly allow a distinction between M. olmonae and northernTrinidadian M. trinitatis, laying to rest arguments that M. olmonae may not be a good species. Inconjunction with chromosome information found in the literature, our data confirm that chromo­some complements of phylogenetically basal dendrobatids can be quite variable, with both 2n = 24and 2n = 22 complements present. Discrepancies in the karyotypes of M. trinitatis from nearCaracas, Venezuela, and from the Northern Range of Trinidad indicate the possibility of crypticspecies within M. trinitatis. The notion that a reduction in chromosome number by up to threechromosome pairs has taken place among Dendrobatidae from a putative ancestral 2n = 24 kar­yotype is shown to be congruent with current molecular hypotheses of dendrobatid relationships.

Key words: Chromosome banding; Colostethus; Dendrobatidae; Mannophryne; Systematics

THE FROG family Dendrobatidae is amonophyletiC lineage with a high profileamong biologists because of the strikingand very diverse, purportedly aposematic(Summers and Clough, 2001), colorationof some of its members (see Gray, 2000for a discussion of problems with applyingthe concept of aposematism to dendroba­tids). Dendrobatid species in the generaAllobates, Dendrobates, Cryptophylloba­tes, Epipedobates, Minyobates, Phoboba­tes, and Phyllobates are often brightly col­ored (e.g., Myers and Daly, 1983) and pro­duce lipophilic alkaloids. They are com­monly known as poison frogs. Theremaining, more enigmatic genera (Aro­mobates, Colostethus, Mannophryne, Ne­phelobates) are drably colored; with fewexceptions, produce no toxins; and havecollectively been considered phylogeneti-

3 PRESENT ADDRESS: Department of Biology, Vic­tor Valley College, Victorville, CA 92392, USA.

4 CORRESPONDENCE: e-mail, kaiserh@VVc.edu

cally basal, with relatively little knownabout their intra- and intergeneric rela­tionships (for an example, see Coloma,1995 and his discussion of the problemswith paraphyly of the genus Colostethus inrelation to the putatively nonbasal Epipe­dobates). In fact, the validity of severalnamed dendrobatid genera (Allobates,Aromobates, Mannophryne, Nephelobates,Phobobates) has been uncertain and a top­ic of controversy. Only through the recentapplication of molecular data to the phy­logenetiC relationships among Dendroba­tidae (Clough and Summers, 2000; Venceset aI., 2000) is this controversy being re­solved. Based on these data, it is apparentthat Phobobates is phylogenetically nestedwithin Epipedobates and should be consid­ered its junior synonym (Vences et al.,2000). Similarly, at least some species pre­viously aSSigned to the genus Minyobatesare phylogenetically nested within the ge­nus Dendrobates and should be includedin that genus (Clough and Summers, 2000;

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TABLE I.-Origins of the 50 dendrobatid frogs used in this study.

[Vol. 59, No.2

Sex (n)

Species Collection locality Male Female

Colostethuschalcopis Martinique, French Antilles

3 km NE Morne Rouge 0 2leopardalis Venezuela

Cordillera de Merida 3 3

Mannophryneherminae Venezuela

Rancho Grande, Nueva Quebrada I 7neblina Venezuela

Rancho Grande, La Trilla 4 4olmonae Tobago

Mile 3.0, Roxborough-Bloody Bay Road 6 7trinitatis Trinidad

3 km N Verdant Vale, Arima Valley 7 7

Vences et al., 2000). Furthermore, there isemerging molecular evidence that Man­noph"Jne and Nephelobates are indeedvalid taxa (M. Vences, personal communi­cation).

Duellman (1967) was the first to reporta chromosome count for dendrobatids,displaying the karyotype of Dendrobatespumilio (2n = 20) in a meiotic and a mi­totic spread. This number was confirmedby Leon (1970), who added the count forD. auratus (2n = 18). In the same year,Bogart (1970) reported the chromosomenumber of Epipedobates (then Phylloba­tes) trivittatus as 2n = 24. More recentstudies by Aguiar-Junior et al. (2002), Bo­gart (1991), Rada de Martinez (1976), Ra­sotto et al. (1987), and Veiga-Manoncelloet al. (2001) indicate that there is both in­ter- and intrageneric karyotype variabilityamong Dendrobatidae and that taxa withmore highly speCialized phenotypic char­acteristics (Myers, 1987) seem to have ex­perienced a reduction in chromosomenumber. There have only been two chro­mosome banding studies on dendrobatids;one involved a single species (Dendrobatesauratus; Schmid, 1980), and the other ex­amined four species of Epipedobates(Aguiar-Junior et al., 2002). We believethat chromosome data, espeCially those in­cluding detailed chromosome morpholo­gies, can be of considerable utility in am­phibian systematics, and we worked withtwo of the most basal dendrobatid genera

to establish baseline chromosome bandingdata. In this paper, we present the mostdetailed chromosome data compiled forthe genera Colostethus and Mannoph"Jneto date.

MATERIALS AND METHODS

SpecimensFrogs were collected under permits to

H. Kaiser (Caribbean) and E. La Marca(Venezuela) on research expeditions dur­ing 1987, 1989, 1992, and 1993 (n = 50;Table 1). Bone marrow suspensions wereprepared locally whenever logistics per­mitted. In 1993, live animals were broughtto the laboratory in Wiirzburg to ensurethat auxilliary preparations could be made(e.g., blood for flow cytometry, tissue forallozyme and DNA analyses). Animalswere treated according to the commonpractices of animal care, as approved byWiirzburg University.

Chromosome PreparationAfter overnight in vivo treatment with

0.03% colchicine, animals were over­anaesthetized using ether or a 1% MS-222solution. Bone marrow was extracted fromfemurs, tibiae, and radioulnae by cuttingacross epiphyses with a scalpel and flush­ing with 0.007 M KOH administered bysyringe and fine gauge needle. Each bonewas flushed several times to ensure releaseof the greatest pOSSible number of bonemarrow cells. Intestines were removed in

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their entirety (stomach--cloaca) and cutinto small pieces. Samples were incubatedin distilled water for 20 min (bone mar­row) or 50 min (intestine). Bone marrowsamples were then centrifuged for 8 minat 1400 rpm, the supernatant liquid re­moved, and ice cold fixative (3:1 methanol:acetic acid) added carefully to avoidclumping. Intestine prepa~ationswere re­moved from liquid and fixative was added.All preparations were kept frozen at - 20C until use.

Microscope slides of mitotic chromo­some spreads were made from bone mar­rowand/or intestinal samples. Bone mar­row preparations were centrifuged, andmost fixative was removed. The bone mar­row sample in the bottom of the test tubewas carefully agitated until a faintly milkyconsistency was achieved. Three to fourdrops of the sample were dropped ontoeach cleaned (using chromic acid) micro­scope slide from about 30 cm height.When bone marrow material was insuffi­cient to accomplish a complete suite ofbanding experiments, small pieces of thefixed intestine were transferred to pre­warmed slides on a slide warmer (40 C),cut into tiny pieces, and treated with adrop of 50% acetic acid. Fragments weresiphoned up and down in the acetic aciddrop using a Pasteur pipette until the tis­sue had disassociated completely to forma cell suspension and evaporation of theacetic acid was complete. Occasionally,more acetic acid was added to keep intes­tinal lumps suspended until they werecompletely diSintegrated. Slides were airdried and frozen at - 20 C until staining.Slides were usually stained within 1 wk ofpreparation. This methodology is modifiedfrom that developed by Schmid (1978).

Chromosome Banding

Conventional chromosome staining(Giemsa, C-banding), fluorescence stain­ing (quinacrine mustard, counterstainingwith distamycin Almithramycin-D.JM),and NOR labeling with AgN03 were per­formed following previously publishedtechniques (Schmid et al., 1983; Schweiz­er, 1976). At least five metaphase plates

were analyzed from each of the differentbanding procedures and AgN03 labeling.

Karyotype Analysis

Microscopic analyses were completedon a Zeiss Axiophot microscope equippedwith HBO 50-W mercury lamp illumina­tion. Images of specific fluorescence stains(quinacrine mustard, Hoechst 33258,mithramycin Aldistamycin) were obtainedby exciting with UV light in the 450-490nm range (filters BP450IFT510/LP520).The DAPI fluorescence was viewed underexcitation with 360--400 nm UV light (fil­ters G365/FT395/LP420). Photographswere taken using Agfaortho 25 ASA film.Mitotic karybtypes for each banding tech­nique were prepared from each animal.For detailed analysis, chromosome cut­outs were prepared of all karyotypes andsorted by chromosome size. Cut-outs werethen affixed to double-sided tape in par­allel rows, to white cardboard for conven­tionally stained chromosomes and to blackcardboard for fluorescence-stained chro­mosomes. This system allows excellentcomparisons of inter-individual metaphasedifferences as well as of differences be­tween individuals and species.

After the final figures of AgN03-stainedkaryotypes had been prepared, they, aswell as figure 3 of Rada de Martinez(1976), were scanned at high resolutionand imported into Adobe Photoshop 6.0on an iMac personal computer. In Pho­toshop, lines were drawn in a separategraphiC layer over the chromosomes in theoriginal scans to obtain accurate length es­timates. The layer with the lines was im­ported into Aldus Superpaint 3.5 for theMacintosh, and idiograms were then handdrawn on the computer. Chromosomelength ratios and relative genome sizes (ascalculated from total chromosome lengths)were determined by comparing the pho­tographs of metaphases with a 10-l-LmZeiss objective micrometer photographedat the same magnification. Throughout, wefollowed the chromosome terminology ofGreen and Sessions (1991).

206 HERPETOLOGICA [Vol. 59, No.2

RESULTS

Gross Chrorrwsomal Morphology

Five of the six karyotyped species had abasic complement of 2n = 24 chromo­somes (Figs. 1-5). Although karyotype ar­rangements differed in the numbers ofmeta-, submeta-, subtelo-, and telocentricchromosomes in each complement, thevirtual absence of telocentric chromo­somes was conspicuous (Tables 2, 3). In­variably, chromosome No.1 was metacen­tric and comprised over 15% of the totalgenome (Table 3). The remaining chro­mosome pairs assorted primarily into thesubmetacentric and metacentric catego­ries, with the exception of chromosomeNo.4, which was subtelocentric in all spe­cies except M. trinitatis. In all examinedspecies, genome distribution was skewed,with the first six chromosomes holding inexcess of 70% of the genetic material (Fig.5, Table 3). The single examined specieswith 2n = 22· chromosomes, one telocen­tric chromosome pair (No.7), and no sub­metacentrics was C. chalcopis.

We observed considerable discrepanciesbetween the karyotypes of the Trinidadianpopulations of M. trinitatis and those fromthe vicinity of Caracas, Venezuela, as fig­ured by Rada de Martinez (1976). Where­as in Trinidadian M. trinitatis, chromo­some No.4, 6, and 8 are submetacentricin the Caracas population it is chromo~some No. 3-6 and 11 (Fig. 6; Tables 2, 3)that have a submetacentric centromereposition. In chromosome No.4, 5, 7, and9, both the short and long arm of the chro­mosomes are longer in the Trinidadianpopulations than in the chromosomes ofthe frogs from Caracas (Fig. 6).

The genomes of M. olrrwnae and M.trinitatis are distinct not only in detailedchromosomal morphologies but also interms of gross morphology. Relative ge­nome size (as expressed by chromosomelength) in M. trinitatis is nearly 21% lessthan in M. olrrwnae. The telocentric chro­mosome No. 4 in M. olmonae has no sub­telocentric counterpart in M. trinitatis,where chromosome No. 4 is submetacen­tric. Furthermore, the two remaining sub­metacentric chromosomes in M. olmonae

(No.3 and 5) are much larger (30.7% ofthe genome; Table 3) than those in Trini­dadian M. trinitatis (No.6 and 8; 19.5%of the genome; Table 3).

Euchromatin and Heterochromatin

Metaphase spreads stained with quina­crine primarily fluoresced uniformly. Twoof the six examined species (C. leopardalis,M. neblina) had brightly fluorescing cen­tromeric regions, indicative of tightly com­pressed constitutive heterochromatin (Fig.2b,e; Table 4). Three other species (M.henninae, M. olmonae, M. trinitatis) hadcentromeric regions fluorescing with thesame intensity as the other portions of thechromosomes (Figs. 1b,e, 2h), and in C.chalcopis, centromeric regions were en­tirely Q- (Fig. Ih). There were only twospecies with Q-band heteromorphisms.Colostethus chalcopis possesses threesmall, brightly fluorescing centric regionson the long arm of chromosome No.1, 4,and 7 (Fig. 1h; Table 4), whereas M. 01­monae had bright Q+ centric regions onthe long arm of chromosome No. 1 and onthe short arm of chromosome No.5 (Fig.Ib; Table 4). None of the other speciespossessed visible Q+ heterochromatic sites.The Q- regions exist in the position of theNORs of M. olmonae, M. henninae, andM. neblina (Figs. Ib, 2e,h; Table 4).

In all six species, centromeric regionsw~re stained darkly during C-banding(FIgS. 1, 2). Heteromorphisms in C-banddistribution were evident in only three ofthe examined species (C. chalcopis, M.henninae, M. neblina; Table 4). In C. chal­copis, C-bands exist in the centromeric re­gion on the long arms of chromosome No.1 and 4 (Fig. Ig). Both M. neblina and M.henninae possess terminal C-bands on thelong arm of chromosome No.6, and M.herminae has an additional band in thecentromeric region of chromosome No.7(Fig. 2d,g).

Nucleolus Organizer Regions (NORs)

There is no uniformity in placement ofNORs among the species studied. TheNOR of M. olmonae (Fig. 3a,a') and Trin­idadian M. trinitatis (Fig. 3c,c') are near

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HERPETOLOGICA

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207

FIG. I.-C-bands (a, d, g), quinacrine mustard fluorescence (b, e, h), and DAPI fluorescence (c, f, i) ofMannophryne olmonae (a, b, c), M. tnnitatis (d, e, 0, and Colostethus chalcopis (g, h, i). Note that thekaryotype of C. cha1copis only comprises 11 chromosome pairs.

208 HERPETOLOGICA [Vol. 59, No.2

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FIG. 2.---C-bands (a, d, g), quinacrine mustard fluorescence (b, e, h), and DAPI fluorescence (c, f, i) ofColostethus leopardalis (a, b, c), Mannophryne neblina (d, e, 0, and M. herminae (g, h, i).

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FIG. 3.-AgN03 stains identiJYing nucleolus organizer regions (a, a', c, c', e, e') and distamycin Almithra­mycin fluorescence (b, b', d, d', f, r) of Mannophryne olmonae (a, a', b, b'), M. trinitatis (c, c', d, d'), andColostethus chalcopis (e, e', f, f').

210 HERPETOLOGICA [Vol. 59, No.2

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FIG. 4.-AgN03 stains identifying nucleolus organizer regions (a, a', c, c', e, e/) and distamycin A/mithra­mycin fluorescence (h, h', d, d', f, f) of Colostethus leopardalis (a, a', h, h'), Mannophryne neblina (c, c', d,d'), and M. henninae (e, e ' , f, f).

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HERPETOLOGICA

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1 2 3 4 5 6 7 8 9 101112

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CHROMOSOME PAIR NUMBER

FIG. 5.-Computer drawn idiograms of two Colostethus species and four Mannophryne species. The y-axisrepresents the percentage of total genome length. In the position of the secondary constriction, nucleolusorganizer regions are indicated by small black circles. Colostethus chalcopis has only 11 chromosome pairs.

the centromere on the long arm of chro­mosome No.5, whereas they are near thecentromere on the short arm of chromo­some No.7 in M. herminae (Fig. 4e,e')and M. neblina (Fig. 4c,c'). Caracas pop­ulations of M. trinitatis have an NOR inthe long arm of chromosome No.6 (Rada

de Martinez, 1976). The NOR in C. chal­copis occupy an interstitial position on theshort arm of chromosome No. 5 (Fig.3e,e'), and they are found in a nearly ter­minal position on the short arm of chro­mosome No. 4 in C. leopardalis (Fig.4a,a').

212 HERPETOLOGICA [Vol. 59, No.2

TABLE 2.-Gross chromosomal morphologies of Colostethus and Mannophryne species karyotyped in thisstudy and of M. trinitatis from Rada de Martinez (1976). Morphotype arrangements follow Green and Sessions(1991) and are abbreviated as follows: t (telocentric), st (subtelocentric), sm (submetacentric), m (metacentric).

Species 2n st sm m

Colostethuschalcopis 22 7 4 1-3,5,6,8-11leopardalis 24 4,5 2,3,6,8,9 1,7,10-12

Mannophrynehenninae 24 4 5,6,9,12 1-3,7,8,10,11neblina 24 4 3,8,10,12 1,2,5-7,9,11olrnonae 24 4 3,5 1,2,6-12trinitatis* 24 4,6,8 1-3,5,7,9-12trinitatis t 24 3-6, 11 1,2,7-10,12

* Specimens from Trinidad (this study).t Specimens from Caracas, Venezuela (Rada de Martinez, 1976).

Fluorescence Staining with DAPIand DA/M

Staining with D.JM confirmed the po­sitions of the NORs in all species withbright fluorescence (Figs. 3b,d,f, 4b,d,f).All centromeres were DA/M-. There ap­peared to be no visible polymorphism afterDJM treatment of the karyotypes. TheDAPI treatment resulted in bright fluores­cence of the centromeres in all species(Figs. lc,f,i, 2c,f,i).

DISCUSSION

Chrorrwsomal Diversity withinPhylogenetically Basal DendrobatidsTaking all known chromosome infor­

mation for frogs in the basal dendrobatidgenera Colostethus and Mannophryne to­gether, two facets of their karyotypes be­come apparent: (1) a high degree of chro­mosome number conformity (2n = 22 or24) with few telocentric chromosomes and(2) occurrence of only limited banding di­versity. We therefore believe the chromo­some complement of basal dendrobatidsto be quite conserved evolutionarily andaltered only slightly, mainly by rearrange­ments (such as inversions) but not muchby chromosome fusions or fissions; thus,telocentric chromosomes are relativelyrare and the chromosome number is con­strained to the 22- or 24-chromosomecomplement. Even in the only species ofColostethus karyotyped to date that pos­sesses a substantial number of telocentric

chromosomes (C. subpunctatus has fivetelocentric chromosomes; Bogart, 1991),the chromosome complement is still 2n =24. This is very different than the situationin some leptodactylids (e.g., Eleutherodac­tylus) , where chromosome complementsrange from all telocentric 2n = 36 karyo­types to those with all metacentric chro­mosomes and 2n = 18 chromosomes (seeBogart, 1991 for a discussion of Eleuther­odactylus karyotypes). The 2n = 22 chro­mosome complement in Colostethus wasreported only recently by Veiga-Manon­cello et ale (2001) for C. caeruleodactylus,C. marchesianus~ and two as yet unde­scribed species with affinities to C. mar­chesianus. The karyotype of C. chalcopisdescribed herein is the first of the 2n =22 karyotype Colostethus to be figured andtreated with fluorescence stains. There isno apparent sex chromosome pair amongany dendrobatid chromosome comple­ments examined to date, and there hasbeen no Significant extracentromeric ac­cumulation of constitutive heterochroma­tin in any of the genomes studied here orreported in the literature. In fact, most ofthe chromosome banding resulted in ahighly uniform stain rather than highlyvariable banding patterns. In the case ofQ-bands, this might be due to the highdegree of contraction of the chromosomesduring metaphase rather than due to a lackof bands (Schmid, 1978).

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TABLE 3.-Comparative haploid genome analyses for two species of Colostethus, four species of Mannophry­ne, and for a Venezuelan population of M. trinitatis reported by Rada de Martinez (1976). Values given arepercentage genome length and centromeric ratio (length of short arm:length of long arm) in parentheses foreach chromosome in the karyotype. Nomenclature of centromeric positions follows Green and Sessions (1991)

and is abbreviated as in Table 2.

Chromo- Colostethus Mannophrynesome

number chalcopis leopardalis neblina henninae olmonae trinitatis* trinitatist

1 16.4 (1.33) 17.9 (1.28) 19.2 (1.17) 18.0 (1.24) 17.8 (1.18) 15.7 (1.29) 16.3 (1.17)m m m m m m m

2 15.8 (1.42) 13.2 (1.99) 14.7 (1.46) 12.7 (1.42) 13.6 (1.27) 13.6 (1.34) 13.8 (1.54)m sm m m m m m

3 12.7 (1.57) 12.6 (2.40) 13.1 (1.89) 11.4 (1.54) 12.3 (2.05) 12.9 (1.59) 12.0 (2.42)m sm sm m sm m sm

4 11.3 (3.45) 11.5 (3.32) 10.7 (5.37) 11.3 (3.21) 12.3 (4.19) 11.6 (2.14) 11.1 (2.18)st st st st st sm sm

5 9.9 (1.33) 10.9 (3.00) 10.7 (1.52) 11.0 (2.26) 11.3 (2.05) 11.2 (1.20) 10.2 (1.74)m sm m sm sm m sm

6 7.9 (1.41) 10.9 (1.96) 10.2 (1.52) 9.8 (1.69) 9.8 (1.28) 9.5 (1.93) 9.9 (1.84)m sm m sm m sm sm

7 6.8 (0) 4.7 (1.39) 4.3 (1.00) 6.1 (1.08) 4.8 (1.28) 5.6 (1.23) 5.7 (1.27)t m m m m m m

8 5.3 (1.61) 4.2 (1.77) 4.2 (1.78) 5.2 (1.29) 4.1 (1.21) 5.0 (2.09) 5.2 (1.11)m sm sm m m sm m

9 4.7 (1.45) 4.0 (2.71) 3.8 (1.50) 4.7 (2.33) 4.1 (1.39) 4.5 (1.34) 4.4 (1.35)m sm m sm m m m

10 4.6 (1.19) 3.7 (1.16) 3.5 (1.86) 4.2 (1.37) 3.6 (1.35) 3.7 (1.24) 4.3 (1.57)m m sm m m m m

11 4.6 (1.24) 3.7 (1.23) 3.1 (1.48) 4.1 (1.30) 3.5 (1.04) 3.7 (1.22) 3.7 (1.68)m m m m m m sm

12 2.8 (1.11) 2.5 (1.70) 3.9 (2.17) 2.8 (1.00) 3.0 (1.00) 3.4 (1.46)m sm sm m m m

* Specimens from Trinidad (this study).t Specimens from Caracas, Venezuela (Rada de Martinez, 1976).

The Problem with Mannophryne trinitatis

We observed unexpected differencesbetween the karyotype of Trinidadian M.trinitatis reported here and populations ofthat species from near Caracas, Venezuela.Some of these differences can be resolvedby careful examination of the chromosom­al nomenclature used by us and by Radade Martinez (1976). In her description ofthe M. trinitatis karyotype, Rada de Mar­tinez (1976) used a nomenclature differentfrom the one proposed by Green and Ses­sions (1991) that we use herein. Rada deMartinez (1976) used the nomenclatureproposed by Levan et al. (1964), whichdoes not define the category CCsubtelocen­tric." Consequently, centromeric ratios(Table 3) are grouped into slightly differ­ent categories. Using our scanning andmeasuring technique, we remeasured thekaryotype presented by Rada de Martinez(1976), recalculated centromeric indices,

and reassigned chromosomal categorieswhere appropriate. This examination re­vealed that six of the centromeric ratiosshown in figure 3 of Rada de Martinez(1976) differ by 5-34% from actual valuesbased on the printed karyotype, thoughthis did not change chromosomal assign­ments. Rada de Martinez (1976) enumer­ated four submetacentric (3-6,11) andeight metacentric pairs, whereas we listonly three submetacentric (4,6,8) and ninemetacentric chromosome pairs (Table 2).Furthermore, Rada de Martinez (1976)figures the secondary constriction (and thelocation of the NOR) on chromosome No.5 in her idiogram; however, this chromo­some is actually the sixth largest chromo­some in that figure. In Trinidadian popu­lations of M. trinitatis, the secondary con­striction is on chromosome No.5 and noton chromosome No. 6 as in the Caracaspopulation. Several other differences be-

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CHRO 050 E PAIR NU BERFIG. 6.-Comparison of computer drawn idiograms of Mannophryne trinitatis from near Caracas, Venezuela

(dark gray) and from the region of the type locality of the species (the Northern Range of Trinidad; lightgray). The y-axis represents the percentage of total genome length. In the position of the secondary constric­tion, nucleolus organizer regions are indicated by small black circles. Centromeric position assignment ac­cording to centromeric ratios (Table 3) is provided underneath the chromosome number for each chromo­some.

TABLE 4.-0ccurrence of Q-bright centromeres, Q- and C-band heteromorphisms, and Ag-NORs among theexamined dendrobatid species and of Mannophryne trinitatis from Rada de Martinez (1976). Abbreviationsare used as follows: p = short arm, q long arm, cen = centric or pericentric, int = interstitial, ter =

terminal.

Species

Colostethuschalcopisleopardalis

Mannophryneherminaeneblinaolnwnaetrinitatis*trinitatist

Q-brightcentromeres

++

+

Q-bandheteromotphisms

lq",., 4q",., 7q",.

lq....,6p",.

C-bandheteromorphisms

lq",.,4q=

6q,en 7p",.6qter

Position ofAg-NOR

7p",.7p=5q",.Sq=6q",•

• Specimens from Trinidad (this study).f Specimens from Caracas, Venezuela (Rada de Martfnez, 1976).

June 2003] HERPETOLOGICA 215

come clear in detailed length comparisons(Fig. 6).

It is evident that there are some signif­icant differences in chromosome mor­phology between the M. trinitatis karyo­type described by Rada de Martinez(1976) and the one we describe. Onemight explain such differences by invokingtechnical challenges that could have pre­vented determination of accurate sizes.However, in giving other chromosomeworkers the benefit of the doubt, we con­sider it more likely that we are observinga real signal in these karyotypes, indicatingpopulation- or even species-level diver­gence: the specimens of Rada de Martinez(1976) came from the vicinity of Caracas,Venezuela, whereas ours came from the is­land of Trinidad, several hundred miles tothe east. Observation of such chromosom­al differences in these disjunct M. trinitatispopulations (and others in the VenezuelanCordillera de la Costa, the mountains ofthe Peninsula de Paria, and the Northemand Central Ranges of Trinidad) raises thelikelihood of multiple cryptic species inwhat is now considered M. trinitatis. Re­gardless, the name applied to Mannophry­ne populations occurring in the NorthemRange of Trinidad will remain M. trinita­tis, as that is the type locality for the spe­cies. Until additional data emerge, we rec­ommend that trinitatis-like populationsfrom other areas, speCifically from Trini­dad's Central Range and from Venezuela'scoastal cordillera, that are currently con­sidered conspecific with M. trinitatis bereferred to as Mannophryne cf. trinitatis.

The Distinctiveness ofMannophryne olmonae

Hardy (1983) applied the name Colos­tethus olnwnae to Mannophryne popula­tions on Tobago, thus separating them tax­onomically from M. trinitatis and identi­fying them as a distinct, single-island, en­demic species. Based on our experiencewith both Trinidad and Tobago popula­tions of Mannophryne in the field, as wellas with their detailed morphology and os­teology, we agree with the distinctivenessof M. olnwnae. However, the initial speciesdescription did not include sufficient com-

parative information, especially with re­spect to the phenotypic variability of To­bago and Trinidad populations of Manno­phryne, to remain uncontroversial. In thatregard, it is also unfortunate that the spe­cies description appeared in a journal thatdoes not routinely submit manuscripts torigorous peer review. In his excellent ac­count of the Trinidad and Tobago herpe­tofauna, Murphy (1997) suggested infor­mally that M. olnwnae might be a juniorsynonym of M. trinitatis, but withheld aformal synonymy pending further evi­dence.

Our chromosome banding data showunequivocally that M. olnwnae deservesrecognition at the species level. Grosschromosomal differences, such as thepresence of the subtelocentric chromo­some No.4, as well as banding differences,such as the presence of Q-band polymor­phisms on chromosome No.1 and 6, aregood indicators that the karyotypes of M.olnwnae and M. trinitatis are evolving in­dependently and divergently. Further­more, the haploid genome of M. olnwnaeis 20.8% larger than that of M. trinitatis,and it is highly unlikely that meiotic pair­ing of karyotypes with such size differenc­es would be successful. Clearly, isolation ofthe Tobago Mannophryne populationsfrom those found on Trinidad has resultedin an accumulation of genetic changes, aswell as some less well developed morpho­logical ones, in a classic case of allopatricspeciation.

The Problem with Colostethus chalcopis

Colostethus chalcopis was described byKaiser et al. (1994), who speCifically statedthat the generic placement of their newtaxon in Colostethus was provisional inview of a suite of morphological and lifehistory characteristics of this unusual frog,including its clutch size and tadpole mor­phology (Kaiser and Altig, 1994). Accord­ing to Veiga-Menoncello et ale (2001), the22-chromosome karyotype might indicatea distinct group among Colostethus. Theonly 22-chromosome dendrobatid previ­ously known was Minyobates (now Den­drobates) ophistomelas (Bogart, 1991).Chromosomally, these data might there-

216 HERPETOLOGICA [Vol. 59, No.2

fore align the nontoxic C. chalcopis witheither a Brazilian assemblage of Coloste­thus or some of the toxic species of Den­drobates (formerly Minyobates); either ofthese options is highly questionable. Den­drobates appears to be a highly derivedtaxon among Dendrobatidae (Clough andSummers, 2000; Vences et al., 2000) withchromsome numbers including 2n = 18,2n = 20, and 2n = 22. Further data areneeded to determine whether 22-chro­mosome Amazonian Colostethus form amonophyletic assemblage, but, for such agroup, C. chalcopis forms a rather improb­able biogeographic outlier on the 1500­km-distant island Martinique. Among spe­cies groups currently recognized withinColostethus (Grant et al., 1997) or amongthe basal dendrobatids, C. chalcopis mightbe most closely affiliated with the morpho­lOgically poorly defined genus Manno­phryne, a genus based on the former C.collaris group (La Marca, 1992, 1994).However, among the characteristics defin­ing Mannophryne are the following: ~~in fe­males presence of a discrete throat collar,accompanied by a white venter and a yel­low throat; in males a dark venter" (H.Kaiser's translation from the original Span­ish in La Marca, 1992). Colostethus chal­copis does not fit this definition since theentire female venter is yellow and sincemales also have a throat collar. Additionaltaxa, such as M. riveroi, have similarlyproblematic coloration with respect to thegeneric definition, as was already pointedout by Myers et aI. (1991). Furthermore,webbing in C. chalcopis is absent exceptfor a very slight remnant between toes IIIand IV (Kaiser et aI., 1994), whereas allspecies of Mannophryne have some de­gree of webbing between all toes (La Mar­ca, 1994). Thus, the placement of C. chal­copis among the nontoxic dendrobatidsshould be considered uncertain.

Chromosome Data and the GeneraColostethus and Mannophryne

Based on gross chromosomal morphol­ogy, the four species of Mannophryne andtwo species of Colostethus examined canbe differentiated by chromosome comple­ment, C. chalcopis being the only species

with 2n = 22 chromosomes. The positionof the NOR is more instructive. Amongspecies of Mannophryne, M. herminae andM. neblina have NORs on chromosomeNo.7, whereas M. olmonae and Trinida­dian M. trinitatis have their NORs onchromosome No.5. In M. cf. trinitatisfrom Caracas, Venezuela (Rada de Marti­nez, 1976), the NOR is on chromosomeNo.6. Furthermore, NOR position in re­lation to the centromere among these fourspecies assorts them identically: in M. her­minae and M. neblina, the NOR is on theshort arm of the chromosome; whereas, inM. olmonae, Trinidadian M. trinitatis, andM. cf. trinitatis from Caracas, the NORslie on the long arm. The NOR position inC. chalcopis and C. leopardalis is also dif­ferent, on chromosome No.5 and 4, re­spectively. Mannophryne herminae and M.neblina can also be grouped by a terminalC-band heteromorphism on the long armof chromosome No.6. Chromosome dataderived from NOR position may thereforeindicate two hitherto unrecognized group­ings in the genus Mannophryne, one com­prising M. herminae and M. neblina andthe other M. olmonae and M. trinitatis. Weconsider M. cf. trinitatis to be part of thelatter grouping based on (1) the positionof the NOR on the long arm of the chro­mosome and (2) the fact that transfers ofsmall amounts of genetic material near thetermini of the chromosome arms duringchrossing-over in meiosis can easily ac­count for the differences between chro­mosome No.5 and 6.

Chromosome Data andDendrobatid Phylogeny

Two dendrobatid phylogenies based onmolecular evidence were published re­cently (Clough and Summers, 2000;Vences et al., 2000). Although the analysisby Clough and Summers (2000) is not in­formative regarding placement of Colos­tethus and Mannophryne, their two ex­amined species of Colostethus (C. mar­chesianus, C. talamancae) form an unre­solved basal polytomy with the remainingdendrobatids. Vences et al. (2000) alsochose C. talamancae and added C. bocageiand C. cf. trilineatus. Their analysis con-

June 2003] HERPETOLOGICA 217

firmed the placement of C. talamancae asa taxon basal to other dendrobatids, butmore closely related to Allobates femoralisthan in the tree of Clough and Summers(2000). Colostethus bocagei nested withinan Epipedobates clade (Vences et aI.,2000), and this group requires some moredetailed analysis.

Based on the chromosomal informationavailable for dendrobatids and in view ofcurrent molecular hypotheses, it is appar­ent that the 2n = 22 karyotype has evolvedat least twice within the family, onceamong the basal taxa (in Colostethus) andonce among the derived, lipophilic-alka­loid producing species (the Dendrobatesbranch including some taxa formerlyplaced in the genus Minyobates and sup­ported by both published molecular phy­logenies). Furthermore, it seems as if theancestral dendrobatid karyotype is bestrepresented by the 2n = 24 chromosomecomplement. This karyotype exists amongColostethus (Bogart, 1991; this study),Epipedobates (Bogart, 1991; Aguiar-Junioret al., 2002), Mannophryne (Rada de Mar­tinez, 1976; this study) and Phyllobates(unpublished data). The dendrobatid cladethat includes the most highly derived den­drobatid frogs (Dendrobates; Vences et aI.,2000) seems to have undergone a reduc­tion in chromosome number. The reducedchromosome numbers neatly support thegroupings derived through the molecularapproach: the chromosome complementof 2n = 22 is characteristic of the cladecomprising the hitherto molecularly ex­amined species of the former genus Min­yobates and several unkaryotyped Dendro­bates (though an analysis of those closelyrelated Dendrobates species is essential toverify this), 2n = 20 lends support to theclade containing D. pumilio and D. his­trionicus (Clough and Summers, 2000),and 2n = 18 strengthens the already wellsupported arrangement of the clade con­taining D. auratus (Clough and Summers,2000; Vences et al., 2000).

Based on our data, it is apparent thatdetailed banding analyses are not very use­ful in elUCidating intergeneric or even in­terspecific relationships within Dendroba­tidae for lack of characters. However, gross

chromosomal morpholOgies are undoubt­edly useful in strengthening some of themolecular hypotheses currently available.As such hypotheses are refined, chromo­somal data may prove instructive instreamlining the process of molecularanalysis by confirming larger groupingsand by prOviding data based suggestionson the scope of taxa to add next to thecontinuing molecular analyses.

Acknowledgments.-We thank E. La Marca and A.Fernandez Badillo for faCilitating the stay of M.Schmid and W. Feichtinger in Venezuela; H. M.Gray, H. H. Schwarten, and T. F. Sharbel for ableassistance in the field; and M. Vences, D. M. Green,and three anonymous reviewers for comments on themanuscript. This research was supported in part bygrants from the Deutsche Forschungsgemeinschaft toH. Kaiser and M. Schmid and by a research fellow­ship from Boehringer Ingelheim Fonds and grantsfrom the College of Arts and Sciences and the Uni­versity Research Council at La Sierra University toH. Kaiser.

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Accepted: 15 October 2002Associate Editor: David Green