Large Metacentric Pair Research Articles

Chromosomes of three gall wasps of the tribe Aylacini (Hymenoptera, Cynipidae).

Chromosomes of two species of the tribe Aylacini (Cynipidae), Isocolus jaceae (Schenck, 1863) and I. scabiosae (Giraud, 1859) (both have 2n = 18) were studied for the first time. In addition, 2n = 20 is confirmed in a member of the same tribe, Aulacidea hieracii (Bouché, 1834). All chromosomes of these gall wasps are biarmed; however, they gradually decrease in size in the case of A. hieracii, whereas a pair of large metacentrics is characteristic of karyotypes of both Isocolus Förster, 1869 species. Chromosomes of the two latter gall wasps are either metacentric or submetacentric, but elements with lower centromeric indices prevail in the karyotype of A. hieracii. Chromomycin A3 (CMA3)/DAPI staining revealed single CMA3-positive bands on a particular pair of chromosomes of all species, and these bands apparently refer to the nucleolus organizing regions (NORs). However, localization of CMA3-positive bands differs substantially between the studied members of Isocolus and Aulacidea Ashmead, 1897. Together with normal haploid and diploid mitotic divisions, several metaphase plates with 2n = 17 containing a peculiar dicentric chromosome were found in a single male specimen of I. scabiosae; this appears to be the first report of an obvious dicentric in the order Hymenoptera in general. Certain aspects of the chromosome diversity and karyotype evolution within the family Cynipidae and the tribe Aylacini in particular are briefly discussed.

A Comprehensive Integrated Genetic Map of the Complete Karyotype of Solea senegalensis (Kaup 1858).

Solea senegalensis aquaculture production has experienced a great increase in the last decade and, consequently, the genome knowledge of the species is gaining attention. In this sense, obtaining a high-density genome mapping of the species could offer clues to the aquaculture improvement in those aspects not resolved so far. In the present article, a review and new processed data have allowed to obtain a high-density BAC-based cytogenetic map of S. senegalensis beside the analysis of the sequences of such BAC clones to achieve integrative data. A total of 93 BAC clones were used to localize the chromosome complement of the species and 588 genes were annotated, thus almost reaching the 2.5% of the S. senegalensis genome sequences. As a result, important data about its genome organization and evolution were obtained, such as the lesser gene density of the large metacentric pair compared with the other metacentric chromosomes, which supports the theory of a sex proto-chromosome pair. In addition, chromosomes with a high number of linked genes that are conserved, even in distant species, were detected. This kind of result widens the knowledge of this species’ chromosome dynamics and evolution.

Karyotype diversity between species of Crenicichla (Perciformes, Cichlidae) from different Brazilian hydrographic basins.

Crenicichla is the largest genus in the Cichlidae family in South America. The genus includes 100 valid species that are popularly known in Brazil as jacundás or joaninhas and are widely distributed in rivers east of the Andes. Cytogenetic analyses were carried out on seven species in this genus. All species showed a diploid number of 48 with interspecific differences in karyotype formulas and AgNORs located in interstitial position on the short arm of the largest metacentric pair, except for the two populations from C. britskii. Population A showed terminal markings on the long arm of the fifth pair of the complement, and population B showed up to two marked chromosome pairs. FISH with an 18S rDNA probe was coincident with AgNORs and CMA3, except for pair 6 from population B of C. britskii that did not presented positive CMA3 sites. This work presents first cytogenetic data for C. haroldoi, C. maculata, and C. punctata, and the results show karyotypic patterns similar to those in the literature. However, the diversity found in populations of C. britskii represents new information about the evolution of the karyotype of the Cichlidae family, which has been conservative. Furthermore, the data could assist in phylogenetic studies of Crenicichla.

G-banding of the genus Rattus (rodent, Muridae) in Tunisia, occurrence of different banding of the sexual chromosome Y in the norvegicus species

The two species of rats Rattus rattus and Rattus norvegicus are the only representatives of the genus Rattus in Tunisia. R. rattus is represented by a karyotye 2n = 38 to 39 chromosomes, it belongs to the oceanic type, whereas R. norvegicus has a karyotype of 2n = 42 chromosomes. The G-bands produced by these two species are specific ; the ideogram representation of the chromosomes shows 136 bands for R. rattus and 189 bands for R. norvegicus, the number of the bands for each species is lower than those identified in Europe. Based on the comparison of the various G-banding profiles, we can confirm that for R. rattus, the integration of the acrocentrics pairs 4/ 7 and 11/ 12 by two robertsonian fusions and the two pericentric inversions of acrocentric pairs 1 and 9, who have infected the asian type, give respectively the two large metacentric pairs and the two subtelocentric pairs (1 and 9) characteristic of the oceanic type and the B chro- mosome is completely heterochromatic. For R. norvegicus, the change of the position between the dark and clear bands of the sexual chromosome Y is the remarkable opposite of the one identified in Europe.

Analysis of the histone cluster in Senegalese sole (Solea senegalensis): evidence for a divergent evolution of two canonical histone clusters.

The Senegalese sole (Solea senegalensis) is commercially very important and a priority species for aquaculture product diversification. The main histone cluster was identified within two BAC clones. However, two replacement histones (H1.0 and H3.3) were found in another BAC clone. Different types of canonical histones H2A and H2B were found within the same species for the first time. Phylogenetic analysis demonstrated that the different types of H1, H2A, and H2B histones were all more similar to each other than to canonical histones from other species. The canonical histone H3 of S. senegalensis differs from subtypes H3.1 and H3.2 in humans at the site of residue 96, where a serine is found instead of an alanine. This same polymorphism has been found only in Danio rerio. The karyotype of S. senegalensis comprises 21 pairs of chromosomes, distributed in 3 metacentric pairs, 2 submetacentric pairs, 4 subtelocentric pairs, and 12 acrocentric pairs. The two BAC clones that contain the clusters of canonical histones were both mapped on the largest metacentric pair, and mFISH analysis confirmed the co-location with the dmrt1 gene in that pair. Three chromosome markers have been identified which, in addition to those previously described, account for 18 chromosome pairs in S. senegalensis.

Molecular and karyological identification and morphological description of cystocercous cercariae of Phyllodistomum umblae and Phyllodistomum folium (Digenea, Gorgoderidae) developing in European sphaeriid bivalves

Two cystocercous gorgoderid cercariae of the genus Phyllodistomum Braun, 1899, Phyllodistomum umblae (Fabricius, 1780) and Phyllodistomum folium (Olfers, 1816), developing in sphaeriid bivalves from Norway and Lithuania, were studied and compared. Our previous molecular studies revealed very close phylogenetic relation of these two species and proved that cystocercous cercariae of P. folium develop in sporocysts in the gills of sphaeriid bivalves. In the present study morphological descriptions are given for the two cercariae, together with karyological and molecular characteristics. Comparative karyological and sequence analysis using ITS2 and 28S rDNA revealed the conspecificity of cercariae emitted from Pisidium hibernicum and Sphaerium corneum with adult P. umblae from Thymallus thymallus and Coregonus albula. The cercariae of P. folium and P. umblae are very similar morphologically with main differences in the structure of the tail. The two species clearly differ in karyotype structure. The diploid set of P. folium is composed of 18 chromosomes, but 16 chromosomes with one pair of large metacentrics were found in mitotic cells of P. umblae. Interspecific karyotypic difference presumably arose from Robertsonian fusion of two uni-armed chromosomes.

First description of the karyotype and localization of major and minor ribosomal genes in Rhoadsiaaltipinna Fowler, 1911 (Characiformes, Characidae) from Ecuador.

Karyotypic features of Rhoadsia altipinna Fowler, 1911 from Ecuador were investigated by examining metaphase chromosomes through Giemsa staining, C-banding, Ag-NOR, and two-color-fluorescence in situ hybridization (FISH) for mapping of 18S and 5S ribosomal genes. The species exhibit a karyotype with 2n = 50, composed of 10 metacentric, 26 submetacentric and 14 subtelocentric elements, with a fundamental number FN=86 and is characterized by the presence of a larger metacentric pair (number 1), which is about 2/3 longer than the average length of the rest of the metacentric series. Sex chromosomes were not observed. Heterochromatin is identifiable on 44 chromosomes, distributed in paracentromeric position near the centromere. The first metacentric pair presents two well-defined heterochromatic blocks in paracentromeric position, near the centromere. Impregnation with silver nitrate showed a single pair of Ag-positive NORs localized at terminal regions of the short arms of the subtelocentric chromosome pair number 12. FISH assay confirmed these localization of NORs and revealed that minor rDNA clusters occur interstitially on the larger metacentric pair number 1. Comparison of results here reported with those available on other Characidae permit to hypothesize that the presence of a very large metacentric pair might represent a unique and derived condition that characterize one of four major lineages molecularly identified in this family.

Atlantic surgeonfishes bear only minor microstructural changes in highly derived karyotypes

Acanthurus is a representative and widespread genus of marine fish that plays a key role in ecological dynamics of coral reefs. Three species are common along coastal reefs of Western Atlantic: A. coeruleus, A. bahianus, and A. chirurgus. The cytogenetic patterns of these species indicated sequential steps of chromosomal rearrangements dating back 19–5 millions of years ago (M.a.) that accounted for their interspecific differences. A. coeruleus (2n=48; 2sm+4st+42a), A. bahianus (2n=36; 12m+2sm+4st+18a), and A. chirurgus (2n=34; 12m+2sm+4st+16a) share an older set of three chromosomal pairs that were originated through pericentric inversions. A set of six large metacentric pairs formed by Robertsonian (Rb) translocations found in A. bahianus and A. chirurgus and a putative in tandem fusion found in A. chirurgus are more recent events. In the present study, new cytogenetic data are being reported for these three Acanthurus species based on mapping of repetitive sequences such as ribosomal 18S and 5S rDNA and telomeric repeats to improve their karyotype evolutionary analyses. The lack of interstitial telomeric sequences (ITS) in spite of several centric fusions in A. bahianus and A. chirurgus might be related to the long period of time after their occurrence (estimated in 5M.a.). Furthermore, the homeologies among the chromosome pairs bearing ribosomal genes, in addition to other structural features, highlight large conserved chromosomal regions in the three species. Our findings indicate that macrostructural changes occurred during the cladogenesis of these species were not followed by conspicuous microstructural rearrangements in the karyotypes.

Chromosomal fusion in Brazilian populations of Trinectes inscriptus Gosse, 1851 (Pleuronectiformes; Achiridae) as revealed by internal telomere sequences (ITS)

Flatfish (Pleuronectiformes) are one of the few examples of marine fish with remarkable chromosomal variation. Nonetheless, cytogenetic data in species of this order are still scarce and population approaches have been neglected. Therefore, a detailed karyotypic survey was carried out for the first time in Trinectes inscriptus along northeastern and southeastern Brazil. All specimens shared 2n=42 with a karyotype composed of eight metacentric/submetacentric and 34 subtelocentric/acrocentric chromosomes, differing from the basal condition of marine teleosteans (48 acrocentric chromosomes). Heterochromatin was restricted to the pericentromeric region of most chromosomes, short arms of a few pairs and coincident with GC-rich sites at NORs. Fluorescence in situ hybridization (FISH) using 18S and 5S rDNA probes revealed synteny between both rDNA classes at pair 5 in all samples. Telomeric sequences were mapped at terminal sites of most chromosomes as well as at centromeric region of the second pair. The derived karyotype macrostructure reported in T. inscriptus (low 2n, presence of large biarmed chromosomes) has probably evolved from chromosomal fusions, as reinforced by the presence of internal telomere sequences (ITS) in a large metacentric pair, besides inversions and heterochromatin accumulation. Since this pattern of genomic organization was stable among collection sites (up to 1000km apart), we suggest these rearrangements have taken place in the beginning of species diversification.

Sequential steps of chromosomal differentiation in Atlantic surgeonfishes: evolutionary inferences.

Surgeonfishes are a species-rich group and a major biomass on coral reefs. Three species are commonly found throughout South Atlantic, Acanthurus bahianus, A. chirurgus, and A. coeruleus. In this paper, we present the first cytogenetic data of these species, revealing a sequential chromosomal diversification. A. coeruleus was characterized by a relatively conserved karyotype evolved by pericentric inversions of some pairs (2n = 48, 2sm + 4st + 42a). In contrast, the karyotypes of A. bahianus (2n = 36) and A. chirurgus (2n = 34) were highly differentiated by the presence of six large metacentric pairs in A. bahianus (12m + 2sm + 4st + 18a) and A. chirurgus (12m + 2sm + 4st +1 6a) probably derived by chromosomal fusions that corroborate their closer relationship. A discernible in tandem fusion represents an autapomorphic character to A. chirurgus. In spite of macrostructure variation, single nucleolar organizer regions (NORs) on short arms of a subtelocentric pair and similar distribution of C-bands were observed in the three species. Overlapping of chromosomal data with molecular phylogeny indicated pericentric inversions which took place nearly at 19 Ma while centric fusions are as recent as 5 Ma. A physical mapping of coding and noncoding sequences in Acanthurus could clarify the role of additional rearrangements during their chromosomal evolution.

Karyological analysis of the shrimp Palaemon serratus (Decapoda: Palaemonidae)

Analysis of metaphases of Palaemon serratus (Pennant, 1777) revealed a diploid number of 2n=56 chromosomes with a karyotype composed of two large metacentric pairs, six subtelocentric pairs and twenty telocentric pairs. Four Ag-NORs and 45S rDNA signals were observed at the telomeres of two small telocentric chromosome pairs. Chromomycin A3 fluorochrome staining revealed GC-rich regions associated with Ag-NORs, and DAPI showed positive bright bands on centromeric or intercalary positions of subtelocentric and telocentric chromosomes.

First report of chromosomes of the parasitoid wasp Trissolcus basalis (Wollaston) (Hymenoptera: Platygastridae: Telenominae)

AbstractHere we present the first data on the chromosome complement of Trissolcus basalis gathered in conjunction with the ongoing whole genome sequencing efforts for this species. The cytogenetic investigation revealed a diploid karyotype with 20 chromosomes, comprising one pair of large metacentrics and nine pairs of acrocentrics that gradually decrease in length.

Atlantic moonfishes: independent pathways of karyotypic and morphological differentiation

Fish of the genus Selene, known as lookdowns or moonfish, are one of the most morphologically derived groups of the family Carangidae, whose phylogenetic relationships are still largely unknown. In this study, we discuss karyoevolutionary aspects of three representatives of this genus from the Western Atlantic: Selene brownii (2n = 48; FN = 48), Selene setapinnis (2n = 46; FN = 48), and Selene vomer (2n = 48; FN = 50). Their body patterns were also investigated and compared to one another and in relation to two other species of different genera. Two mechanisms of karyotypic evolution seem to have acted in the diversification of this genus, namely pericentric inversions and centric fusions. Mapping of rDNA sequences showed that chromosome pairs bearing 5S rDNA sites are similar, whereas those bearing 18 rDNA sites are morphologically distinct while apparently also exhibiting interspecies synteny. Although the nucleolar organizer-bearing chromosomes are extremely efficient cytotaxonomic markers among Selene species, others cytogenetic patterns of these species are relatively conserved. Hybridization with telomeric probes (TTAGGG)n did not exhibit interstitial telomeric sites (ITS), especially in S. setapinnis, where, along with a reduction in diploid number, a large metacentric pair derived from centric fusion is present. Data obtained by geometric morphometrics enable a clear morphological distinction among the three species, as well as in relation to two other species of the genus Caranx and Oligoplites. Data obtained suggest that morphologic evolution in Selene species was primarily dissociated from visible changes that occurred at the chromosomal level.

Cytogenetic Mapping of rRNAs and Histone H3 Genes in 14 Species of Dichotomius (Coleoptera, Scarabaeidae, Scarabaeinae) Beetles

Standard cytogenetic analyses and chromosomal mapping of the genes for 18S and 5S rRNAs and histone H3 were performed in 14 species of beetles of the genus Dichotomius (Coleoptera, Scarabaeidae, Scarabaeinae). Conserved karyotypes with 2n = 18 and biarmed chromosomes were observed in all species. Moreover, the presence of a large metacentric pair (pair 1) was characteristic in the studied species, evidencing a remarkable synapomorphy for this genus, which probably originated by an ancient fusion of 2 autosomes while the ancestral sex-chromosome pair remained conserved. FISH showed that the 5S rRNA and histone H3 genes are located in the proximal region of pair 2, with the 2 genes co-located. However, the major rDNA cluster probed by the 18S rRNA gene mapped to 1–3 bivalents, being exclusively autosomal, associated with sex elements, or both. In most species, the major rDNA cluster was observed in pair 3, and it was frequently (64.3%) located in the distal region regardless of the chromosome. The conserved number and position of the 5S rDNA/H3 histone cluster seems to be an ancient pattern shared by all of the studied species. In contrast, the major rDNA clusters apparently tolerate distinct patterns of diversification in the karyotypes of the species that could be associated with small inversions, ectopic recombination, and transposition. Moreover, we reinforced the association/co-localization between the 5S rRNA and histone H3 genes in this group contributing thus to the knowledge about the chromosomal organization and diversification patterns of multigene families in beetles and insects.

Cytogenetic characterization of Brycon amazonicus (Spix et Agassiz, 1829) (Teleostei: Characidae) from Caicara del Orinoco, Venezuela

A cytogenetic analysis by conventional Giemsa staining, silver staining, C-banding, and fluorescence in situ hybridization (FISH) was carried out on Brycon amazonicus from Caicara del Orinoco, Venezuela. The karyotype of this species is characterized by the presence of 2n = 50 chromosomes, a karyotypic formula 22m+14sm+14st, and a fundamental number of 100 chromosomal arms. Nucleolar organizer regions (NORs) and 18S rDNA genes are located in the terminal regions of the long arms of the second pair of subtelocentric chromosomes, corresponding to pair 13. C-banding revealed heterochromatin distribution in only seven chromosome pairs. The largest metacentric pair (number 1) possesses a paracentromeric block equilocally distributed in both chromosome arms, whereas in pairs 12 to 17 positive C band blocks were located in the paracentromeric region of the long arm, close to the centromere. Analysis performed with 5S rDNA gene revealed a terminal site located on the short arm of one small submetacentric chromosome (pair 15) corroborating previous studies with this repetitive gene showing an apparent conservation of 5S rDNA in the genome of these fish species. The data obtained in this study reinforce the chromosomal conservativeness in the species of the genus Brycon, related not only to the macro-chromosomal structure (diploid number, karyotypic formula, and fundamental number), but also to the repeated DNAs, such as heterochromatic regions and ribosomal DNAs. These data will contribute to a better understanding of chromosomal evolution in both Brycon and Characidae fishes.

Mapping of Ribosomal DNA by FISH, EcoRI Digestion and Replication Bands in the Cardinalfish Apogon americanus (Perciformes)

Summary Reef fishes of the order Perciformes represent an interesting group for both genetic and cytogenetic studies as their evolutionary pattern, usually unclear, might range from dynamic to quite stable. A karyotype with 2n� 48 acrocentric chromosomes is considered basal for this group, whereas karyotypes with other formulae are regarded as derived. Some Perciformes families present a high karyotypic diversification such as Apogonidae, although cytogenetic reports in this family along South Atlantic were absent so far. In the present work, cytogenetic analyses were carried out in Apogon americanus from Northeastern Brazil, revealing a modal diploid number of 2n� 36 (12m� 6sm� 16st� 2a; FN� 70) and a remarkable presence of large metacentric pairs. The AgNORs were located on short arms of the 8th pair, at telomeric position, coincident with hybridization signals obtained by FISH using 18S rDNA probes. The 5S minor ribosomal genes are distributed in 2 chromosomal pairs, not syntenic to the major ribosomal subunits. Heterochromatic blocks are located mainly at pericentromeric position in most of chromosomal pairs. The early replication pattern showed longitudinal bands with both early and late replication in the heterochromatin segments, being very similar to the pattern obtained after chromosomal digestion with EcoRI restriction enzyme. The present data, coupled with previous reports in other species, suggest a karyotypic evolution mainly driven by Robertsonian fusions (Rb) and pericentric inversions. In spite of the structural rearrangements, some features in the DNA composition and specific regions, such as 5S and 18S rDNA sites, reflect symplesiomorphic karyotype traits, shared by other clades in Perciformes.

The karyotype ofCabassous unicinctus(Dasypodidae, Xenar-thra)

Abstract Armadillos was belonged to the Xenarthra Order, Dasypodidae family. This family has been comprising the largest number of genera and species among the Xenarthrans; eight (8) and 21, respectively. Two adult males of the species Cabassous unicinctus were analyzed in this study. Lymphocyte cultures from whole blood were used and the cells were then submitted to conventional staining by C- and Ag-NOR banding. Data regarding the number of chromosomes showed discrepancies among the species described in the literature. The 46 chromosomes observed were distributed in six large metacentric pairs, five medium submetacentric pairs, five medium and small metacentric pairs and six acrocentric pairs. The Y chromosome was classifed such as the smallest acrocentric of the group. The X chromosome was classifed as medium submetacentric, it considered atypical for mammals, because X was generally large size metacentric when it compared to the remaining karyotype. The diploid number reduction from 62 to 46 chromosom...

CYTOLGICAL AND ISOZYMES STUDIES OF GARLIC CULTIVATED IN EGYPT.

Generally It has been accepted that the apomictic nature of nature of garlic clones could affect its cytological and genetical features as a result of accumulation of certain somatic mutations. Thus, the aim of this study is to examine some of the cytological (karyotyping, mitotic index and mitotic abnormalities) and isozyme characteristics of five garlic genotypes cultivated in Egypt (Baladi , Chinese , Chinese , 30-4 , Chinese 31-1and Chinese 31-3).Relative chromosome lengths and centromere position of these genotypes revealed insignificant difference in their karyotype formula (5 large , 2 medium from submetacentric pair and one large metacentric pair no. 5). The percentage of mitotic index of Baladi genotype (8.28%) was not very much different from that of Chinese (12.08%) whereas it was significantly lower than those of Chinese 30-4 (15.6%),Chinese 31-1(18.18%)and Chinese 31-3 (16. 8%). Means of total chromosomal abnormalities (including anuepliody, break and stickiness) were remarkably lower in genotypes Baladi (7.64%) and Chinese (9.38%)than those of Chinese 30-4 (11.94%), Chinese 31-3 (11.2%). Statistical analysis revealed that there was a high correlation (0.77) between the mitotic index and the percentage of mitotic chromosome abnormalities. Isozymes [Acid phosphatase, Glutamate Oxaloacetate Transaminase (GOT), Peroxidase and Esterase) banding patterns were qualitatively compared. The banding patterns of esterase (2bands) and GOT (one band) were similar in all five genotypes. As for Pyroxidase, the four genotypes of the Chinese origin had two bands, whereas only one band was shown by the genotype Baladi that was similar in molecular weight to small band of Chinese genotypes. All studies genotypes of acid phosphatase showed one band, which was of a low molecular weight in all except in Chinese genotype where it was a high molecular weight and Chinese 31-1 in which both low and high molecular weight bands were found.

Comparative karyological analysis of three members of Allocreadiidae (Digenea): taxonomic and phylogenetic implications

Chromosome number and morphology were studied in three allocreadiid species from mitotic metaphases of intramolluscan stages using karyometric analysis. Bunodera luciopercae has a diploid number 2n=14, with two large pairs of metacentrics, one pair of submeta-subtelocentrics, and four pairs of acrocentrics. Karyotype of Allocreadium isoporum, 2n=14, with two large pairs of metacentrics and five pairs of acrocentrics was recognized to be closely related to that of B. lucioperca. The most conspicuous interspecific difference occurs in the centromere position of the chromosomes of the pair 3. The possible reasons of the discrepancies between data reported therein and the karyological information previously published on these two species are discussed. Crepidostomum sp. has 2n=12, with one pair of large metacentrics and five pairs of acrocentrics and is more distantly related karyologically to the other two members of the family. Evolutionary considerations based on comparison of the chromosomes of Allocreadiidae and other studied families of the suborder Xiphidiata suggest that allocreadiids are karyotypically distinct. The placement of Allocreadiidae and Opecoelidae in the superfamily Allocreadioidea is questioned.

Karyotypic Variation in the Australian Gecko Diplodactylus Tessellatus, with the Description of a New Karyotypic Complement for Diplodactyline Geckos

Abstract We report the discovery of a 2n = 30 karyotype race in the Australian diplodactyline gecko Diplodactylus tessellatus. The new chromosome race has four large metacentric pairs and 11 acrocentric pairs, a unique complement among diplodactyline geckos. Three karyotypic races are now known from D. tessellatus. One retains the ancestral Diplodactylus 2n = 38 all-acrocentric karyotype, whereas the 2n = 30 and the 2n = 28 races appear to have been derived from a 2n = 38 karyotype via fusion of four or five pairs of acrocentrics. Diplodactylus tessellatus and its relatives in the Diplodactylus vittatus species complex are now known to possess five different diploid number complements; 2n = 38, 36, 34, 30, and 28, more variation than any other diplodactyline gecko genus or species group for which data are available.