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Casa de Lletres Academic Editing Services [email protected] www.casadelletres.eu Evolution of herbivory in Drosophilidae linked to loss of behaviors, antennal responses, odorant receptors, and ancestral diet

Benjamin Goldman-Huertasa, Robert F. Mitchellb,c, Richard T. Lapointa,c, Cécile P. Faucherb,d, John G. Hildebrandb,1, and Noah K. Whitemana,1

Departments of aEcology and Evolutionary Biology and bNeuroscience, and cCenter for Insect Science, University of Arizona, Tucson, AZ 85721; and dLife Science Solutions, Thermo Fisher Scientific, 64293 Darmstadt, Germany

Contributed by John G. Hildebrand, January 4, 2015 (sent for review August 29, 2014; reviewed by Ewald Grosse-Wilde, Benjamin Prud’homme, and Michael G. Ritchie) Herbivory is a key innovation in insects, yet has only evolved in one- been rarely shown but occurs in nematodes, although their olfactory third of living orders. The evolution of herbivory likely involves systems are distinct from insects (14). Families of mammalian ol- major behavioral changes mediated by remodeling of canonical factory receptor proteins have been remodeled during transitions to chemosensory modules. Herbivorous flies in the genus Scaptomyza flight, aquatic lifestyles, and frugivory (15–18). Similarly, the evo- (Drosophilidae) are compelling species in which to study the geno- lution of diet specialization in Drosophila species correlates with mic architecture linked to the transition to herbivory because they chemoreceptor gene losses (19–21), and hematophagous flies have recently evolved from microbe-feeding ancestors and are closely lost gustatory receptors that detect sweet compounds (22). More related to Drosophila melanogaster. We found that Scaptomyza profound changes such as the evolution of new protein families are flava, a leaf-mining specialist on plants in the family (Brassicaceae), associated with major evolutionary transitions such as the evolution was not attracted to yeast volatiles in a four-field olfactometer of flight in insects (23). Although gene loss is unlikely to be a assay, whereas D. melanogaster was strongly attracted to these driving force of innovation, loss-of-function mutations may be volatiles. Yeast-associated volatiles, especially short-chain aliphatic exaptations that allow novel behaviors to evolve by disrupting an- esters, elicited strong antennal responses in D. melanogaster,but cestral attractions. If detection of different chemical cues becomes weak antennal responses in electroantennographic recordings from selected in a novel niche, then loss through relaxed constraint may S. flava. We sequenced the genome of S. flava and characterized indicate which chemical cues have changed during a trophic shift. this species’ odorant receptor repertoire. Orthologs of odorant re- The chemosensory repertoires of many drosophilid species ceptors, which detect yeast volatiles in D. melanogaster and medi- have been functionally annotated. The genus Drosophila includes ate critical host-choice behavior, were deleted or pseudogenized in 23 species with published genome sequences (24–27), and the genome of S. flava. These genes were lost step-wise during D. melanogaster presents the most fully characterized insect olfac- the evolution of Scaptomyza. Additionally, Scaptomyza has expe- tory system (28), allowing potential linkage of receptor remod- rienced gene duplication and likely positive selection in paralogs eling to a mechanistic understanding of behavioral change. of Or67b in D. melanogaster. Olfactory sensory neurons express- Most drosophilids feed on yeast and other microbes growing ing Or67b are sensitive to green-leaf volatiles. Major trophic shifts on decaying plant tissues (29). Adult female D. melanogaster and in insects are associated with chemoreceptor gene loss as recently evolved ecologies shape sensory repertoires. Significance plant–herbivore interactions | gene loss | olfaction | The evolution of herbivory in animals is rare but has resulted in Drosophila melanogaster | Scaptomyza flava major adaptive radiations. Its rarity suggests that there are barriers to colonization of plants. Behavioral adaptations, involving nderstanding the origins and consequences of trophic shifts, host plant finding, are likely the first to evolve during the tran- Uespecially the transition to herbivory, has been a central sition to herbivory. A recently evolved herbivorous fly species problem in evolutionary biology. The paleontological record was derived from yeast-feeding ancestors. This herbivorous fly, suggests that evolutionary transitions to herbivory have been rare unlike its yeast-feeding relatives, lost attraction to yeast volatiles, in insects (1), and the first transitions to herbivory in vertebrates the ability to detect yeast volatiles, and three genes that encode occurred long after the colonization of land (2). However, species olfactory receptors critical for detecting yeast volatiles in Dro- radiations result from herbivorous transitions in insects and ver- sophila melanogaster. Loss-of-function mutations may play a role tebrates, suggesting that herbivory is a key innovation (3, 4). in the transition to herbivory in insects, which account for nearly Identifying functional genomic changes associated with the 25% of all species of life. evolutionary transition to herbivory could yield insight into the mechanisms that have driven their success. However, the origins of Author contributions: B.G.-H., R.F.M., R.T.L., C.P.F., J.G.H., and N.K.W. designed research; the most diverse clades of herbivorous insects are ancient and date B.G.-H., R.F.M., R.T.L., and C.P.F. performed research; B.G.-H., R.F.M., R.T.L., J.G.H., and N.K.W. to the Jurassic or earlier (5), limiting meaningful genomic com- analyzed data; and B.G.-H., R.F.M., R.T.L., C.P.F., J.G.H., and N.K.W. wrote the paper. parisons. In contrast, herbivory has evolved more times in Diptera Reviewers: E.G.-W., Max Planck Institute for Chemical Ecology; B.P., CNRS; and M.G.R., University of St. Andrews. than in any other order (3). The Drosophilidae is an excellent The authors declare no conflict of interest. system to study the evolution of herbivory from a functional ge- Data deposition: The sequences reported in this paper have been deposited in the GenBank nomic perspective because it includes several transitions to her- database (accession nos. KM278522–KM278527, KM277412–KM277433). Odorant receptor bivory, and the genomic model Drosophila melanogaster (6, 7). (OR) sequences from the S. flava and S. pallida genomes are provided in Datasets S1 and S2. – The transition to herbivory involves adaptations in physiology (8 See Commentary on page 2927. 10), morphology (11), and behavior (12). The evolution of sensory 1To whom correspondence may be addressed. Email: [email protected] or repertoires could reinforce or even precipitate these adaptations [email protected]. through adaptive loss or relaxation of functional constraint sub- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sequent to a trophic shift (13). Adaptive loss of chemoreceptors has 1073/pnas.1424656112/-/DCSupplemental.

3026–3031 | PNAS | March 10, 2015 | vol. 112 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1424656112 distantly related species innately prefer yeast chemical cues to those hypothesized that gravid ovipositing S. flava females would not be produced by the fruit on which they oviposit (30, 31). D. melanogaster attracted to yeast volatiles. Second, we hypothesized that the ol- SEE COMMENTARY detects volatiles with chemoreceptors of several different protein factory sensory organs of S. flava would have a decreased ability families, but especially receptors from the odorant receptor (OR) to detect individual yeast volatiles and volatile mixtures. Third, gene family, some of which, such as Or42b, are highly conserved chemoreceptor genes from the OR gene family implicated in across species (31). Or42b is necessary for attraction and orien- detection of yeast volatiles would be lost in the S. flava genome. tation to vinegar and aliphatic esters (32–34). Similar compounds Finally, we predict chemoreceptor genes potentially mediating activate Or42b across many Drosophila species (35), suggest- detection of plant volatiles would show evidence of positive se- ing that volatile cues for yeast, and the associated receptors, are lection and possibly, neofunctionalization. conserved across the Drosophilidae. Results and Discussion The ancestral feeding niche for the genus Scaptomyza (Droso- philidae) is microbe-feeding, but Scaptomyza use decaying leaves Characterization of the Olfactory Capabilities of an Herbivorous and stems rather than the fermenting fruit used by D. melanogaster Drosophilid. Olfaction is used by insects to find resources, mates, and other members of the subgenus Sophophora (29, 36). The close and oviposition substrates (37). We tested the hypothesis that S. flava D. melanogaster association of Scaptomyza with decaying plant tissues may have is not attracted to yeast volatiles, whereas is attracted to yeast volatiles. We used a four-field olfactometer precipitated the evolution of herbivory <20 MyBP (Fig. 1; ref. 36). assay (38, 39) in which filtered air blown through four corners of Adult females of the species S. flava feed and oviposit on living a diamond-shaped arena establishes four independent airfields. leaves of many cruciferous plants (Brassicales) including Arabi- Two of the four fields were exposed to yeast volatiles from dopsis thaliana. Females puncture leaves with serrated ovipositors to Saccharomyces cerevisiae cultures. The presence of gravid adult fe- create feeding and oviposition sites, and larvae mine and pupate males of both species in either yeast or control fields was recorded within the living leaves (7). every 6 s for 10 min. D. melanogaster flies spent 82.4 ± 18.2% Here, we use Scaptomyza as a model to test the hypothesis that SD of the assay time in yeast-volatile fields (Wilcoxon signed rank, functional loss of chemosensory genes has played a role in a major W = 295, P < 0.0001, n = 25) and more time in yeast-volatile fields ecological transition to herbivory in insects. We hypothesized that the than S. flava (Mann–Whitney U = 669, P < 0.0001, n = 25, n = 31) conserved detection of yeast volatiles would be lost in the herbivorous (Fig. 2A). S. flava did not spend more time in yeast-volatile fields Scaptomyza lineage. We tested this loss by comparing D. melanogaster (Wilcoxon signed rank W = 266.5, n = 31, P = 0.49) and divided and S. flava at behavioral, physiological, and genetic levels. First, we residence time evenly between yeast and control fields (51.5 ± 24.5% SD), consistent with a loss of attraction to yeast volatiles in S. flava flies. Because S. flava flies failed to increase their residence time in olfactometer quadrants exposed to yeast volatiles, we hypothesized that S. flava antennal olfactory sensory neurons (OSNs) were deficient in their ability to detect yeast volatiles. We addressed this hypothesis by conducting electroantennogram (EAG) measurements in adult D. melanogaster and S. flava flies of both sexes 4–20 d after eclosion, exposed to the same yeast volatiles used in the olfactometer assays and to crushed rosette leaves of the host plant of S. flava flies in our laboratory colonies (Arabidopsis thaliana accession Col-0). EAG responses are driven by the aggregate depolarization of OSNs in the antennae and scale with the concentration and identity of stimulants (40). We found no difference between sexes and combined data for male and female flies. We recorded consistently lower EAG signals in S. flava flies compared with D. melanogaster, preventing inter- specific comparisons of signal amplitude, possibly due to differences in electrical properties of antennae (40). The antennae of S. flava were more strongly stimulated by Arabidopsis volatiles than by yeast (P < 0.01; Fig. 2B), whereas the antennae of D. melanogaster were more responsive to volatiles from yeast than those from Arabidopsis (P < 0.0001; Fig. 2C). We concurrently measured responses to a small panel of three volatiles associated with A. thaliana [(Z)-3-hexenol, myrcene, phenethyl isothiocyanate; ref. 41] and two with S. cerevisiae (2-phenylethanol, ethyl acetate; ref. 42). Antennae of both species detected all volatiles compared with a negative control (S. flava, P < 0.0001; D. melanogaster, P < 0.0001; Fig. 2 B and C). The antennae of S. flava were most responsive to (Z)-3-hexenol (Fig. 2B), a volatile produced by damaged leaves of many plant species (43), and were also highly attuned to phenethyl iso- Fig. 1. Time-calibrated Bayesian phylogeny of Drosophila and Scaptomyza thiocyanate, a hydrolyzed product of glucosinolates, which are the species with herbivorous taxa in dark green. Scaptomyza flies are nested major defensive compound in host plants of S. flava. Responses to phylogenetically within the Drosophila genus. (Inset) Adult female S. flava myrcene and 2-phenylethanol were not in the expected direction, fly with green-pigmented abdomen after feeding on Arabidopsis thaliana. although 2-phenylethanol, as a widespread floral volatile (44), Arrowhead indicates serrated ovipositor used to create feeding and ovipo- Scaptomyza sition holes within leaf tissue. Node labels indicate posterior clade proba- may remain an important chemical cue for adults. bility (PP). Unlabeled nodes have PP = 1. Error bars are 95% highest posterior Antennae of S. flava were less responsive to yeast and the yeast-associated volatile ethyl acetate than to plant-related vol-

density interval. Pie graphs indicate probability of change to herbivory EVOLUTION (green) or microbe-feeding (white) traits reconstructed at each node. Her- atiles, but these relative comparisons were insufficient to prove bivorous taxa are in brackets indicated by leaf. that the detection threshold for yeast volatiles had decreased in

Goldman-Huertas et al. PNAS | March 10, 2015 | vol. 112 | no. 10 | 3027 in D. melanogaster (reviewed in ref. 28), and members of subfamily A H OR genes in particular (20) are highly conserved and enriched in receptors for aliphatic esters (35), a group of compounds S. flava detected poorly. To characterize changes in the OR gene repertoire in S. flava associated with the olfactory phenotypes, we sequenced the genome of S. flava and annotated OR genes by using reciprocal tBLASTn searches (47) of previously annotated Drosophila OR protein sequences (SI Appendix, Table S1) against this de novo S. flava genome assembly. All significant BLAST hits and their B homology assignments are listed in SI Appendix, Table S2 and S. flava sequences are in Dataset S1. We found 65 full-length ORFs for OR genes in S. flava (Dataset S2). Consistent with previous OR gene-naming conventions (48–50), ORs were named after the D. melanogaster ortholog or the most closely related gene, with the exception of OrN1 and OrN2 orthologs, which are not present in D. melanogaster (50). Protein translations of S. flava genes were included in a phy- C logeny of D. melanogaster, Drosophila virilis, Drosophila mojavensis, and Drosophila grimshawi OR protein sequences to assess homology (SI Appendix, Fig. S2). The latter three species are the closest relatives of Scaptomyza with fully sequenced genomes (Fig. 1). S. flava retains duplicates of Or42a, Or67a, Or74a, Or83c, Or98a, and OrN2 found in other sequenced Drosophila species (SI Appendix,Fig.S2). Scaptomyza also has duplications not shared with close relatives, although nine of these genes are pseudogenized. The majority of paralogs (56%) were found on the same scaffold in tandem arrays (SI Appendix,TableS2). The functional significance of these gene duplications is not yet clear, but it is suggestive that Or67b, with three copies in S. flava, is in single copy in nearly all Fig. 2. Herbivorous S. flava have diminished behavioral and antennal sequenced Drosophila.InD. melanogaster, neurons expressing responses to yeast volatiles, consistent with a loss of attraction to yeast. Or67b respond to green leaf volatiles such as (Z)-3-hexenol (51), to (A) Gravid adult female S. flava (Sfla) and D. melanogaster (Dmel) flies were which S. flava also has a robust antennal response (Fig. 2B). introduced into a four-field olfactometer arena. Half the fields were ex- Only four widely conserved ORs were uniquely lost (Or22a posed to volatiles from cultures of Sa. cerevisiae yeast or to a control solu- and Or85d) or pseudogenized (Or9a, Or42b) in the Scaptomyza tion. Boxplots of electroantennographic (EAG) recordings of adult S. flava (B) lineage (Table 1). Syntenic regions flanking OR losses were re- and D. melanogaster (C) flies exposed to volatiles of crushed A. thaliana – leaves, yeast cultures, single compounds, and mineral oil controls. Green covered in the genome assembly (Fig. 3 B E). Orthologs of Or9a, boxplots indicate A. thaliana-associated volatiles; purple are yeast-associated Or22a, and Or42b are intact in 23 Drosophila species with ge- volatiles. Letters indicate significance of FDR-corrected post hoc Wilcoxon nome sequences, and Or85d is missing only in the Drosophila rank-sum test. Boxplot whiskers are 1.5× interquartile range. albomicans and Drosophila rhopaloa genome assemblies. As pre- dicted, orthologs of ORs that persist in microbe-feeding Dro- sophila species and are lost in S. flava, function in yeast-volatile Scaptomyza. We therefore tested the sensitivity of S. flava and detection (Table 1). Or42b is highly conserved in sequence (31) D. melanogaster flies to this and other short-chain aliphatic esters and receptor function (35) among Drosophila species, and the by exposing females to half-log dilution series of ethyl acetate, ethyl receptor is highly attuned to aliphatic esters at low concentrations propionate, and isobutyl acetate (SI Appendix,Fig.S1). We defined (32). Knockouts of Or42b in adult D. melanogaster result in failure sensitivity as the first concentration increase that generated an to orient in flight toward aliphatic ester odor plumes (33), and increased antennal response. S. flava was insensitive to ethyl rescuing these neurons restores attraction to yeast volatiles (34). acetate at the concentrations tested (P > 0.05 in all comparisons, Similarly, no sequences similar to Or22a were present in the Wilcoxon rank sum, adjusted by false discovery rate). S. flava was S. flava assembly, although we recovered conserved intergenic also less responsive to ethyl propionate (minimum threshold regions in S. flava that flank Or22a in other Drosophila species − − 10 1.5;W= 2.2; P < 0.05) and isobutyl acetate (10 1;W= 2.2, P < (Fig. 3B). Or22a also detects aliphatic esters (56) and in the − 0.05) compared with D. melanogaster (ethyl propionate at 10 2.5; specialist species Drosophila erecta and Drosophila sechellia, − W = 2.5; P < 0.05; isobutyl acetate at 10 2;W= 2.3; P < 0.05). Or22a detects volatiles produced by host fruit (57, 58). Both Scaptomyza is considerably less sensitive to short aliphatic esters, Or22a and Or42b are activated by floral volatiles of Arum pal- which may account for differences in signal strength in response to estinum, which mimics yeast fermentation volatiles and attracts plant and yeast volatile mixtures and the lack of attraction to yeast a diversity of drosophilids (31). Finally, Or85d orthologs were not volatiles by S. flava. This unresponsiveness is consistent with the detected in the S. flava genome by BLAST or by inspection of fact that deficits in the production of aliphatic esters in a yeast genome regions flanking Or85d in other species (Fig. 3E). Or85d strain decreased attractiveness to D. melanogaster flies (45). is expressed in the maxillary palps (59) and in D. melanogaster is responsive to the yeast metabolites 2-heptanol, ethyl acetate, OR Gene Annotation and Phylogenetics. The lack of attraction and and isoamyl acetate (59). Or85d is highly sensitive to phenethyl minimal EAG response to yeast volatiles in S. flava suggested acetate, a common volatile of many yeast species (54). In D. that chemosensory genes have been lost or changed in herbivo- melanogaster, Or9a is activated by a broad range of ketone-, al- rous Scaptomyza species. ORs are expressed in the dendrites of cohol-, and carboxylic acid-containing ligands (60). Some of OSNs in the antennae and maxillary palps and are the primary these ligands, such as acetoin, are common yeast volatiles and receptors by which most neopteran insects detect odors in their strong attractants (30). The consequences of Or9a pseudogeni- environments (46). The OR family has been functionally annotated zation will require further study.

3028 | www.pnas.org/cgi/doi/10.1073/pnas.1424656112 Goldman-Huertas et al. Table 1. OR genes lost or with signatures of positive selection in S. flava lineage SEE COMMENTARY OR genes with significant Ortholog expression pattern Known ligands of D. melanogaster OR gene branch-site test* in D. melanogaster (52, 53) orthologs (51)

Or9a pseudogene NA Antennal basiconic 8B OSNs 3-hydroxy-2-butanone, 2,3-butanediol Or22a deleted NA Antennal basiconic 3A OSNs Ethyl hexanoate, Methyl hexanoate, Ethyl butyrate Or42b pseudogene NA Antennal basiconic 1A OSNs Ethyl acetate, Ethyl butyrate Larval dorsal organ † ‡ § Or67b-1,2 ω2 = ∞ (FDR P = 0.023) Antennal basiconic 9B OSNs (Z)3-hexenol Larval dorsal organ

Or67b-3 ω2 = 8.79 (FDR P = 0.024) Antennal basiconic 9B OSNs (Z)3-hexenol Larval dorsal organ Or85d deleted NA Palp basiconic 9B OSNs 2-heptanone, isoamyl acetate, phenethyl acetate (54)

Or88a ω2 = 8.80 (FDR P = 0.006) Antennal trichoid 4C OSNs Female and male conspecific volatiles (55)

*Additional parameters for all OR gene branch-site models listed in Table S7. NA, not applicable. † Branch ancestral to S. flava Or67b-1 and Or67b-2 paralogs. ‡ Estimated ratio in foreground branch for codons with dN/dS > 1 consistent with episodic positive selection. §False discovery rate corrected P value.

Timing of the Evolution of Herbivory in Scaptomyza. A time-calibrated of herbivory in Scaptomyza. Losses were shared by herbivorous phylogeny of the family Drosophilidae suggests that herbivory congeners (Fig. 3A). Or22a, while lost in S. flava, is intact in the evolved in Scaptomyza ca.13.5 million years ago (95% highest pos- microbe-feeding species Scaptomyza apicata and S. pallida and is terior density 10.02–17.48 million years ago), overlapping with age also lost in two other herbivorous species, Scaptomyza nigrita and ranges inferred from previous analyses (36, 61) (Fig. 1). Ancestral Scaptomyza graminum. state reconstructions were performed in the APE package (62) by Specialist, microbe-feeding Drosophila species, such as using an equal rates model. This analysis indicated that microbe D. sechellia and D. erecta have an accelerated rate of chemore- feeding is ancestral in Drosophila and Scaptomyza (99.7% proba- ceptor gene loss (19, 20), but this pattern could also be due to bility) and that herbivory evolved once within the genus Scaptomyza. nearly neutral processes (49, 63). S. flava feeds almost exclusively We hypothesized that OR gene losses would coincide with the on plants within the Brassicales, and we hypothesized that this evolution of herbivory. We developed and used degenerate PCR species has experienced an accelerated rate of chemosensory primers (SI Appendix, Table S3) from genomes of multiple gene loss compared with other microbe-feeding Drosophila spe- Scaptomyza and Drosophila species that targeted exonic se- cies. We tested this hypothesis by coding homologous groups of quences of Or22a and Or9a (Fig. 3 B and C), and conserved, ORs as present or absent in S. flava, D. virilis, D. mojavensis and flanking, intergenic sequences of Or42b and Or85d (Fig. 3 D and D. grimshawi (the closest Drosophila relatives of Scaptomyza;Fig.1 E and SI Appendix, Table S4). Sequences were deposited in and SI Appendix,TableS5), and inferred two models of gene loss in GenBank (KM277412–KM277433). the Brownie software package (64). We found no evidence for the Gene losses in S. flava were confirmed by PCR screen in three alternative model of increased rate of loss in Scaptomyza (AICc = natural populations (SI Appendix, Table S4), with the exception 2.14411), but cannot rule out that there were insufficient loss events of SflaOr9a-1, which appeared to be present in a functional copy to parameterize the more complex model or that other chemore- in a population from Arizona. A preliminary genome assembly of ceptor gene families have undergone accelerated loss in S. flava. Scaptomyza pallida was consistent with PCR screening results for Also, S. flava is oligophagous, feeding on many plant species in the OR loss patterns in this species (Dataset S1). We reconstructed Brassicales, and it is less specialized than D. sechellia and D. erecta. the presence/absence of S. flava gene losses along ancestral nodes and found that three of the four OR gene losses in S. flava Evidence for Episodic-Positive Selection in S. flava. Because the shift (Or22a, Or85d, Or42b) coincided with or preceded the evolution to herbivory in Scaptomyza likely involved many changes in olfactory

Fig. 3. OR genes critical for yeast volatile detection in ABCDED. melanogaster are lost in step-wise fashion in Scap- tomyza phylogeny. Or22a loss is coincident with the evolution of herbivory. (A) OR gene losses mapped onto the Scaptomyza phylogeny using maximum likelihood ancestral state reconstruction based on PCR screens and genomic data. (B–E) Diagram of genes flanking Or9a (B), Or22a (C), Or42b (D), and Or85d (E)lossesinS. flava (B and C, Left; D and E, Left and Right)andD. grimshawi (B and C, Right; D and E, Center). ORs colored as in A. Homologous genes are indicated by solid connecting lines, pseudogenes by Ψ in both species and the location of diagnostic primers by the long tick marks along the scale bar. bp, base pairs; kb, kilobase pairs. Or22a (C)is duplicated in the D. grimshawi genome, but deleted in S. flava.TwoS. flava scaffolds show homology to each of the Or42b and Or85d regions in D. grimshawi (D and E). The Or42b region (D)inbothS. flava and D. grimshawi is

tandemly duplicated, whereas in S. flava, Or42b genes EVOLUTION are pseudogenized while in D. grimshawi flanking Or42a genes are pseudogenized and Or42b intact.

Goldman-Huertas et al. PNAS | March 10, 2015 | vol. 112 | no. 10 | 3029 cues, we hypothesized that some S. flava OR genes should bear to herbivory may be common (5). The genetic changes that signatures of episodic positive selection, as flies adapted to a novel underlie host-finding remain to be identified, but recently dupli- environment. To test this hypothesis, we inferred null and alter- cated ORs, such as the unique triplication of Or67b in Scapto- native (branch-site) models in PAML 4.7a where subsets of codons myza, are likely candidates for further functional study. Subtle, in extant S. flava ORs could evolve under (i) purifying or neutral targeted remodeling of chemoreceptor repertoires may be a gen- selection or (ii) purifying, neutral, or positive selection, relative to eral mechanism driving changes in behavior, facilitating trophic 12 Drosophila species (25). We used a phylogeny-aware alignment shifts and ultimately diversification in animals. program, PRANK (65), to identify regions where indels were prob- able while minimizing sensitivity to alignment errors. Alignments Materials and Methods where more than one taxon had an inferred indel in greater than Olfactometer Assay. Adult female D. melanogaster and S. flava were exposed two regions were trimmed by using Gblocks (66) to remove columns to volatiles produced by Sa. cerevisiae yeast cultures in 1% sucrose in non- with ambiguous homology (SI Appendix,TableS6and Dataset S3). adjacent quadrants of a four-field olfactometer arena. Individual flies were After correcting for false discovery (67), we found two ORs in introduced and allowed to walk throughout the arena and recorded for which the branch-site model consistent with episodic positive selec- 10 min. Four independent airfields are created in the olfactometer arena by tion was more likely than the null model (Table 1 and SI Appendix, directing compressed air through flowmeters into the four corners of the arena and exiting through a central hole in the bottom of the device (SI Table S7). Or88a had the strongest statistical support for the branch- < Appendix, Fig. S3). Preference or aversion to yeast volatiles was determined site model (FDR-adjusted P 0.01). In D. melanogaster, Or88a by residence time in yeast-exposed quadrants over control quadrants. Assays functions in recognition of male and virgin female conspecifics were performed under infrared light in a dark room to remove visual cues. (55). Two other branches among the S. flava Or67b paralogs also Details of the olfactometer assay are in SI Appendix, Materials and Methods. supported the branch-site model (FDR adjusted P < 0.05, P < 0.05): an ancestral branch preceding a Scaptomyza-specific duplication Electroantennography. Headspace volatiles from the yeast cultures tested in event and a branch leading to Or67b-3. Homologs of this gene in the olfactometer assays and crushed leaves of A. thaliana host plants were D. melanogaster encode ORs that respond to the green-leaf vol- used as olfactory stimuli in EAG analyses along with five compounds found atile (Z)-3-hexenol (51), one of the most salient ligands found in in either yeast or A. thaliana headspace. A recording microelectrode was our EAG studies of S. flava (Fig. 2B). Experimental, functional, placed against the ventral base of the third antennomere of S. flava and and population-based tests are needed to verify whether positive D. melanogaster adult males and females, and a ground electrode was selection has fixed amino acid changes in the Scaptomyza lineage. inserted into the eye. Each headspace sample and individual compound was tested 10 times, in seven flies for each species and sex. An additional EAG experiment was run on four S. flava and five D. melanogaster flies that Conclusions − − tested a half-log dilution series (10 3.5 to 10 1) of three short-chain aliphatic Trophic transitions in the history of animal life, such as herbiv- esters. Details of EAG methods are in SI Appendix, Materials and Methods. ory, may be mediated by genetic changes in chemosensory repertoires. The majority of Drosophilidae feed on microbes (29), Gene Annotation. OR genes were annotated in S. flava by running a tBLASTn and distantly related drosophilid lineages are attracted by the search with the full-length, OR protein sequences of multiple Drosophila same yeast-mimicking floral scent produced by A. palestinum species against the S. flava genome assembly, and manual curation. (31). A subset of the ORs stimulated by this scent are highly Orthology and paralogy were assigned by aligning translations all OR genes conserved in other drosophilids, which may be part of a homol- of S. flava and four other Drosophila species and inferring the evolutionary ogous and conserved olfactory circuit used to find fermenting history of the gene family with ML and Bayesian methods in RAxML (69) and host substrates across the family (31). We hypothesized that MrBayes (70), respectively. Details on annotation are in the SI Appendix, Materials and Methods. mutations disrupting the function of OR homologs in this conserved olfactory circuit could mediate the evolution of herbivory Evolution of Olfactory Receptor Genes in Scaptomyza. PCR primers were de- or other novel food preferences. veloped targeting S. flava gene losses. Primers were developed for either S. flava, an herbivorous drosophilid, has lost orthologs of ORs exonic sequences or conserved sequences flanking genes and used to am- involved in this generalized yeast olfactory circuit. Consistent plify fragments of yeast-feeding and herbivorous Scaptomyza, including with these findings, S. flava did not respond to yeast volatiles in laboratory and natural populations of S. flava. Gains and losses were map- a behavioral assay. Antennae of S. flava were weakly activated by ped onto a time-calibrated Drosophila and Scaptomyza phylogeny by using active yeast cultures and short-chain aliphatic esters, key com- maximum likelihood methods in Mesquite v2.75 (mesquiteproject.org). Tests pounds found in yeast volatile blends and known ligands of ORs of changes in gene loss rate were performed based on S. flava gene counts in D. melanogaster lost in S. flava. However, retention of some in Brownie (64) according to methods in McBride et al. (20). Finally, branch- ORs implicated in yeast-volatile detection, such as Or92a and site tests were performed on S. flava OR genes, with S. flava genes in Or59b, implies that S. flava may retain the ability to detect some foreground, and 12 Drosophila species in the background in PAML v4.7a (71). Details are in SI Appendix, Materials and Methods. Herbivory and untested yeast compounds (31). microbe-feeding were reconstructed as ancestral states along the above We hypothesized that OR genes would be intact in non- species phylogeny by using the ace function in APE (62). herbivorous Scaptomyza and gene losses would coincide with the transition to herbivory. Or22a loss did coincide with the evolu- ACKNOWLEDGMENTS. We thank Sammy Dominguez, Daniel Papaj, and tion of herbivory, but losses of Or42b and Or85d likely predate Lynne Oland for loan of materials and useful discussion on study design, the evolution of plant feeding. These more ancient losses of and Carolina Reisenman for initial EAG tests. We acknowledge support from the National Science Foundation (NSF) through Graduate Research conserved yeast-volatile receptors suggest ancestral Scaptomyza Fellowships Program DGE-0646147, Integrative Graduate Education and may have already evolved novel olfactory pathways that were Research Traineeship DGE-0654435 (to B.G.-H.), and NSF DEB-1256758 (to later co-opted by herbivorous lineages, and in fact, many Scap- N.K.W.); a University of Arizona Foundation Faculty Seed Grant (to N.K.W.) tomyza species feed on microbes living within decaying leaves or and University of Arizona Center for Insect Science Faculty Seed Grant (to N.K.W.); John Templeton Foundation Grant 41855 (to N.K.W.); National in leaf mines produced by other insects (68). Sister groups of Institutes of Health (NIH) Postdoctoral Excellence in Research and Teaching many major herbivorous insect lineages also feed on detritus Fellowship K12-631 GM000708 PERT (to R.F.M. and R.T.L.); and NIH Grant and fungi, suggesting that the transition from microbe feeding R01-DC-02751 (to J.G.H.).

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Goldman-Huertas et al. PNAS | March 10, 2015 | vol. 112 | no. 10 | 3031 Descriptive paper

Casa de Lletres Academic Editing Services [email protected] www.casadelletres.eu Anais da Academia Brasileira de Ciências (2016) 88(2): 829-845 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-3765201620150288 www.scielo.br/aabc

Late Pleistocene echimyid rodents (Rodentia, Hystricognathi) from northern Brazil

THAIS M.F. FERREIRA1,2, ADRIANA Itati OLIVARES3, LEONARDO KERBER4, RODRIGO P. DUTRA5 and LEONARDO S. AVILLA6

1Programa de Pós-Graduação em Geociências, Instituto de Geociências, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, 91501-970 Porto Alegre, RS, Brasil 2Seção de Paleontologia, Museu de Ciências Naturais, Fundação Zoobotânica do Rio Grande do Sul, Av. Salvador França, 1427, 90690-000 Porto Alegre, RS, Brasil 3Sección Mastozoología, División Zoología Vertebrados, Museo de La Plata, UNLP, CONICET, Paseo del Bosque, s/n, 1900 La Plata, Buenos Aires, Argentina 4Centro de Apoio à Pesquisa Paleontológica da Quarta Colônia/CAPPA, Universidade Federal de Santa Maria, Rua Maximiliano Vizzotto, 598, 97230-000 São João do Polêsine, RS, Brasil 5Programa de Pós-Graduação em Zoologia, Departamento de Zoologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Pampulha, 31270-901 Belo Horizonte, MG, Brasil 6Laboratório de Mastozoologia, Departamento de Zoologia, Instituto de Biociências, Universidade Federal do Estado do Rio de Janeiro, Av. Pasteur, 458, sala 501, Urca, 22290-240 Rio de Janeiro, RJ, Brasil

Manuscript received on May 8, 2015; accepted for publication on June 29, 2015

ABSTRACT Echimyidae (spiny rats, tree rats and the coypu) is the most diverse family of extant South American hystricognath rodents (caviomorphs). Today, they live in tropical forests (Amazonian, coastal and Andean forests), occasionally in more open xeric habitats in the Cerrado and Caatinga of northern South America, and open areas across the southern portion of the continent (Myocastor). The Quaternary fossil record of this family remains poorly studied. Here, we describe the fossil echimyids found in karst deposits from southern Tocantins, northern Brazil. The analyzed specimens are assigned to Thrichomys sp., Makalata cf. didelphoides and Proechimys sp. This is the first time that a fossil of Makalata is reported. The Pleistocene record of echimyids from this area is represented by fragmentary remains, which hinders their determination at specific levels. The data reported here contributes to the understanding of the ancient diversity of rodents of this region, evidenced until now in other groups, such as the artiodactyls, cingulates, carnivores, marsupials, and squamate reptiles. Key words: Caviomorphs, Echimyidae, fossil record, Quaternary, South America.

INTRODUCTION porcupines (Hystricidae) and the New World caviomorphs distributed in 13 families (Huchon The living members of Hystricognathi (Rodentia: and Douzery 2001, Woods and Kilpatrick 2005, Hystricomorpha) include three lineages: the African Upham and Patterson 2012, Patton 2015a). The phiomorphs, represented by four extant families Echimyidae (spiny rats, tree rats and the coypu) is (the three classical families plus Heterocephalidae; the most diverse family of caviomorphs (Emmons Patterson and Upham 2014), the Old World 2005, Patton 2015a). These rodents have a small

Correspondence to: Thais Matos Pereira Ferreira body size (with an exception for Myocastor Kerr, E-mail: [email protected] 1792) and brachydont or protohypsodont cheek

An Acad Bras Cienc (2016) 88 (2) 830 THAIS M.F. FERREIRA et al. teeth (sensu Mones 1968). The taxonomic history 2005, Hadler et al. 2008, Roth et al. 2008, Castro of the echimyids has been complex, even at the and Langer 2011, Ferreira et al. 2012, Oliveira et al. subfamily and genus levels (Patterson and Pascual 2013, Kerber et al. 2014, 2016), Bolivia (Marshall 1968, Patterson and Wood 1982, Galewski et al. and Sempere 1991), and Argentina (Vucetich et al. 2005, Emmons 2005, Fabre et al. 2014, Emmons 1997, Olivares and Verzi 2015). Although this fossil et al. 2015a, Patton 2015a). Recent molecular record is impoverished, a sample of the astonishing and morphological phylogenies reveal traditional diversity achieved by this group in the tropical subfamilies to be non-monophyletic (Leite and and subtropical areas of lower latitudes of South Patton 2002, Carvalho and Salles 2004, Galewski America (Verzi et al. 2014) provides important data et al. 2005, Upham and Patterson 2012, Fabre et al. for diversity analyses and paleogeographic and 2012, Verzi et al. 2014, 2016, Olivares and Verzi paleoclimatic inferences. We present here the first 2015). data on the late Pleistocene diversity of echimyids Echimyids have an extended fossil record, but from the Gruta dos Moura limestone cave in Aurora some controversies remain about the age of the de Tocantins, low latitudes of South America. family. Molecular studies date their origin back to the early Miocene (Upham and Patterson 2012, MATERIALS AND METHODS

Fabre et al. 2012, Voloch et al. 2013) while the Locality and Geology fossil record could be even older (e.g. late Eocene, Frailey and Campbell 2004; late Oligocene, Verzi The studied material was collected from a carbonate et al. 2014). It is also worth noting that Paleogene deposit, which was found in a block at the wall of echimyids require cladistic analyses to assess their the main room of the Gruta dos Moura limestone relationships with modern forms (Carvalho and cave in Aurora de Tocantins (12º42’47” S and Salles 2004, Verzi et al. 2014, 2016). 46º24’28” W), State of Tocantins, northern Brazil The modern distribution of echimyids includes (Figure 1). The carbonate rocks from Aurora do predominantly tropical forests (i.e. Amazonian, Tocantins are part of the Speleological Province coastal and Andean forests) and occasionally of the Bambuí Group, where numerous caves are more open and xeric habitats in the Cerrado and located (Zampaulo and Ferreira 2009). The geology Caatinga in northern South America (Hershkovitz of this area is poorly known. The predominant rocks 1958, Vucetich and Verzi 1999, Galewski et al. in the region are rhythmic limestones and siltstones 2005, Upham and Patterson 2012, Olivares and from the Paraopeba Subgroup of Neoproterozoic Verzi 2015). Myocastor is the only extant echimyid age, although alluvial deposits might occur locally distributed in southern South America (Woods et (Dardene 1978, Dardene and Walde 1979). al. 1992, Patton 2015b). Additionally, part of the The information provided by the Serviço Ge- history of the family (late Miocene to Pleistocene) ológico do Brasil on the geology of the municipal- took place in open habitats of southern South ity of Aurora de Tocantins report carbonate and ter- America (Olivares and Verzi 2015). The youngest rigenous deposits (CPRM 2006). The lower portion fossil echimyids are mostly from the Pleistocene of area is represented by the Sete Lagoas Forma- to Holocene of Brazil (Table I; Winge 1887, tion, which is composed of thick deposits of mud- Ameghino 1907, Paula Couto 1950, Souza-Cunha stones, calcareous and dolomites presenting stro- and Guimarães 1978, Guerin et al. 1993, Emmons matolites. The Sete Lagoas Formation is covered and Vucetich 1998, Salles et al. 1999, 2006, Toledo by siltstone and laminate siltstones of the Serra de et al. 1999, Rodrigues and Ferigolo 2004, Chahud Santa Helena Formation. This carbonate-terrige-

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Figure 1 - Location of Aurora de Tocantins, which contains the Gruta dos Moura limestone cave. nous conjunction of rocks is superimposed by dark ground level in high portions of the plateaus. The calcarenites and marls with organic material from caves originated during a period of formation of the Lagoa do Jacaré Formation. Superimposed over karst relief in which the rocks that now compose the the Bambuí Group are the Cretaceous sediments of plateaus were below the ground level. The current the Urucuia Formation. The caves were developed outcrops were developed not by the uplift of the mainly in the limestones of Lagoa do Jacaré For- plateaus, but by the different types of erosion of the mation, which consist in slightly weathered dark limestones and the erosive retraction of the Urucuia gray metacalcarenites, massive or with horizontal sandstones that once covered the region and today lamination, with sparry calcite (generally in veins), occur to the east of the study area. Access to the micritic calcite, ooids and small amounts of silica. plateaus occurs mainly through vicinal roads and In some outcrops there are interbedded metacal- paths that cut the vegetation. Currently, the region carenites, mudstones and calcilutites (Avilla et al. is situated mainly within the Cerrado biome. 2013a). Millimeter- to centimeter-wide whitish The Gruta dos Moura cave is epigenic, gray intraclasts occur throughout the predominant solutional, ramiform to network cave with some limestones (CPRM 2006). branchwork passages (sensu Palmer 1991) and The limestones often form plateaus that rise has great vertical and horizontal development. from the rest of the terrain and comprise a partially The entire length of the cave has not yet been active karst system. Most caves occur above the completely mapped. The Gruta dos Moura cave

An Acad Bras Cienc (2016) 88 (2) 832 THAIS M.F. FERREIRA et al. originated in a phreatic zone and was later invaded METHODOLOGY and enlarged by vadose water. There is a notable The fossil remains of Echimyidae were collected difference between the upper and lower levels of the cave. The higher levels show galleries and fissures from a block of calcareous rock that was screen- with many speleothems, including travertines, washed by four sieves with meshes of 10 mm, 5 mm, draperies, stalactites, stalagmites and coraloids, 2.5 mm and 1 mm. Fossil materials and small pieces showing high activity of vadose waters. The fossils of rocks kept on the sieves were dried and sorted in were found in the lower levels, which consist of the laboratory with the help of a stereomicroscope. phreatic passages and rooms with few speleothems. The identification and diagnosis of the Slightly consolidated sedimentary deposits outcrop specimens from Gruta dos Moura cave were made at these levels, composed of massive, very fine by direct morphological comparison of molars and to coarse, immature and poorly sorted sands with dentaries of Echimyidae specimens housed at the clayey matrix. In some parts of the cave, a relict Setor de Mastozoologia of the Museu Nacional, carbonate precipitation layer can be seen that is a Universidade Federal do Rio de Janeiro, Rio de rich in millimeter- to centimeter-sized fragments of Janeiro, Brazil (MN/UFRJ); Museo Argentino de bones and teeth. This layer seems to have covered Ciencias Naturales ‘Bernardino Rivadavia’, Buenos the sedimentary deposits in a period of decreased Aires, Argentina (MACN); Museo de La Plata, sedimentation and reduced water flow, causing La Plata, Argentina (MLP); Museo de Ciencias the precipitation of carbonates and concentrating Naturales de Mar del Plata ‘Lorenzo Scaglia’, Mar sediment (Avilla et al. 2013a). del Plata, Argentina (MMP); Museum of Vertebrate The occurrence of sedimentary deposits Zoology, University of California, Berkeley, CA, covered by carbonate incrustation lenses is also USA (MVZ); Museu de Zoologia, Universidade observed in many other caves in the region of Aurora Federal de Bahia, Bahia, Brazil (UFBA); Museu do Tocantins. These sediments may correspond to a de Zoologia, Universidade de São Paulo, São large-scale event of increased humidity that caused Paulo, Brazil (MZUSP); Universidade de Brasília, changes in the pattern of recharge from the land Brasília, Brazil (UnB) (Appendix); and also by surface and consequently in the entire environment. comparisons with descriptions in the literature Similar occurrences were studied by Auler et al. (Emmons 2005). Dental nomenclature follows (2009), who identified three processes (i.e. clastic sediment input, erosion and calcite deposition) Marivaux et al. (2004) and homologies of crests that are linked to distinct palaeoenvironmental and are based on Verzi et al. (2014), Olivares and Verzi climatic conditions. (2015). The episodes of clastic input would be Abbreviations: MDL, mesiodistal lenght; LLW, related to a drier climate, sparse vegetation and Linguolabial width; UNIRIO-PM, Coleção de intense sediment yield due to runoff, while the Paleontologia de Mamíferos da Universidade precipitation of calcite would be related to wetter Federal do Estado do Rio de Janeiro, Brazil. conditions (Auler and Farrant 1999, Brain 1995, Brook et al. 1997). Sediment erosion inside those RESULTS caves can be interpreted as cause of intermediate Systematic Paleontology climatic conditions, which were not humid enough for speleothem deposition and not dry enough to Rodentia Bowdich, 1821 allow transportation of sediment into the caves Hystricomorpha Brandt, 1855 (Auler et al. 2009). Hystricognathi Tullberg, 1899

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Octodontoidea Simpson, 1945 Description: This taxon is the most abundant in Echimyidae Gray, 1825 our sample. Thrichomys sp. is represented by 19 Thrichomys Trouessart, 1880 upper and lower teeth with distinct wear stages and Thrichomys sp. a dentary with dp4-m2 corresponding to a juvenile Material: UNIRIO-PM 2753, right dentary with specimen (m2 unworn and unerupted m3). dp4-m2; UNIRIO-PM 2754, left M1 or M2; The upper cheek teeth show distinct wear UNIRIO-PM 2755, left dp4; UNIRIO-PM 2756, stages. Right DP4 (UNIRIO-PM 4795: Figure 2 c) left dp4; UNIRIO-PM 4795, right DP4; UNIRIO- shows little wear (juvenile specimen sensu Neves PM 4796, left dp4; UNIRIO-PM 4797, left dp4; and Pessôa 2011) and it is trilophodont, but the UNIRIO-PM 4798, left M1 or M2; UNIRIO-PM union between of the last loph and the hypocone is 4799, left M1 or M2; UNIRIO-PM 4800, left M1 or wide, evidencing the coalescence of the mesoloph- M2; UNIRIO-PM 4801, left M1 or M2; UNIRIO- ule with the posteroloph + metaloph. The enamel PM 4802, left M1 or M2; UNIRIO-PM 4803, left layer of the anterior face of this loph is oblique and M1 or M2; UNIRIO-PM 4804, left M1 or M2; antero-posteriorly wide as in Thrichomys pachy- UNIRIO-PM 4805, right M1 or M2; UNIRIO-PM urus (Wagner, 1845) (see Neves and Pessôa 2011: 4806, right M1 or M2; UNIRIO-PM 4807, right M3; fig. 2A, B). There is no evidence of a fossette in the UNIRIO-PM 4808, right m1 or m2; UNIRIO-PM last loph, distinguish it from Thrichomys inermis 4809, left m1 or m2; UNIRIO-PM 4810, left m3. Pictet, 1841, in which the mesolophule appears as

Figure 2 - Thrichomys sp. from the late Pleistocene of Tocantins. a-b, Right dentary with the dp4-m2 series (UNIRIO-PM 2753) in lateral (a) and occlusal views (b). c, Right DP4 (UNIRIO-PM 4795) in occlusal view. d, Left M1 or M2 (UNIRIO-PM 2754) in occlusal view. e, Left dp4 (UNIRIO-PM 2756) in occlusal view. Scale bar: 1 mm. (See the colors in the online version).

An Acad Bras Cienc (2016) 88 (2) 834 THAIS M.F. FERREIRA et al. an independent crest in juvenile specimens (Neves material of the Quaternary of Ceará (Oliveira et al. and Pessôa 2011: fig. 3A-C). 2013: fig. 6B) in which the mesoflexid is shorter. The M1 or M2 correspond to sub-adults and The dentary is slender and the notch for the tendon adults with distinct wear stages. The sub-adult of the infraorbital part of the medial masseter specimens (sensu Neves and Pessôa 2011) resemble muscle (i.e. masseteric notch) is a semicircular T. pachyurus, Thrichomys laurentius Thomas, 1904 step at level of dp4, incorporated into the origin and Thrichomys sp. from Tocantins (Rocha et al. of the masseteric crest as in the remaining species 2011: fig. 19), in which the protoloph is uniformly of the genus (Olivares and Verzi 2015). The dental wide, and differ from the other species where the measurements among species of Thrichomys are labial portion of this loph is wider than the medial very similar. However, T. pachyurus is slightly portion (M1-M2 of T. pachyurus in Neves and larger than T. inermis and T. apereoides (Oliveira Pessôa [2011: fig. 2F] compared to Figure 2d). et al. 2013: table 4). The material described here, in In these molars, all lophs are of similar length some cases, is still a little bigger than T. pachyurus. compared to T. laurentius, Thrichomys apereoides Measurements: See Table II. (Lund, 1839) and Thrichomys sp. (Rocha et al. Commentaries: Thrichomys has cursorial habits 2011: fig. 19) but not inT. pachyurus and T. inermis and lives in open areas from Caatinga, Cerrado in which the protoloph is slightly longer (Neves and Pantanal (Oliveira and Bonvicino 2006). The and Pessôa 2011). The tetralophodont molars of alfa taxonomy of this genus is not well known (see the sample could correspond to the M2 because Pessôa et al. 2004, Reis and Pessôa 2004, Oliveira they are longer than wide. These preserve the and Bonvicino 2006). Four species are recognized: metafossette in advanced wear stages (molars with Thrichomys apereoides (Lund, 1839), T. inermis para- and mesofossette), such as in the T. inermis (Pictet, 1843), T. pachyurus (Wagner, 1845), and and T. pachyurus (Neves and Pessôa 2011: figs. 2G T. laurentius Thomas, 1904 (Pessôa et al. 2015). and 3G). In a recent study, the morphological distinctions among T. laurentius, T. inermis and T. pachyurus The upper molars of this sample are similar have been recognized (Neves and Pessôa 2011). to the material recently described from the late D’Elía and Myers (2014) recognized the validity of Quaternary of Ceará and Piauí (Oliveira et al. 2013, Thrichomys fosteri Thomas, 1903 for populations Kerber et al. 2016). However, the anteroloph of from Paraguay and Mato Grosso do Sul. Rocha et the DP4 of Ceará (Oliveira et al. 2013: fig. 6) is al. (2011) identified a distinct species in the western more transversely oriented than in the DP4 of the region of Tocantins, different from T. inermis and sample of Tocantins. The upper molars of Ceará T. pachyurus (see also Braggio and Bonvicino correspond to a juvenile (sensu Neves and Pessôa 2004). The materials recovered from the caves 2011), whereas in the sample of Tocantins there is are more similar to T. pachyurus, T. inermis and only one DP4 and molars belong to sub-adults and Thrichomys sp., of the western region of State of adults, which difficult their comparison. Tocantins (Rocha et al. 2011) than the remaining Lower cheek teeth are mostly of juvenile living species of the genus. Currently, T. pachyurus specimens, as seen in UNIRIO-PM 2753 (Figure is distributed to the east and south of the studied 2a, b), in which the tetralophodont dp4 shows area, while T. aff. inermis is found to the north little wear and preserves the lophids and flexids (Nascimento et al. 2013). Fossils of Thrichomys or newly formed fossettids (UNIRIO-PM 2756; have been found in the intertropical region of Figure 2e). All labial flexids of dp4 are similar in Brazil (Table I), including the late Quaternary of length, which is different from T. laurentius and the the States of Mato Grosso do Sul (Salles et al.

An Acad Bras Cienc (2016) 88 (2) LATE PLEISTOCENE ECHIMYID RODENTS FROM NORTHERN BRAZIL 835 TABLE I TABLE PE, Pernambuco; PI, Piauí; RS, Rio Grande do Sul; SP, São Paulo; TO, Tocantins; †, extinct taxon. Tocantins; TO, São Paulo; PE, Pernambuco; PI, Piauí; RS, Rio Grande do Sul; SP, (Winge, 1887)(Winge, 1887)(Winge, Grutas de Lagoa Santa/MG Sítio Garivaldino/RS Quaternary Holocene Paula Couto (1950) Hadler et al. (2008) (G. Fisher, 1814)(G. Fisher, Grutas de Iporanga, Iporanga/SP Quaternary Ameghino (1907) (Wagner, 1845)(Wagner, 1845)(Wagner, Grutas de Iporanga, Iporanga/SP Grutas de Lagoa Santa/MG Quaternary Quaternary Ameghino (1907) Paula Couto (1950) (Desmarest, 1817) Gruta dos Moura/TO Late Pleistocene This work Hadler et al. 2008 Sítio Garivaldino/RS Holocene Hadler et al. (2008) (Desmarest, 1817) Serra dos Carajás/PA Holocene et al. (1999) Toledo (Lund, 1840) Grutas de Lagoa Santa/MG Quaternary Paula Couto (1950) (Lund, 1841)(Lund, 1841)(Lund, 1841)(Lund, 1841) MG Emperaire (P.L), Vermelha G.A. da Lapa Serra da Bodoquena/MS Holocene Serra da Mesa/GO Grutas de Lagoa Santa/MG Souza-Cunha and Guimarães (1978) Quaternary Quaternary Quaternary Salles et al. (2006) Paula Couto (1950) Salles et al. (1999) Goeldi, 1901 Sítio Deobaldino Marques/RS Holocene Roth et al. (2008) (Desmarest, 1821) Grutas de Lagoa Santa/MG Quaternary Paula Couto (1950) (Thomas, 1909) Grutas de Lagoa Santa/MG Quaternary Paula Couto (1950) Jentink, 1891Jentink, 1891 Abismo Ponta de Flecha/SP Abismo Ponta de Flecha/SP Quaternary Quaternary Chahud (2005) Castro and Langer (2011) didelphoides J. A. Allen, 1899A. J. Allen, 1899A. J. Allen, 1899A. J. Serra da Mesa/GO Serra dos Carajás/PA Gruta dos Moura/TO Quaternary Holocene Late Pleistocene Salles et al. (1999) This work et al. (1999) Toledo Lund, 1839 Serra da Capivara/PI Late Pleistocene/Holocene Kerber et al. (2016) Lund, 1839Buíque/PEAlcobaça, Sítio Holocene Ferreira et al. (2012) Lund, 1839 Abismo Ponta de Flecha/SP Quaternary Castro and Langer (2011) Lund, 1839 Sítio Deobaldino Marques/RS Holocene Roth et al. (2008) Lund, 1839 Sítios Sangão and Garivaldino/RS Holocene Hadler et al. (2008) Lund, 1839 Emperaire (P.L)Vermelha G.A. da Lapa Holocene Souza-Cunha and Guimarães (1978) cf. Thomas, 1921 Abismo Ponta de Flecha/SP Quaternary Castro and Langer (2011) Cuvier, 1809Cuvier, Serra dos Carajás/PA Holocene et al. (1999) Toledo Euryzygomatomys mordax Euryzygomatomys mordax Euryzygomatomys mordax Clyomys riograndensis Quaternary fossil records of Echimyidae from Brazil. Abbreviations: BA, Bahia; CE, Ceará; GO, Goiás; MG, Minas Gerais; MS, Mato Grosso do Sul; PA, Pará; do Sul; PA, BA, Bahia; CE, Ceará; GO, Goiás; MG, Minas Gerais; MS, Mato Grosso Abbreviations: Brazil. of Echimyidae from Quaternary fossil records Trinomys setosus Trinomys Trinomys † † Euryzygomatomys spinosus Euryzygomatomys Clyomys laticeps † Phyllomys Phyllomys Phyllomys Phyllomys Phyllomys Dactylomys dactilynus Kannabateomys amblyonyx Kannabateomys amblyonyx Kannabateomys Kannabateomys Phyllomys brasiliensis Phyllomys Taxa sulcidens Carterodon sulcidens Carterodon sulcidens Carterodon sulcidens Carterodon Proechimys Proechimys Proechimys Echimys Makalata Provenance Age Reference

An Acad Bras Cienc (2016) 88 (2) 836 THAIS M.F. FERREIRA et al. TABLE I (continuation) TABLE Winge, 1887Winge, Sítio Deobaldino Marques/RS Holocene Roth et al. (2008) (Lund, 1839)(Lund, 1839)(Lund, 1839)(Lund, 1839) Grutas de Lagoa Santa/MG Serra da Mesa/GO Serra da Bodoquena/MS Serra da Capivara/PI Quaternary Quaternary Quaternary Paula Couto (1950) Late Pleistocene/Holocene Guerin et al. (1993) Salles et al. (2006) Salles et al. (1999) Winge, 1887Winge, 1887Winge, 1887Winge, Grutas de Lagoa Santa/MG Sítios Sangão and Garivaldino/RS Grutas de Iporanga /SP Holocene Quaternary Quaternary Hadler et al. (2008) Paula Couto (1950) Ameghino (1907) (Molina, 1782)(Molina, 1782)(Molina, 1782)(Molina, 1782)(Molina, 1782)(Molina, 1782) Cavernas de Lagoa Santa/MG(Molina, 1782) do Ossos/BA Toca Plataforma continental, B. Hermenegildo/RS Sítio Sangão/RS Pleistocene Passo /RSTouro Arroio Sanga da Cruz /RS Quaternary Arroio Chuí /RS Rodrigues and Ferigolo (2004) Quaternary (1887); Kerber et al. (2014) Winge Late Pleistocene Holocene Late Pleistocene Kerber et al. (2014) Kerber et al. (2014) Late Pleistocene Kerber et al. (2014) Hadler et al. (2008) Kerber et al. (2014) fossor Kerr, 1792 Kerr, Serra da Capivara/PI Late Pleistocene/Holocene Kerber et al. (2016) aff. aff. Emmons and Vucetich, 1998Vucetich, Emmons and Lapa do Capão Seco, Lagoa Santa/MG Quaternary (1998) Vucetich Emmons and Trouessart, 1880Trouessart, 1880Trouessart, 1880Trouessart, 1880Trouessart, 1880Trouessart, Buíque/PEAlcobaça, Sítio Emperaire (P.L)/MGVermelha G.A. Lapa Parque Nacional de Ubajara, Ubajara/CE Serra da Capivara/PI Holocene Gruta dos Moura/TO Holocene Holocene Souza-Cunha and Guimarães (1978) Oliveira et al. (2013) Late Pleistocene/Holocene Ferreira et al. (2012) Kerber et al. (2016) Late Pleistocene This work Thomas, 1916 Serra da Bodoquena/MS Quaternary Salles et al. (2006) Myocastor Dicolpomys fossor Dicolpomys fossor Dicolpomys fossor Dicolpomys TaxaClyomys Thrichomys apereoides Thrichomys apereoides Thrichomys apereoides Thrichomys apereoides Thrichomys Thrichomys Thrichomys Thrichomys Thrichomys † † Provenance† † Callistomys Myocastor coypus Myocastor coypus Myocastor coypus Myocastor coypus Myocastor coypus Myocastor coypus Myocastor coypus Agecf. Eumysopinae indet. Reference Plataforma continental, B. Hermenegildo/RS Pleistocene Rodrigues and Ferigolo (2004)

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2006), Goiás (Salles et al. 1999), Minas Gerais TABLE II (Paula Couto 1950, Souza-Cunha and Guimarães Measurements (in mm) of the late Pleistocene equimyids from Tocantins. MDL, mesiodistal length; LLW, 1978), Piauí (Kerber et al. 2016), Ceará (Oliveira labiolabial width. et al. 2013) and Pernambuco (Ferreira et al. 2012). Taxon/specimen MDL LLW Thrichomys sp. Proechimys Allen, 1899 UNIRIO-PM 4795 - DP4 1.81 2.18 Proechimys sp. UNIRIO-PM 2754 - M1 or M2 3.36 2.30 UNIRIO-PM 4798 - M1 or M2 2.31 2.03 UNIRIO-PM 4799 - M1 or M2 2.65 2.35 UNIRIO-PM 4800 - M1 or M2 2.23 2.02 UNIRIO-PM 4801 - M1 or M2 2.73 2.00 UNIRIO-PM 4802 - M1 or M2 2.05 1.94 UNIRIO-PM 4803 - M1 or M2 1.75 1.70 UNIRIO-PM 4804 - M1 or M2 2.38 1.66 UNIRIO-PM 4805 - M1 or M2 2.16 1.80 UNIRIO-PM 4806 - M1 or M2 2.22 2.22 Figure 3 - Proechimys sp. from the late Pleistocene of UNIRIO-PM 4807 - M3 2.07 2.40 Tocantins. Right M1 or M2 (UNIRIO-PM 2757) in occlusal UNIRIO-PM 2755 - dp4 2.84 1.58 view. Scale bar: 1 mm. (See the colors in the online version). UNIRIO-PM 2756 - dp4 2.50 1.83 UNIRIO-PM 4796 - dp4 1.45 2.25 Material: UNIRIO-PM 2757, right M1 or M2. UNIRIO-PM 4797 - dp4 1.40 1.78 Description: The specimen is a right M1 or M2 UNIRIO-PM 4808 - m1 or m2 2.25 2.49 with advanced stage of wear. All labial flexi are UNIRIO-PM 4809 - m1 or m2 2.23 2.27 UNIRIO-PM 4810 - m3 1.55 1.84 closed to form fossettes, and the hypoflexus is UNIRIO-PM 2753 - dp-m2 series 8.11 2.61 still open. The presence of three fossettes equally Proechimys sp. penetrating in the occlusal surface differentiate UNIRIO-PM 2757- M1 or M2 2.76 2.51 UNIRIO-PM 2757 from Trinomys Thomas, 1921, Makalata cf. didelphoides Mesomys Wagner, 1845 and Isothrix Wagner, 1845, UNIRIO-PM 2751 - dp4 3.53 2.03 in which the mesoflexus (and the corresponding UNIRIO-PM 2752 - M1 3.16 2.33 mesofossette) is less penetrating than the remaining flexi (fossettes). UNIRIO-PM 2757 shows a Cerrado. In the living fauna of Tocantins, P. roberti well-developed metafossette (Figure 3); this is recorded (Weksler and Bonvicino 2010, Rocha feature differentiates it from Thrichomys, which et al. 2011). Although the material described herein in a similar wear stage has the metafossette more is similar to this species (see Rocha et al. 2011: fig. reduced or absent. Among the variation in lophs 17), the information of a single molar is limited to pattern showed by Proechimys, an occlusal design differentiate it from the other species. of M1-2 with four lophs as that in UNIRIO-PM The fossil record of Proechimys includes 2757, is the most frequent (Patton 1987). materials assigned to Proechimys sp. from the Measurements: See Table II. Plio-Pleistocene of Venezuela (Rincón et al. Commentaries: Proechimys is the most speciose 2009), Holocene of Pará (Toledo et al. 1999) and living echimyid with more than twenty species Quaternary of Goiás (Salles et al. 1999) (Table I). recognized across a wide geographic range (Patton The assignment of “Eumysops” ponderosus, from 1987, Patton and Leite 2015). This genus occupies the Late Miocene of Argentina, to Proechimys (see diverse environments in Amazonian forest and Bond 1977) is contingent on a revision (Reig 1989).

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Makalata Husson, 1978 IP 267), the lingual margin of protocone area Makalata cf. didelphoides (Desmarest, 1817) is straight, and the entire area of protocone plus protoloph is shaped like a triangle, and the anterior side of posteroloph has a slight medial convexity (cf. Fig. 4a and Iack-Ximenes 2005, fig. 11D). The stage of UNIRIO-PM 2751 indicates that is not an adult individual because this pattern is similar to specimens of Makalata with unerupted or scarcely worn M3 (e.g. MN/UFRJ 21499, IP 267). The specimen UNIRIO-PM 2751 (Fig. 4b) is a left dp4 with a general occlusal morphology similar to that of Makalata, Echimys Cuvier, 1809 and Toromys Iack-Ximenes, Vivo and Percequillo, 2005. The metalophulid I (or anterolophid) and metalophulid II (e.g. MN/UFRJ 21499, 21494) form a curved lamina (Emmons 2005) that is in contact with the mesolophid lingually. This lamina has a strong posterior fold because of the metalophulid II is interrupted leaving the anterior fossette opened backwards. The mesolophid is slightly sigmoid, different to that of Echimys and Toromys, which is straight. The presence of a shallow fold in the anterior margin of this lophid, combined with the curved morphology of the anterior lamina, is shared Figure 4 - Makalata cf. didelphoides from the late with specimens of Makalata didelphoides. The last Pleistocene of Tocantins. a, Right M1 (UNIRIO-PM 2752) in occlusal view. b, Left dp4 (UNIRIO-PM 2751) in occlusal two lophids, hypolophid and posterolophid, are view. Scale bar: 1mm. (See the colors in the online version). labially connected. UNIRIO-PM 2751 (Table II) is similar in size to the dp4 of Makalata (MDL – 3.45 Material: UNIRIO-PM 2751, left dp4; UNIRIO- mm), and smaller than those of Echimys (MDL – PM 2752, right M1. 4.43 mm) and Toromys (MDL – 4.95 mm). Detailed Description: UNIRIO-PM 2752 (Figure 4a) is a complementary morphology of UNIRIO-PM right M1. The anteroloph and the protoloph are 2751 and UNIRIO-PM 2752 is equivalent to that lingually connected and there is no indication observed in the specimen Makalata didelphoides of mure, as it occurs in the anterior part of M1 MZUSP 899 (Iack-Ximenes et al. 2005, fig. 11D). of Makalata didelphoides and Santamartamys Measurements: See Table II. rufodorsalis (J.A. Allen, 1899). As in M1 of the Commentaries: The specimens reported here latter two species, these lophs (and the paraflexus) have laminar cheek teeth with a morphology are slightly anterolabially oriented while distinctive of Echimyinae (Emmons 2005). This mesolophule and posteroloph (and mesoflexus) are group within Echimyidae, as defined by Emmons more transverse. The metaflexus is more penetrating (2005), includes Echimys, Phyllomys Lund, 1839, than the paraflexus and is open only labially. As Makalata, Diplomys Thomas, 1916, Callistomys in Makalata didelphoides (e.g. MN/UFRJ 21499, Emmons and Vucetich, 1998, Pattonomys

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Emmons, 2005, and Santamartamys Emmons, is scarce and its alpha taxonomy and chronology 2005. Toromys was recently included within this remains poorly understood. Most of Brazilian subfamily (Emmons et al. 2015a). records are from Quaternary sediments of caves Makalata includes arboreal echimyids that are (Table I) and have limited temporal resolution. restricted to the Amazon region where they occupy The fossils material derived from old collections, areas near bodies of water (Emmons 2005, Oliveira such as the classic fauna of Lagoa Santa, clearly and Bonvicino 2006). The taxonomic status of liv- need a review. In this sense, the collection of new ing Makalata species has been in state of flux. At specimens with stratigraphic control associated to least eight species were recognized by Woods and numerical ages is important to understand the past Kilpatrick (2005). According to Emmons (2005), diversity of the group and how such diversity was living representantives are divided into two groups: affected by Pleistocene climatic oscillations. “didelphoides group” and “grandis group”. The Based on the stratigraphic control of the mate- IUCN redlist recognizes at least four valid species rial described herein, we can reliably assume that - Makalata didelphoides, Makalata macrura (Wag- specimens from Gruta dos Moura cave represent a ner, 1842), Makalata rhipidura (Thomas, 1928) synchronous sample. The calcareous block where and Makalata obscura (Wagner, 1840) - (Patton et the echimyid fossil remains were found belongs al. 2008a, b, c, Zeballos et al. 2008). Iack-Ximenes to the same stratigraphic level found throughout et al. (2005) assigned one of the species of “grandis one of the secondary tunnels of Gruta dos Moura group” to a genus of its own, Toromys, i.e. Toromys cave. The sedimentological features, such as color grandis. M. rhipidura is considered Toromys rhipi- and granulometry, also suggest that the calcareous durus, but this is pending a more thorough review blocks had the same genesis (Avilla et al. 2013a). (Emmons et al. 2015b). Accordingly, Makalata is Disaggregation of the block revealed that restricted only to “didelphoides group” (i.e. M. di- there was a taphonomic selection by size, where delphoides, M. macrura and M. obscura; Emmons only fragments of small vertebrates, especially and Patton 2015). mammals such as marsupials (Villa Nova et al. The presence of Makalata in Gruta dos Moura 2015), bats and rodents, were preserved. The only implies the first mention of this genus in the fossil exception was a fragment of a maxillary bone with record (Table I). As noted above, the occlusal an almost complete upper tooth row of the tayassuid morphology of the recovered teeth resembles M. Catagonus stenocephalus (Lund in Reinhardt, didelphoides, the living representative of the genus 1880), an extinct mammal from the South American currently inhabiting Tocantins (Patton et al. 2008a). Pleistocene which dated by ESR (Electron Spin This species is distributed in the islands of Trinidad Resonance) to 20.000 (± 2000) years before and Tobago, Venezuela, French Guiana, Suriname, present (YBP; Avilla et al. 2013a). In accordance, Guyana, southwest to northeastern of Bolivia we consider here that this dating applies to the and Brazil (States of Amazonas, Roraima, Pará, entire fossil assemblage found in the calcareous Amapá, Mato Grosso and Tocantins) (e.g. Emmons blocks of the secondary tunnel of Gruta dos Moura and Patton 2015). cave. Thus, the diversity of echimyids described

DISCUSSION here, considering that Makalata is recorded as a fossil also for the first time, represents part of the Brazil hosts the largest diversity of living echimyids. fauna that inhabited the region around the Gruta Both in this key area and other zones within the dos Moura cave during the Last Glacial Maximum current distribution of this family, the fossil record (LGM). The LGM occurred at approximately

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20.000 YBP, and represents the coldest and drier and Amazonia (Makalata) recorded together for climatic period of South America of the last million the first time (Table I). Similarly, some fossil years. The presence of Catagonus stenocephalus didelphids of Gruta dos Moura are associated in these sediments indicates the development of with open and deciduous forest areas and others relatively open and dry environments. The ancestral with humid forest (Villa Nova et al. 2015). Such limits of the Chacoan subregion could be traced an interaction could result from the presence of analyzing its fossil record and other taxa with gallery forests, which act as dispersal corridors similar ecological requirements (e.g. Reithrodon for rainforest mammals (e.g. Redford and Fonseca auritus G. Fisher, 1814 - Tobelém et al. 2013, 1986, Mares et al. 1989, Costa 2003), or by the Avilla et al. 2013a). Remarkably, the taxonomic presence of ecotones between these biomes (e.g. composition of the echimyids fossil samples from Rocha et al. 2011). Alternately, this pattern, which Gruta dos Moura is similar, at least at generic level, includes taxa with distinct environmental affinities to the current assemblage from the Mid-Araguaia absent at present in the region of Gruta dos Moura, River basin in the west of State of Tocantins (Rocha could be associated with oscillations of the climatic et al. 2011). The last area represents an ecotone pulses that occurred in the past during the interval between Cerrado and Amazonia in which the typical recorded in this region. In any case, the presence Cerrado’s echimyid Thrichomys sp. coexists with of Amazonian species could help in understanding the Amazonian Makalata didelphoides (Rocha et al. the dynamics of ecotones or dispersal corridors 2011). The other representative of this community, between Amazonian and Cerrado. Proechimys, is the most widely distributed echimyid The data here reported contributes to the genus inhabiting lowland rainforest habitats in understanding of the ancient diversity of vertebrates Central America, cis- and trans- Amazonia, dry of that region, evidenced until now in other groups, forest in Bolivia and Paraguay, and the Cerrado of such as the artiodactyls, cingulates, carnivores, central Brazil (Patton and Leite 2015). The Gruta cervids, marsupials and squamates (e.g. Avilla et dos Moura cave is located in the current Cerrado, al. 2013a, b, Castro et al. 2013, Hsiou et al. 2013, farther east than Mid-Araguaia River basin. Rodrigues et al. 2014, Villa Nova et al. 2015). The fossil record of Echimyidae in Brazil during the Quaternary is represented, on the one ACKNOWLEDGMENTS hand, by typical Amazonian genera recorded at We would like to thank Anselmo Rodrigues, Wag- Serra dos Carajás in the Holocene of Pará (Toledo ner Moura, the Municipality of Aurora do Tocan- et al. 1999) and typical Cerrado genera registered tins and the staff of the Sociedade Brasileira de at Serra da Bodoquena (Salles et al. 2006), and on Espeleologia, who invited us and gave us logistic the other hand, it is represented by both Cerrado help in Aurora do Tocantins. We appreciate all help (e.g. Thrichomys and Clyomys Thomas, 1916) (and from the group involved in the prospection and Caatinga – Thrichomys) and Atlantic forest genera collection of the material. We also thank S. Maia (Phyllomys and Callistomys) recorded together at Vaz, C. Bonvicino, D. Flores, D. Romero, J.L. Pat- sites such as Lagoa Santa (Winge, 1887, Emmons ton, E. Lacey, C. Conroy, J. Oliveira, W. Kliem and and Vucetich 1998), Sangão and Garivaldino M. de Vivo for granting access to materials under (Hadler et al. 2008), Sítio Alcobaça (Ferreira et their care. We especially thank D.H. Verzi for the al. 2012), and Serra da Capivara (Kerber et al. helpful discussion about the systematics and evolu- 2016). The fossil echimyid assemblage studied tion of echimyids described here and for valuable herein includes genera from Cerrado (Thrichomys) comments on an earlier version of this manuscript.

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Trinomys, T. dimidiatus UnB 723, 739; MN-UFRJ Thrichomys, T. apereoides UnB 188 – 190, 62273, 62275, 62278; T. yonenagae UFBA 00467 T. inermis MMP 1242 – 1245, 1295, 1296, T. – 00473. laurentius MMP 1246, 1293, 1294, 1297, MN/ Proechimys, P. brevicauda MVZ 153623; P. UFRJ 14511, 13664, 42505, 42411, 42395,42403, cuvieri MZUSP 21257; P. goeldii MZUSP 26621; 42451, 42483, 42458, 42446, 42386, 42457, T. P. poliopus MLP 22.II.00.8, 22.II.00.7; P. roberti pachyurus MN/UFRJ 46896; 616599 (T. fosteri?), MVZ 197578, UnB 316, 326; P. semispinosus Thrichomys sp. MMP USB 542, MMP 1247, 1292, MZUSP 2002; Proechimys sp. MZUSP 27551. 150, MN/UFRJ 34406.

An Acad Bras Cienc (2016) 88 (2)

Methods paper

Casa de Lletres Academic Editing Services [email protected] www.casadelletres.eu International Journal of Environmental Research and Public Health

Article Measurement of Reduced Gingival Melanosis after Smoking Cessation: A Novel Analysis of Gingival Pigmentation Using Clinical Oral Photographs

Tomotaka Kato 1,*, Hiroya Takiuchi 1, Seiichi Sugiyama 2, Michiko Makino 1, Satoshi Noguchi 1, Tomoko Katayama-Ono 3, Takashi Hanioka 4 and Toru Naito 1,*

1 Section of Geriatric Dentistry, Department of General Dentistry, Fukuoka Dental College, Fukuoka 814-0193, Japan; [email protected] (H.T.); [email protected] (M.M.); [email protected] (S.N.) 2 Sugiyama Dental Clinic, Yachiyo 276-0027, Japan; [email protected] 3 Oro-facial Plastic Medical Center, Department of Oral & Maxillofacial Surgery, Fukuoka Dental College, Fukuoka 814-0193, Japan; [email protected] 4 Section of Oral Public Health, Department of Preventive and Public Health Dentistry, Fukuoka Dental College, Fukuoka 814-0193, Japan; [email protected] * Correspondence: [email protected] (T.K.); [email protected] (T.N.); Tel.: +81-92-801-0411 (T.K.); +81-92-801-0411 (T.N.)

Academic Editor: Paul B. Tchounwou Received: 31 March 2016; Accepted: 9 June 2016; Published: 16 June 2016

Abstract: Background: Due to moisture and the anatomical complexity of the oral mucosa, it is difficult to measure the extent of gingival melanosis in an optical manner. Therefore, we developed a new quantitative method using clinical oral photographs and compared the extent of gingival melanosis before and after smoking cessation. Methods: A new analysis method, which we named the gingival melanosis record (GMR), is a quantitative analysis method using clinical oral photographs. We obtained 659 clinical photographs from 263 patients from 16 general dental offices in Japan. Standardized measuring sites were automatically spotted on the screen, and the presence of gingival melanosis was determined at the measuring sites. We assessed the validity of the GMR with the previously reported Hedin’s classification using Spearman’s rank correlation and intraclass correlation coefficients. Results: The GMR showed a significant association with Hedin’s classification (p < 0.01, correlation coefficient = 0.94). The GMR also showed excellent reproducibility of the substantial repeated agreement intraclass correlation coefficients (ICC) (1,1) and ICC (2,1), p > 0.61). The longitudinal loss of gingival melanosis was confirmed by a change in the GMR among patients who successfully achieved smoking cessation for a mean of 4.5 years. Conclusion: The GMR is an effective method to assess gingival melanosis. The loss of gingival melanosis after smoking cessation can be objectively confirmed with the use of the GMR.

Keywords: gingival melanosis; smoking; clinical oral photographs

1. Introduction It is well known that cigarette smoking is a major health problem [1]. Smoking is an apparent risk for periodontal disease [2] and oral cancer [3,4]. Oral healthcare professionals, who have relatively long teachable moments with patients, should work toward developing smoking cessation programs in their own clinics. In 1977, Hedin et al. ﬁrst reported that smokers showed more pigmented gingiva than non-smokers [5]. They referred to this gingival pigmentation as “smoker’s melanosis”. There is an ethnic difference for gingival melanosis, with a prevalence of 100% in the Black population [6],

Int. J. Environ. Res. Public Health 2016, 13, 598; doi:10.3390/ijerph13060598 www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2016, 13,598 2of8

Int. J. Environ. Res. Public Health 2016, 13, 598 2 of 8 40% in the Asian population [7], and 5%–10% in the Caucasian population [8,9]. The pigmentation of in the Asian population [7], and 5%–10% in the Caucasian population [8,9]. The pigmentation of the the human gingival tissue is derived from melanin granules, which are synthesized in melanosomes human gingival tissue is derived from melanin granules, which are synthesized in melanosomes of of melanocytes [10,11]. The nicotine present in tobacco activates melanocytes to promote melanin melanocytes [10,11]. The nicotine present in tobacco activates melanocytes to promote melanin secretion [12,13]. Thus, the melanin pigmentation in gingival tissues has a strong correlation with secretion [12,13]. Thus, the melanin pigmentation in gingival tissues has a strong correlation with smokingsmoking habits habits [5 ,[5,14].14]. A previousA previous study study also also suggested suggested thatthat thethe severityseverity of gingival pigmentation pigmentation decreased decreased after after smokingsmoking cessation cessation and and was was related related to to the the number number ofof yearsyears afterafter smoking cessation cessation [9,12]. [9,12]. These These findingsfindings indicate indicate that that there there is is a a bi-directional bi-directional associationassociation between tobacco tobacco smoking smoking and and melanin melanin pigmentationpigmentation in in the the gingiva gingiva and and the the possibility possibility ofof recoveryrecovery of normal gingival gingival pigmentation pigmentation by by smokingsmoking cessation. cessation. MonitoringMonitoring the the gingival gingival color color may may provideprovide anan indicatorindicator of of the the biological biological reaction reaction to to smoking, smoking, andand may may thus thus be abe tool a tool for for instruction instruction and and supporting supporting smoking smoking cessation. cessation. We We previously previously reported reported the precisethe measurementprecise measurement of gingival of pigmentationgingival pigmentation with a non-contact with a type non-contact of dental spectrophotometer,type of dental andspectrophotometer, demonstrated the and relationship demonstrated between the relationship smoking status between and smoking gingival status color and [15]. gingival However, color this procedure[15]. However, requires this special procedure equipment. requires Therefore, special weequipment. developed Therefore, a new quantitative we developed method a new using clinicalquantitative oral photographs method using and clinical compared oral photograph the extents of and gingival compared melanosis the extent before of gingival and after melanosis smoking cessationbefore and with after this smoking technique. cessation The purpose with this of techniqu this studye. The was purpose to validate of this the study new quantitativewas to validate method the andnew to clarifyquantitative the relationship method and between to clarify smoking the cessationrelationship and between the reduction smoking in gingival cessation melanosis. and the reduction in gingival melanosis. 2. Materials and Methods 2. Materials and Methods 2.1. Patients and Ethical Considerations 2.1. Patients and Ethical Considerations Longitudinal clinical oral photographs and treatment records were obtained with patient consent from 16Longitudinal general dental clinical offices oral in Japan. photographs Patients and who treatment visited these records dental were offices obtained for regular with check-upspatient andconsent had series from of 16 oral general photographs dental offices were in enrolled Japan. inPa thistients study. who Twovisited hundred these dental and eighty-three offices for regular patients check-ups and had series of oral photographs were enrolled in this study. Two hundred and with 900 oral photographs were enrolled in this study; all patients provided their informed consent. eighty-three patients with 900 oral photographs were enrolled in this study; all patients provided To maintain anonymity, patients were given unique ID numbers and the personal information was their informed consent. To maintain anonymity, patients were given unique ID numbers and the kept at the clinic. This study protocol was approved by the Ethics Committee for Clinical Research at personal information was kept at the clinic. This study protocol was approved by the Ethics Fukuoka Dental College (permission number 194). Committee for Clinical Research at Fukuoka Dental College (permission number 194). 2.2. Measurement of Gingival Pigmentation 2.2. Measurement of Gingival Pigmentation Gingival pigmentation was measured using frontal oral photographs taken by a non-contact-type Gingival pigmentation was measured using frontal oral photographs taken by a dentalnon-contact-type camera. We employeddental camera. commonly We employed used digital commonly cameras used and digital standardized cameras and photograph standardized taking techniques.photograph All taking the dentists techniques. and dental All the hygienists dentists wereand dental took standardized hygienists were oral took photo standardized taking class. oral Oral photographsphoto taking were class. reviewed Oral photogra with aphs liquid were crystal reviewed display with (Multi a liquid Sync crystal LCD display 2490WUXi2, (Multi Sync NEC LCD Corp., Tokyo,2490WUXi2, Japan), and NEC the Corp., target Tokyo, site was Japan), plotted and on the the target screen. siteThe was target plotted site on was the plottedscreen. The in the target following site mannerwas plotted (Figure in1 the): following manner (Figure 1):

FigureFigure 1. 1.Measurement Measurement of of gingival gingival pigmentationpigmentation (gingival melanosis melanosis record). record).

Int. J. Environ. Res. Public Health 2016, 13,598 3of8

Int. J. Environ. Res. Public Health 2016, 13, 598 3 of 8 (1) A baseline was drawn horizontally on the maxillary gingiva at the crown length of the maxillary(1) rightA baseline lateral was incisor. drawn horizontally on the maxillary gingiva at the crown length of the maxillary(2) Between right thelateral right incisor. and left maxillary canines, a vertical line was drawn from the baseline to the cervical(2) line Between of each the tooth. right and left maxillary canines, a vertical line was drawn from the baseline to the(3) cervical Nine pointsline of each were tooth. plotted to separate eight equal parts on the vertical line, and it was adopted that measuring(3) Nine points sites were were theplotted points to inseparate the attached eight equal gingiva. parts To on mark the vertical the reference line, and mesh it was on the attachedadopted gingiva, that measuring we employed sites were Adobe the Illustrator. points in the Horizontal attached lines gingiva. were To placed markusing the reference the length mesh of the lateralon the incisor, attached and gingiva, divided intowe employed nine points, Adobe and theIllu verticalstrator. linesHorizontal were placed lines were at mesial placed and using central the and distallength points of the of thelateral frontal incisor, teeth. and These divided mesh-generating into nine points, procedures and the were vertical done lines with were a batch placed system at to avoidmesial the and technical central assistant's and distal bias points and of obtain the fron measurementtal teeth. These reliability. mesh-generating procedures were done with a batch system to avoid the technical assistant's bias and obtain measurement reliability. (4) The presence of pigmentation at the measuring sites was evaluated and the percentile with (4) The presence of pigmentation at the measuring sites was evaluated and the percentile with pigmentation was calculated for the gingival melanosis record (GMR). pigmentation was calculated for the gingival melanosis record (GMR). To quantitatively investigate the effect of smoking cessation on gingival melanosis, the patients To quantitatively investigate the effect of smoking cessation on gingival melanosis, the patients were divided into the current smoker group and the smoking cessation group. The GMR was used to were divided into the current smoker group and the smoking cessation group. The GMR was used evaluateto evaluate the presence the presence or absence or absence of gingival of gingiv pigmental pigment at the assessment at the assessment sites, and itsites, was representedand it was as a percentagerepresented by as dividinga percentage the pigmentedby dividing assessmentthe pigmented sites assessment by all assessment sites by all sites. assessment sites. 2.3. Validation of GMR 2.3. Validation of GMR ToTo assess assess the the validity validity of of thethe GMR,GMR, we compared the the GMR GMR with with Hedin’s Hedin’s classification classification (Figure (Figure 2), 2), whichwhich is is the the gold gold standard standard of of gingival gingival melanosismelanosis classification.classification. In In addition, addition, intraclass intraclass correlation correlation coefficientscoefficients (ICC) (ICC) were were calculated calculated toto assessassess the GM GMRR reproducibility. reproducibility. ICC ICC (1,1) (1,1) was was used used to toevaluate evaluate repeatabilityrepeatability by by the the same same examiner, examiner, andand ICC (2,1) (2,1) wa wass used used to to evaluate evaluate stability stability between between examiners. examiners. ICCICC (1,1) (1,1) was was calculated calculated by by the the resultsresults ofof one ev evaluatoraluator who who performed performed two two analyses analyses with with the the same same 2020 oral oral photographs photographs at at a a one-week one-week interval.interval. ICC ICC (2,1) (2,1) was was calculated calculated by by the the results results of of20 20randomly randomly selectedselected oral oral photographs photographs usingusing GMR and and Hedin’ Hedin’ss classification classification analyzed analyzed by bythree three dentists. dentists.

FigureFigure 2.2. Hedin’s classification classiﬁcation (degrees (degrees 1–4). 1–4).

2.4. Statistical Analysis 2.4. Statistical Analysis Statistical analyses were performed using the IBM SPSS Statistics ver. 22.0 software package Statistical analyses were performed using the IBM SPSS Statistics ver. 22.0 software package (International Business Machines Corp., Armonk, NY, USA). The relationship between the GMR and (International Business Machines Corp., Armonk, NY, USA). The relationship between the GMR and Hedin’s classification was analyzed using Spearman’s rank correlation coefficient. Differences in the Hedin’schange classification in the GMR wasbetween analyzed the current using Spearman’s smoker group rank and correlation the smoking coefficient. cessation Differences group were in the changeanalyzed in the by GMR the Mann-Whitney between the current U test. smoker In addition group, the and relationship the smoking between cessation duration group wereof smoking analyzed bycessation the Mann-Whitney and the GMR U test.was analyzed In addition, with the Spearman’s relationship rank between correlation duration coefficient. of smoking The significance cessation and thelevel GMR was was set analyzed at 5%. with Spearman’s rank correlation coefficient. The significance level was set at 5%.

Int. J. Environ. Res. Public Health 2016, 13,598 4of8 Int. J. Environ. Res. Public Health 2016, 13, 598 4 of 8

3.3. ResultsResults

3.1.3.1. Subjects Subjects TheThe characteristics characteristics ofof thethe studystudy subjects are are shown shown in in Table Table 1.1 .Two Two hundred hundred and and sixty-three sixty-three patientspatients (mean (mean age age 45.9 45.9 years;years; rangerange 19–7519–75 years, 134 134 current current smokers smokers and and 129 129 former former smokers) smokers) who who attendedattended the the baseline baseline examination examination completed completed this this study. study. Due Due to unreadable to unreadable photographs photographs or the or absence the ofabsence routine of checkups, routine checkups, 20 patients 20 were patients excluded were inexcluded this study. in this No study. statistically No statistically signiﬁcant significant differences betweendifferences the currentbetween smokers the current and smokers former smokers and former were smokers observed were regarding observed the regarding baseline characteristicsthe baseline orcharacteristics GMR. or GMR.

TableTable 1.1. Patient demographics at at baseline. baseline.

FormerFormer SmokerSmoker Current Current Smoke Smokerr Total Total p-Valuep-Value Number of subjects 134 129 263 Number of subjects 134 129 263 Male/Female 89/45 81/47 170/92 0.30 Male/Female 89/45 81/47 170/92 0.30 Age (years) 46.8 ± 13.4 * 45.0 ± 12.9 * 45.9 ± 13.2 * 0.60 Age (years) 46.8 ˘ 13.4 * 45.0 ˘ 12.9 * 45.9 ˘ 13.2 * 0.60 GMR (%) 25.6 ± 18.6 * 26.7 ± 21.0 * 26.2 ± 19.8 * 0.89 GMR (%) 25.6 ˘ 18.6 * 26.7 ˘ 21.0 * 26.2 ˘ 19.8 * 0.89 * Mean ± SD. * Mean ˘ SD.

3.2.3.2. Validation Validation of of GMR GMR Nine hundred photographs were analyzed to evaluate gingival melanosis with the GMR and Nine hundred photographs were analyzed to evaluate gingival melanosis with the GMR and Hedin’s classification at baseline. Figure 3 shows a scatter diagram of the GMR and Hedin’s Hedin’s classification at baseline. Figure 3 shows a scatter diagram of the GMR and Hedin’s classification. A significant correlation was found between the GMR and Hedin’s classification classification. A significant correlation was found between the GMR and Hedin’s classification (p < 0.01, correlation coefficient = 0.94). For reproducibility, the ICC (1,1) of GMR was 0.781. The ICC (p < 0.01, correlation coefficient = 0.94). For reproducibility, the ICC (1,1) of GMR was 0.781. The (2,1) of GMR (0.783) was higher than that for Hedin’s classification (0.720) (Table 2). Both ICCs for ICC (2,1) of GMR (0.783) was higher than that for Hedin’s classification (0.720) (Table 2). Both ICCs for the GMR indicated a “substantial agreement” according to the reference of Landis and Koch [16]. the GMR indicated a “substantial agreement” according to the reference of Landis and Koch [16].

FigureFigure 3.3. ScatterScatter diagram of of the the GMR GMR and and Hedin’s Hedin’s classification. classiﬁcation.

TableTable 2. 2.Validation Validation ofof thethe GMRGMR and Hedin’s cl classiﬁcationassification (ICC (ICC (1,1) (1,1) and and ICC ICC (2,1)). (2,1)).

ICC (1,1) (95% CI *) ICC (2,1) (95% CI *) ICC (1,1) (95% CI *) ICC (2,1) (95% CI *) GMR 0.78 (0.53–0.91) 0.78 (0.56–0.90) Hedin’sGMR classification 0.87 0.78(0.703–0.945) (0.53–0.91) 0.780.72 (0.56–0.90)(0.37–0.89) Hedin’s classiﬁcation 0.87 (0.703–0.945) 0.72 (0.37–0.89) * 95% CI: 95% confidence interval. * 95% CI: 95% conﬁdence interval.

Int. J. Environ. Res. Public Health 2016, 13, 598 5 of 8 Int.Int. J. Environ. J. Environ. Res. Res. Public Public Health Health 20162016, 13, 13598,598 5 of 5of8 8 3.3. Smoking Cessation and Gingival Melanosis 3.3. Smoking Cessation and Gingival Melanosis 3.3. SmokingThe mean Cessation follow-up and Gingivalperiod was Melanosis 4.5 years for the smoking cessation group and 3.8 years for the currentThe mean smoking follow-up group. period The progre was 4.5ssion years of forthe the GMR smoking in each cessation group is group shown and in Figure3.8 years 4. Thefor the GMR currentin theThe smoking current mean group. follow-upsmoking The group periodprogre decreased wasssion 4.5 of yearsthe from GMR for 26.7% the in smokingeach to 19.9%, group cessation andis shown that group inin theFigure and smoking 3.8 4. yearsThe cessationGMR for the in currentthegroup current decreased smoking smoking group. from group The25.6% progressiondecreased to 12.8%. from ofA thesignificant 26.7% GMR to in difference each19.9%, group and was isthat shown observed in the in Figuresmoking between4. Thecessation the GMR current in groupthesmoking currentdecreased and smoking fromsmoking group25.6% cessation decreasedto 12.8%. groups A from significant ( 26.7%p < 0.01). to difference 19.9%, There and waswas that aobserved insignificant the smoking between negative cessation the correlationcurrent group smokingdecreasedbetween and the from smoking reduction 25.6% cessation to in 12.8%. the GMR groups A significant and ( pthe < duration0.01). difference There of wassmoking was observed a significant cessation between (Figurenegative the current5). correlationAdditionally, smoking betweenandthe smokingreduction the reduction cessation in the in GMR the groups GMRwas (psignificantly and< 0.01). the duration There correla was ofted asmokingsignificant with the cessation duration negative (Figure of correlation smoking 5). Additionally, cessation between the( p = thereduction 0.01).reduction in in the the GMR GMR and was the significantly duration of smokingcorrelated cessation with the (Figure duration5). Additionally, of smoking cessation the reduction (p = in 0.01).the GMR was significantly correlated with the duration of smoking cessation (p = 0.01).

Figure 4. Amount of GMR change (mean GMR for current smoker vs. former smoker). Figure 4. Amount of GMR change (mean GMR for current smoker vs. former smoker). Figure 4. Amount of GMR change (mean GMR for current smoker vs. former smoker).

Figure 5. Figure 5.Scatter Scatter diagramdiagramof ofthe theGMR GMRand andyears yearsof of smoking smoking cessation. cessation. Figure 5. Scatter diagram of the GMR and years of smoking cessation. 4.4. Discussion 4. Discussion InIn thisthis study,study, wewe performedperformed aa newnew quantitativequantitative analysisanalysis ofof gingivalgingival melanosismelanosis usingusing oraloral photographs.photographs.In this study, Our Our we quantitative performed aanalysis, new quantitative the GMR, analysis waswas significantlysignificantly of gingival associated associatedmelanosis withwithusing Hedin’s Hedin’soral photographs.classification,classification, Our whichwhich quantitative isis thethe goldgold analysis, standardstandard the for forGMR, analyzing analyzing was gingival significantlygingival melanosis. melanosis. associated TheThe with GMRGMR Hedin’s showedshowed aa classification,substantialsubstantial agreement whichagreement is the by by ICCgold ICC (1,1) standard(1,1) and and ICC forICC (2,1). analyzing (2,1). Additionally, Additionally, gingival significant melanosis. significant differences Thedifferences GMR in the showed in GMR the wereGMR a substantial agreement by ICC (1,1) and ICC (2,1). Additionally, significant differences in the GMR

Int. J. Environ. Res. Public Health 2016, 13,598 6of8 observed between current smokers and former smokers. Moreover, there was a positive correlation between reductions in the GMR and the duration of smoking cessation. Gingival melanosis and its classification were first reported by Hedin et al. in 1977 [5]. This classification evaluates gingival melanosis in a subjective manner [17], and other subjective evaluation methods were reported by these authors [9,18–20]. Thereafter, quantitative evaluation methods were attempted using a spectrometer or spectrophotometer [21–24] Recent research has reported a relationship between smoking and gingival melanosis using a spectrophotometer [15]. However, the repeatability of this method is unclear. Moreover, the method requires a particular photometer to evaluate gingival melanosis. In this study, we used oral photographs to evaluate the GMR. In addition, we demonstrated that the GMR was significantly associated with Hedin’s classification and the GMR had a high repeatability to evaluate gingival melanosis. Therefore, we suggest that the GMR is a versatile, ideal method for objective analysis of gingival melanosis as compared to a subjective analysis method such as Hedin’s classification. Hedin et al. reported the relationship between smoking and gingival melanosis and demonstrated that smoking cessation resulted in a reduction in gingival melanosis [12]. Specifically, smoking cessation resulted in a reduction within a few months and gingival melanosis disappeared within six months. However, their studies used subjective methods to evaluate gingival melanosis. Additionally, the amount of melanosis reduction was unclear, and the effect of smoking cessation in smokers could not be evaluated due to the lack of a longitudinal study. In addition, it was a synchronic study in which a quantitative investigation about the relationship between smoking and gingival melanosis was carried out using a spectrophotometer [15]. In the present study, we conducted a longitudinal study which compared current smokers to former smokers, and the two groups were similar at baseline. Thus, the effect of smoking cessation was quantitatively evaluated. Few investigations have reported the relationship between the duration of smoking cessation and gingival melanosis in a quantitative manner. Hedin et al. reported the relationship between the duration of smoking cessation and oral melanin pigmentation using a subjective analysis [12]. They reported that smoking cessation resulted in a reduction within a few months and gingival melanosis disappeared by six months of smoking cessation. Additionally, they demonstrated a negative correlation between the GMR and the duration of smoking cessation, and considered that 4.5 years of smoking cessation reduced gingival melanosis by half. The study reported by Hedin [12] was a synchronic study, and they measured only the presence or absence of the oral melanosis using a special chart subjectively. In addition, they evaluated the gingiva, buccal mucosa, palate and lip. For these reasons, our study differed from the study of Hedin et al. In the present study, it was interesting that the GMR decreased in the current smoker group and gingival melanosis demonstrated an improvement. Previous studies reported that the quantity of smoking was correlated with gingival melanosis. Araki et al. reported that the prevalence of gingival melanosis was significantly higher in smokers who smoked more than 10 cigarettes per day [14]. Additionally, the absence of oral melanosis was recognized in a patient who initially smoked four cheroots per day but reduced the amount to one cheroot per day for nine years [12]. Taken together, these findings suggest that the quantity of smoking reduction might lead to a reduction in gingival melanosis. For future studies, the effect of smoking amount on gingival melanosis should be evaluated.

5. Conclusions The GMR indicated that smoking cessation can reduce gingival melanosis and may be an ideal quantitative analysis for gingival melanosis.

Acknowledgments: We gratefully thank the members and staff of the Japan Health Care Dental Association for providing and formatting the oral photographs and records. We also express our appreciation to Hidetoshi Akimoto for coordinating the survey. Part of this work was supported by a Grant-in-Aid for Young Scientists (B: No. 26861826) from the Japan Society for the Promotion of Science. Int. J. Environ. Res. Public Health 2016, 13,598 7of8

Author Contributions: Tomotaka Kato, Michiko Makino, Satoshi Noguchi, Tomoko Katayama-Ono and Toru Naito performed the experiments. Tomotaka Kato, Hiroya Takiuchi, Satoshi Noguchi and Tomoko Katayama-Ono analyzed the data. Seiichi Sugiyama managed the collected data. Tomotaka Kato, Takashi Hanioka and Toru Naito wrote the paper. Tomotaka Kato and Toru Naito conceived and designed the experiments. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations The following abbreviations are used in this manuscript: GMR Gingival melanosis record ICC Intraclass correlation coefﬁcients

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© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). Case Report

Casa de Lletres Academic Editing Services [email protected] www.casadelletres.eu The Turkish Journal of Pediatrics 2015; 57: 409-412 Case Report

A case of xanthogranulomatous pyelonephritis mimicking Wilms tumor

Gülen Tüysüz1, Funda Tayfun1, Fetiye Canpolat2, Hikmet Zeytun2, Cemil Goya3, Ayşe Nur Keleş4, Nihal Özdemir5 1Division of Pediatric Hematology and Oncology, Diyarbakir Children’s Hospital, Departments of 2Pediatric Surgery, 3Radiology and 4Pathology, Dicle University Faculty of Medicine, Diyarbakir, 5Division of Pediatric Hematology and Oncology, Department of Pediatrics, İstanbul University Cerrahpasa Faculty of Medicine, İstanbul, Turkey. E-mail: [email protected] Received: 29 September 2014, Revised: 14 November 2014, Accepted: 26 November 2014

SUMMARY: Tüysüz G, Tayfun F, Canpolat F, Zeytun H, Goya C, Keleş AN, Özdemir N. A case of xanthogranulomatous pyelonephritis mimicking Wilms tumor. Turk J Pediatr 2015; 57: 409-412. Xanthogranulomatous pyelonephritis (XGPN) is a very rare, unusual variant of pyelonephritis characterized by destruction of renal parenchyma. It usually occurs in adults with a history of recurrent urinary tract infections. The condition is rare in children and the disease can imitate renal tumors. Here, we describe a 12-year-old boy who presented with abdominal pain. He did not have any history of urinary tract infection. Computed tomography and magnetic resonance imaging showed a cystic lesion in the left upper kidney. The patient underwent radical nephrectomy with a provisional diagnosis of Wilms tumor however histopathological examination of specimen revealed XGPN. Xanthogranulomatous pyelonephritis should be kept in mind in the differential diagnosis of renal lesions in childhood, during surgery if any suspicion from the diagnosis, a frozen biopsy should have been taken. Key words: pyelonephritis, Wilms tumor, children, magnetic resonance imagining, computed tomography.

Xanthogranulomatous pyelonephritis (XGPN) history of the patient was unexceptional; he did is a rare form of chronic renal infection with not have any previous history of hospitalization, unknown pathogenesis. It is usually associated unexplained fever or urinary tract infection with recurrent urinary tract infections and before. His body temperature was normal. renal calculus causing obstructive uropathy. General and systemic examination of patient The disease process is characterized by the was unremarkable except tenderness on left destruction of the renal parenchyma and upper quadrant at deep palpation. The blood surrounding tissues and replacement with pressure was within the normal limits. Blood lipid-laden macrophages. Xanthogranulomatous chemistry tests were normal except the mildly pyelonephritis can involve the entire kidney or elevated C-reactive protein (value: 10.34 mg/ may be localized, mimicking a renal tumor. It is dl, normal: < 0.5 mg/dl). Complete blood a challenge in the differential diagnosis of renal count was unremarkable: white blood cell tumors. We report a case of XGPN in a 12-year- count, 9710/mm3 of which 60.2% neutrophils, old boy, who underwent radical nephrectomy 28.7% lymphocytes, 9.89% monocytes, 1.21% due to left kidney upper pole located cystic eosinophils. The urine analysis was normal. lesion with the provisional diagnosis of Wilms Ultrasonography of the abdomen showed a tumor. The histopathological examination of renal lesion on left upper lobe of kidney (Fig. 1). the removed kidney showed XGPN. The patient was subsequently hospitalized for further investigation. An intravenous contrast Case Report enhanced computed tomography (CT) scan was A twelve-year-old boy presented with abdominal performed which revealed a renal mass, 54x47 pain ongoing for the last 17 days. Medical mm sized, originating from the left upper pole 410 Tüysüz G, et al The Turkish Journal of Pediatrics • July-August 2015 of the kidney and extending to the medial incidence above 10 years of age, the patient part. There was thickening and mild density was clinically diagnosed as Wilms tumor. A increase at anterior pararenal fascia. Both of total nephrectomy was planned according to the the kidneys were in normal size and location national Wilms tumor staging (NWTS) protocol. (Fig. 2). A T1 weighted abdominal magnetic Total nephrectomy was done. Pathological resonance imagining (MRI) showed 60x58 examination of the specimen showed upper mm sized cystic lesion containing a few septa middle pole located, predominantly cystic, with an 8 mm thickness wall on left upper 4.5x3.5x3.5 cm sized, a brown colored mass pole of the left kidney. Around the lesion, containing 2.8x1x1 cm solid area on the wall there was diffusion restriction on diffusion in 10.5x6x5 cm sized left kidney operation weighted scan and contrast enhancement at material. Microscopic examination of the mass septa and at the wall of the lesion (Fig. 3). showed macrophage predominant mixed type The patient was evaluated at tumor council; on inflammatory zone lining the cyst wall and the basis of radiologic findings, despite the rare a restrictive fibrous zone characterized by connective tissue surrounding the inflammatory zone (Fig. 4) which revealed the presence of xanthogranulomatous pyelonephritis. He is doing well now with no complaints or any signs of urinary tract infection at three-month follow-up after surgery.

Fig. 3. Axial T1-weighted fat-suppressed MRI image with contrast showed cystic lesion with septa on upper pole of left kidney.

Fig. 1. Ultrasonography showed anechoic cystic lesion with a thick irregular wall on upper pole of left kidney.

Fig. 4. Pathological examination of renal mass (H&E X100) showed macrophage predominant mixed type Fig. 2. Abdominal contrast CT scan showed cystic lesion inflammatory zone lining the cyst wall and a restrictive with enhancing septa and wall at upper pole of left kidney fibrous zone characterized by connective tissue surrounding in axial plan. the inflammatory zone. Volume 57 • Number 4 Xanthogranulomatous Pyelonephritis 411

Discussion into 3 stages, depending on the extension of Xanthogranulomatous pyelonephritis is a severe the inflammation: in stage I (nephric XGPN) form of chronic renal infection characterized the lesion is confined to kidney, in stage II by destruction of renal parenchyma and (perinephric XGPN) the lesion extends to replacement by granulomatous tissue containing Gerota’s fascia and in stage III (paranephric lipid filled (xantoma cells) macrophages1. These XGPN) extends to paranephric space and other 13 macrophages form the special yellow color of retroperitoneal structures . In the focal form, the disease. Although chronic bacterial infection like in our patient, ultrasonography shows a accompanied with urinary tract obstruction is the non-enhancing or minimally enhancing localized common pathology, exact etiology still remains renal mass that can mimic renal tumors and unknown2,3. The urinary tract obstruction is this form is usually combined with staghorn usually secondary to nephrolithiasis, uretero- stones. Although radiologic images are suitable pelvic junction obstructions, renal tumors with Wilms tumor in our patient, renal cell or severe vesicoureteral reflux. The most carcinoma, renal abscess, infected renal cystic common causative organisms for infection are disease and tuberculosis should be kept in Escherichia coli, Proteus miribalis and Pseudomonas mind in the differential diagnosis of these aeruginosa (4). Other factors that might have cystic lesions of kidney. If any suspicion from an effect in development of disease are renal the diagnosis, during the surgery, a frozen ischemia, arterial insufficiency, lymphatic biopsy should be taken even though this can blockage, bacteremic seeding, alterations in split the tumor to abdomen in case it turns lipid metabolism and altered immune response out to be Wilms tumor. in renal transplant recipients5-7. The management of XGPN differs in literature The disease is mostly seen in middle aged according to the dissemination of disease. women but can occur at any age. In adults, In diffuse forms, nephrectomy is usually the male to female ratio is 1:28. In pediatric cases, standard treatment. In focal forms partial it is very rare; girls and boys are equally nephrectomy, drainage of perirenal/renal abcess affected, the age of onset varies but usually and concomitant antibiotic therapy has been patients are diagnosed before age of 59. recommended but most of the cases had gone Xanthogranulomatous pyelonephritis usually under renal explorations because focal XGPN affects one kidney, but rarely can involve resembles renal tumors14,15. Because there is no both kidneys. Clinical symptoms and signs clear guideline on the management of XGPN, are nonspecific and include abdominal pain, conservative and surgical treatments should be flank mass, fever of unknown origin, weight considered for each individual case. loss, pyuria and rarely hematuria8,10. These In our case, there was no history of urinary tract symptoms are reported in most of the cases. infection or unexplained fever. Urine sample Laboratory tests are usually non-diagnostic like and CBC tests showed no abnormality. Blood mild anemia, leukocytosis, elevated erythrocyte chemistry and systemic inflammatory reactants sedimentation rate and increased C-reactive were unremarkable except the increased 9,11 protein levels . Pathologic urine sediment C-reactive protein level. Ultrasonography and and positive urine cultures are prognostic for CT showed a cystic lesion originating from the 12 XGPN but are found only in 70% of patients . left pole of kidney but there weren’t any renal Radiologic investigations including calculi. The preoperative distinction between ultrasonography and contrast-enhanced XGPN and malignant kidney tumor is often computed tomography can help in identifying difficult especially in focal forms and in the XGPN. Radiologic image of XGPN differs absence of previous urinary tract complaints. according to the type of the disease. In rather Here, we wanted to emphasize that XPGN frequently seen, diffuse form; there are more should be kept in mind in the differential characteristic lesions like diffuse enlargement diagnosis of renal masses even in the absence of of kidney, loss of renal architecture, perirenal predisposing factors. A high index of suspicion spread of infectious process and accompanying and clinical awareness is needed to accomplish fistulae in CT. Diffuse forms can be divided preoperative diagnosis. 412 Tüysüz G, et al The Turkish Journal of Pediatrics • July-August 2015

REFERENCES 8. Dwivedi US, Goyal NK, Saxena V, et al. Xanthogranulomatous pyelonephritis: our experience 1. Goldman SM, Hartman DS, Fishman EK, with review of published reports ANZ J Surg 2006; Finizio JP, Gatewood OM, Siegelman SS. CT of 76: 1007-1009. xanthogranulomatous pyelonephritis: radiologic- pathologic correlation. AJR Am J Roentgenol 1984; 9. Gupta S, Araya CE, Dharnidharka VR. 142: 963-969. Xanthogranulomatous pyelonephritis in pediatric patients: case report and review of literature. J Pediatr 2. Cakmakci H, Tasdelen N, Obuz F, Yilmaz E, Kovanlikaya Urol 2010; 6: 355-358. A. Pediatric focal xanthogranulomatous pyelonephritis: . dynamic contrast-enhanced MRI findings. Clin Imaging 10. Khalid S, Zaheer S, Zaheer S, Ahmad I, Khalid M. 2002; 26: 183-186. Xanthogranulomatous pyelonephritis: rare presentation of a rare disease. South Asian J Cancer 2013; 2: 4. 3. Al-Ghazo MA, Ghalayini IF, Matalka II, Al-Kaisi NS, Khader YS. Xanthogranulomatous pyelonephritis: 11. Kuo CC, Wu CF, Huang CC et al. Xanthogranulomatous pyelonephritis: critical analysis of 30 patients. Int Urol analysis of 18 cases. Asian J Surg 2006; 29: 257-261. Nephrol 2011; 43: 15-22. 4. Dell’Aprovitola N, Guarino S, Del Vecchio W, et al. 12. Zugor V, Schott GE, Labanaris AP. Xanthogranulomatous Xanthogranulomatous pyelonephritis mimicking a renal pyelonephritis in childhood: a critical analysis of 10 cell carcinoma: a unique and challenging case. Acta cases and of the literature. Urology 2007; 70: 157-160. Radiol Short Rep 2014; 3: 1-4. 13. Khalid M, Ahmad M, Siddiqui MA, Khalid S. 5. Zaid UB, Porten SP, Cinman NM, Sanford TH, Breyer Diagnostic difficulties in diffuse xanthogranulomatous BN. Xanthogranulomatous pyelonephritis presenting pyelonephritis: A case report and review of the with a left flank mass. Case Rep Med 2013;11: 1-3. literature. Kidney 2010; 19: 73-75. 6. Mnif MF, Kamoun M, Kacem FH et al. Complicated 14. Nandedkar SS, Malukani K, Sakhi P. urinary tract infections associated with diabetes Xanthogranulomatous pyelonephritis masquerading mellitus: Pathogenesis, diagnosis and management. as a tumor in an infant. Indian J Urol 2014; 30: 354- Indian J Endocrinol Metab 2013;17: 442-445. 356. 7. Singh R, Gupta G, Kumar P, Kotasthane DS. 15. Shah K, Parikh M, Gharia P, Modi PR. Xanthogranulomatous pyelonephritis in a male child- Xanthogranulomatous pyelonephritis-mimicking renal -a case report with a brief review of literature. J Indian mass in 5-month-old child. Urology 2012; 79: 1360- Med Assoc 2012; 110: 739-740. 1362. CASE REPORT

Small Cell Carcinoma of the Gall Bladder Max Haid, MD; Badri Ganju, MD; Craig Schulz, MD; David Sterner, MD; Steven Falconer, MD

ABSTRACT With regard to surgical margins, tumor was Small cell carcinoma of the gall bladder (SCCGB) is a rare condition, with only 53 prior cases present on the serosal surface of the gall reported in the world literature when our case was first diagnosed. Our patient was found to bladder, but the cystic duct margin was neg- have limited stage disease and was treated with sequential laparoscopic cholecystectomy, eto- ative for tumor. Two gallstones were found poside/carboplatin chemotherapy followed by consolidating loco-regional radiation therapy. She within the gall bladder. On immunoperoxi- is alive and well without evidence of disease more than 132 months since diagnosis. We describe dase staining, the tumor cells were positive here our experience in the diagnosis, staging workup, treatment, and surveillance of a case of for synaptophysin, AE1-AE-3, cytokeratin SCCGB and review the published literature. Treated aggressively with currently available meth- CAM5.2, high molecular weight cytokera- ods, patients with limited stage SCCGB can have an excellent prognosis. The authors’ intent is to tin and cytokeratin 7. The tumor cells were provide a reasonable plan of treatment for other physicians facing such an unusual patient. negative for chromogranin, carcinoembryonic antigen, cytokeratin 20, epithelial membrane antigen (EMA) and vimentin. In order to complete her tumor staging, CASE PRESENTATION a positron emission tomographic (PET) scan with fused comput- Our patient is a white woman who was 63 years old when she erized axial tomographic (CT) imaging was performed (Figures presented to the emergency department with chief complaints 4 and 5). Two lesions in the gastrophepatic ligament measuring of extreme abdominal pain and emesis. Her social history was 3.7 x 2.3 cm and 1.5 x 1.6 cm were noted. They had maximum remarkable for smoking cessation 3 years earlier. An evaluation standardized uptake values (SUV) of 17.2, while lesions poste- including ultrasonography showed her to have cholelithiasis and rior in the head of the pancreas had a maximum SUV of 14.2. cholecystitis as well as a thickened area within the lumen of the These maximal SUV values strongly suggested a malignant etiol- gall bladder (Figures 1 and 2). ogy rather than an infectious or inflammatory process. There was Laparoscopic cholecystectomy was performed the following no evidence of distant metastases. day, after which she made an uneventful recovery. The significant Chemotherapy was initiated 30 days after the operation. It pathologic findings were: a 7.1 x 4.5 x 2.6 cm tan-red, hemor- consisted of 4 courses of etoposide (100 mg/m2 daily x 3) + car- rhagic mass lying primarily within the body of the gallbladder boplatin (area under the curve [AUC] x 6) beginning on day 1 and extending into the infundibulum, cholecystitis, cholelithiasis, of each 21-day cycle. Following completion of the chemother- and a small perforation of the gall bladder. The histology is shown apy, she received consolidation radiation therapy. The plan was in Figure 3. The cystic duct was involved and the serosa had been designed to deliver 45 Gray units (Gy) while sparing appropriate perforated by the tumor. Lymphovascular invasion also was noted. volumes of kidney mass, liver, and spinal cord to keep the likelihood for normal tissue toxicities to a negligible level. This was very well accomplished. Forty percent of her right kidney received • • • > 18 Gy; about 35% of the left kidney received > 18 Gy; 40% of Author Affiliations: Vince Lombardi Cancer Center Clinic, Sheboygan, Wis her liver received > 30 Gy. Her clinical target volume received 45 (Dr Haid is now at Sheboygan Acupuncture LLC, Sheboygan, Wis); Aurora Gy. Sheboygan Clinic, Sheboygan, Wis (Ganju); Columbia St. Mary’s Hospital; Milwaukee, Wis (Schulz); Aurora Sheboygan Memorial Medical Center; The patient experienced expected side effects of nausea and Sheboygan, Wis (Sterner, Falconer). fatigue. However, her nausea was not effectively controlled with aggressive antiemetics until the daily dose was reduced to 1.25 Corresponding Author: Max Haid, MD, Sheboygan Acupuncture LLC; 703 N 8th St; Ste 201, Sheboygan, WI 53081; phone 920.693.2002; e-mail Gy. After that, her tolerance was excellent and she completed [email protected]. therapy without significant impact on her weight or oral intake.

96 WMJ • APRIL 2016 Figure 1. Parasagittal View of the Gall Bladder Figure 2. Transverse Ultrasound of the Gall Bladder Showing the Intraluminal Mass

Ultrasound image shows cholelithiasis and a significant size intraluminal mass, which ultimately proved to be small cell carcinoma upon histologic evaluation.

At the conclusion of therapy, she had no uncontrolled toxicities, no significant pain, and was taking oral nourishment and fluids Figure 3. Gall Bladder Wall well. She did not require readmission to the hospital for symptom management. She was followed with physical examination, complete blood count, and comprehensive metabolic profile every 3 months for the first 2 years, and every 6 months for the next 3 years, then annually. CT scanning was repeated at those same intervals. Her surveillance computerized axial tomographic scans have remained normal. She remains alive and asymptomatic more than 132 months from diagnosis.

DISCUSSION Small cell carcinoma of the gall bladder (SCCGB) is rare. A search of the English medical literature showed only 53 reported cases.1 The prognosis is poorer than that of differentiated car- Photomicrograph specimen showing small cell carcinoma which, perfo- cinomas of the gall bladder. It tends to affect an older patient rated the serosa (hematoxylin-eosin, original magnification x200). The population occurring at a median age of 65 years, with a female cystic duct margin was negative for tumor. Lymphovascular invasion was present. The cells demonstrated positivity for synaptophysin, AE1-AE3, 2 preponderance of 76%. It is frequently associated with chole- cytokeratin CAM5.2, high molecular weight cytokeratin and cytokeratin 7. lithiasis. Metastasis to lymph nodes occurs in 88%, liver 88%, It was negative for chromogranin, carcinoembryonic antigen, cytokeratin 20, EMA and vimentin. lung 23%, and peritoneum 19%.2 The 5 patients treated with surgery and chemotherapy by Moskal et al2 had a median survival of 13 months. This represents an improvement over historical reports. Those with pure small cell tumors had a median sur- such as synaptophysin, chromogranin, or neuron-specific eno- vival of 9 months, while those with mixed tumors had a median lase. Immunoperoxidase stains showed focal carcinoembryonic survival of only 4.5 months.2 antigen in 3 of 11 tumors studied by Albores-Saavedra et al.3 Pathologic characteristics may include large size at diagno- Molecular changes have been identified as well. On electron sis, extensive necrosis, and propensity for submucosal growth. microscopy, neurosecretory granules are sometimes present.4 Histologically, these tumors are composed predominately of Significantly, frequent occurrences of p53 (75%), p16INK4a small round cells usually mixed with spindle cells, both of which (33%), and K-RAS codon (17%) mutations have been identi- have hyperchromatic nuclei, inconspicuous nucleoli, and scan fied.4,5 cytoplasm. The growth pattern is usually diffuse. However, focal Our experience demonstrated that this disease can exhibit nesting or festoon patterns can be seen. Most of these tumors FDG hyper-avidity on PET scanning (Figures 4 and 5). show neuroendocrine differentiation by immunohistochemistry As recently as the year 2000, Mithal et al reported a case in

VOLUME 115 • NO. 2 97 Figure 4. PET Scan of Gall Bladder Bed Showing Area of Intense Uptake Figure 5. PET Scan of Gall Bladder

The multiple areas of 2,3-fluorodeoxyglucose avidity were interpreted to be areas of likely gastrohepatic ligament involvement by the carcinoma. That finding coupled with the absence of distant spread on the PET scan led to the conclusion that the patient had limited stage disease.

Table. Summary of Chemotherapy and Radiation Therapy Regimens Employed, Responses, and Survival

Responders/ Type of Chemotherapy Survival Reference No. Cases No. LTD/No. EXT Total No. Treated and Radiation Therapy (RT) (months) Fujii et al1 Case 1 0/1 1/1 cis-DDP + VP-16 no RT 12+ Review 35 12/23 2/19 no chemotherapy specified 7/13 Albores-Saavedra et al3 19 1/18 2/19 CAV + nitrososurea, no RT 7, 13 Lane et al7 1 0/1 1/1 carbo + VP-16, 45 Gy in 25 fractions 11 Okamoto et al8 1 0/1 1/1 cis-DDP + gem, no RT 7 Bahadur et al9 2 0/2 1/1 cis-DDP + VP-16, no RT 5, 9 Pavithran et al10 1 0/1 1/1 5FU + cis-DDP—>docetaxel + carbo, No RT 13 Ron et al11 1 0/1 1/1 cis-DDP + VP-16, no RT 6+

Abbreviations: LTD, limited stage disease; EXT, extensive stage disease; cis-DDP, cis-Platinum; CAV, cyclophosphamide, doxorubicin (Adriamycin), vincristine; carbo, carbo- platinum; gem, gemcitabine; VP-16, Etoposide; 5FU, 5-fluorouracil; Gy, Gray. a 25-year-old woman.6 They asserted that chemotherapy is not VP-16. This is currently typical treatment of small cell carci- known to improve survival. Our patient has shown a totally noma of the lung. The stage of disease was not apparent in the different possibility. Her therapy was based upon principles of other patients. The addition of local, consolidating radiation treatment promoted by the National Comprehensive Cancer therapy after completion of the chemotherapy may be a dis- Network guidelines. We chose to apply consolidating radiation criminating difference between our patient and others reported. therapy after the course of chemotherapy out of concerns for Had extensive stage disease been present at the time of diagnosis, increased hepatotoxicity from simultaneous application of these then we would not have applied radiation therapy with cura- modalities. It appears that, just as in the treatment of small cell tive intent. Based upon our experience, a patient with SCCGB carcinoma of the lung, only patients with limited stage disease should undergo rigorous staging to include PET/CT scanning. are capable of complete remission and long-term survival. If localized or even loco-regional disease allows a designation of A summary of the different chemotherapy regimens and limited stage disease, we recommend sequential chemotherapy radiation therapy employed is shown in the Table. The most with VP-16 and carboplatin followed by loco-regional radiation commonly used regimens employed a platinum derivative plus therapy with curative intent. This approach can offer hope to

98 WMJ • APRIL 2016 these rare patients. Since so little literature exists regarding this 3. Albores-Saavedra J, Soriano J, Larraza-Hernandez O, et al. Human Pathology. 1984;15(7):639-646. disease, our report is offered in the spirit of providing guidance 4. Johnstone AK, Zuch RH, Anders KH. Oat cell carcinoma of the gall bladder. A rare and cautious optimism to physicians faced with the dilemma of and highly lethal neoplasm. Arch Pathol Lab Med. 1993;117(10):1009-1012. treating such an unusual patient. 5. Maitra A, Tascilar M, Hruban RH, et al. American Journal of Surgical Pathology. 2001;25(5)595-601. 6. Mithal U. Heroor A, Khan K, et al. Small cell carcinoma of gall bladder. Indian Acknowledgments: Dr Haid would like to thank Dr Al Benson III of Journal of Gastroenterology. 2000;19(1):33. Northwestern University and Dr Bernard Levin, formerly of M.D. Anderson 7. Lane JE, Walker AN, Ayers GW, et al Small-cell undifferentiated Carcinoma of Cancer Center (Houston, Texas), for graciously discussing this case when Neuroendocrine Type Originating in the Gallbladder. Current Surgery. 2002;59(5);495- the diagnosis was first made. 497. 8. Okamoto H, Miura K, Ogawara T, et al Small-cell carcinoma manifesting systemic Funding/Support: None declared. lymphadenopathy combined with adenocarcinoma in the gallbladder: aggressiveness and sensitivity to chemotherapy. J Gastroenterol. 2003;38(8):801-805. Financial Disclosures: None declared. 9. Bahadur S, Shaukat A, Gibbs J, et al Cisplatin and gemcitabine for small cell carcinoma of the gall bladder. American J Clinical Oncol. 2005;28(4):425-426. 10. Pavithran K, Doval DC, Vaid AK, et al Small cell carcinoma of the gall bladder: REFERENCES case report and review of literature. Tropical Gastroenterol. 2001;22(3):170-171. 1. Fujii H, Antake T, Horiuchui T, et al. Small Cell Carcinoma of the Gallbladder: A 11. Ron IG, Wigler N, Ilie B, Chaitchik S. Small cell carcinoma of the gallbladder: Case Report and Review of 53 Cases in the Literature. Hepatogastroenterology. clinical course and response to chemotherapy. Tumori. 1992;78(3):207-210. 2001;48(42):1588-1593. 2. Moskal TL, Zhang PJ, Nava HR. Small Cell Carcinoma of the Gallbladder. J Surgical Oncology. 1999;70(1):54-59.

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