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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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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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11 12
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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).
June 2003)
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M. herminae
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M. neblina
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1 2 3 4 5 6 7 8 9 101112
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1 2 3 4 5 6 7 8 9 1011 12
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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-~- 4J:...C)Z~ 0W
ozW 4C)
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2m
3 4smlm sm
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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