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Dry-season Breeding of a Characin in a Neotropical Mountain RiverAuthor(s): Mauricio Torres-Mejia and Martha P. Ramírez-PinillaSource: Copeia, 2008(1):99-104. 2008.Published By: The American Society of Ichthyologists and HerpetologistsDOI: http://dx.doi.org/10.1643/CP-06-256URL: http://www.bioone.org/doi/full/10.1643/CP-06-256

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Dry-season Breeding of a Characin in a Neotropical

Mountain River

Mauricio Torres-Mejia1,2 and Martha P. Ramırez-Pinilla1

The annual reproductive cycle of Creagrutus guanes (Teleostei, Characidae) was studied in a tropical mountain

river, a type of habitat where reproductive biology of fishes has been scarcely investigated. Analysis was made

on adults, 70 males and 135 females, which were captured in 13 monthly samplings. The analysis was based on

macro- and microscopic observations of gonads, and on weight of gonads, mesenteric fat, and liver. Weight and

microscopic observation of ovaries and testes, as well as macroscopic observation of ovaries, suggest

reproductive activity was concentrated in periods of low rainfall. In males macroscopic observations did not

reflect the maturity state revealed by histology. Fat reserves also showed a seasonal variation related with

rainfall pattern. Given that dry season reproduction is relatively uncommon in tropical freshwater fishes,

ultimate factors determining such a pattern are discussed.

THE tropics are often perceived as stable environmentswhen compared with temperate zones, regardingtemperature and day length. However, extensive

evidence showing seasonality in the tropics has beengathered, mainly associated with fluctuations of rainfall(Payne, 1986; Morales-Nin and Panfili, 2005). In tropicalfreshwater fishes, there is another generalization, thatseasonal reproduction is associated with heavy rains (Me-nezes and Vazzoler, 1992). This impression may be theproduct of bias toward studying floodplain river species(Kramer, 1978). Those species spawn during the high waterperiod when the overflowed river slowly invades the floodplains richer in nutrients and habitat structure (Welcomme,1979; Lowe-McConnell, 1987). As an example of thisreseach bias, of the 64 studies on Characiformes listed inMunro’s (1990) review on reproductive cycles of tropicalfreshwater fishes, 67% corresponded to flooding riverspecies, the great majority (98%) of which bred during therainy season.

There are tropical habitats where the flood-pulse phe-nomenon is mild or does not occur at all. One example issmall lowland tropical rivers, where floodings of theriverside plains occur immediately after rainfalls in a smallcatchment area, and water level subsides quickly. Otherexamples are mountain and piedmont tropical rivers, whichhave steep banks that prevent the lateral flooding of watersafter rains, resulting in sudden, dramatic and short-livedwater level risings (Chapman and Kramer, 1991). Suchfloodings have a flushing-out effect on riparian forest(Swanson et al., 1998), plankton (Humphries et al., 2002),benthos (Fisher et al., 1982), and fishes (Chapman andKramer, 1991). Therefore, rainy seasons could inducedifficult conditions for mountain river fishes, in contrastto what occurs to floodplain river species. Fish reproductionin mountain rivers has been scarcely studied. For instance,in the subsample of studies from Munro’s (1990) reviewcorresponding to Characiformes, only two of 64 studies weremade in tropical mountain rivers. Therefore, the mainobjective of this study was to describe the annual reproduc-

tive activity of another mountain river fish, and its relationwith the rainfall pattern.

Creagrutus guanes was recently described from mountainrivers of the Rıo Magdalena basin, Colombia (Torres-Mejiaand Vari, 2005). Creagrutus guanes has a small standardlength (SL, maximum 5 77 mm), and is generally found insmall embayments along river banks and wide portions offast-current rivers, areas where water flow is minimal or null.We used histological and macroscopic observations todetermine the breeding pattern of C. guanes, and comparedthat pattern with reproductive and ecological characteristicsof other freshwater fish species.

MATERIALS AND METHODS

Study site and sampling.—Samplings were made between thetowns of San Gil and Charala, Santander, Colombia. Fishwere collected in the Rıo Fonce and 500 m upstream fromthe mouth of one of its tributaries, the Rıo Mogoticos(6u259–319N and 73u79–99W, between 1100 and 1350 maltitude). The Rıo Fonce drains a region of the west side ofthe Eastern Cordillera of Colombia, and it is part of the RıoMagdalena system. The Rıo Fonce watershed is primarilyforested and agricultural, and its soil is constituted mainly ofsedimentary rocks. The rainfall average shows a bimodalpattern (Figs. 1, 2), with two low-rainfall seasons (Decemberto March and June to August) and two high-rainfall seasons(April to May, and September to November).

Twenty to 30 C. guanes longer than 20 mm SL werecollected monthly from November 2003 to November 2004,in field campaigns of two days toward the middle of eachmonth. Only adults, 70 males and 135 females, wereincluded in the analysis. Capture methods included castnets, seines, dip nets, and hooks with worms as bait,depending on water conditions. Specimens were fixed inbuffered formalin (10%), transferred to ethanol (70%) 15 to30 days later, and were deposited in the ichthyologicalcollection of Universidad Industrial de Santander, Colom-bia.

1 Laboratorio de Biologıa Reproductiva de Vertebrados, Grupo de Estudios en Biodiversidad, Universidad Industrial de Santander, A. A. 678,Bucaramanga, Colombia; E-mail: (MTM) rtorr006@ucr.edu; and (MPRP) mpramir@uis.edu.co. Send reprint requests to MTM.

Submitted: 24 October 2006. Accepted: 16 July 2007. Section Editor: E. Schultz.F 2008 by the American Society of Ichthyologists and Herpetologists DOI: 10.1643/CP-06-256

2 Present address: Department of Biology, University of California, Riverside, California 92521.

Copeia 2008, No. 1, 99–104

Laboratory procedures.—Preserved specimens were dissectedunder a stereo microscope. Prior to dissection the standardlength (60.02 mm) and the total weight (60.001 g) of eachspecimen were measured. During the dissection the gonadswere photographed and observed under a stereo microscopeto record their macroscopic characteristics. Gonads, liver,mesenteric fat, and eviscerated body weights (60.001 g)were recorded for each specimen. We assumed that fatweights were underestimated due to dilution in alcohol toan unknown extent, although such error would have beenconstant for all samples because they were fixed andpreserved similarly.

Ovaries were classified macroscopically in three stages:immaturity, non-reproductive maturity, and reproductivematurity. Immature ovaries were oval, translucent andflaccid, with increasing length, opacity and firmness withsize, and occupied 5–30% of the abdominal cavity. Theywere whitish or yellowish, never with visible oocytes ofmature size. Non-reproductive ovaries were long, sometimeswith lobes, typically flaccid, and occupied 10–30% of theabdominal cavity. Their color was usually yellowish-trans-lucent, except for a few white, yellow or brown oocytesvisible in 50–75% of cases. Reproductive ovaries were long,with lobes, and extended around viscera, occupying 20–60%

of the abdominal cavity. They were always opaque, whitishwith yellow points (oocytes) to completely yellow. Theirconsistency was firm, but they were easily broken apart.Because testes with different microscopic stages showed verysimilar macroscopic characteristics, the development of amacroscopic maturity scale for males was not attempted.

Microscopic observations were made on slides prepared bystandard histological methods (Luna, 1968). To determine thecharacteristics of oocytes, transects were made through thegonads of 20 females chosen randomly. Along transects, themean diameter of more than 50 oocytes per gonad wererecorded following Tomkiewicz et al. (2003). The oocytes werestaged microscopically using an adaptation of the maturityscale of Wallace and Selman (1981) as modified by Matkovicand Pisano (1989): oogonia (stage I), pre-vitellogenic oocytes(stages II and III), cortical-alveoli oocytes (stage IV), early

vitellogenic oocytes (stage V), late vitellogenic oocytes (stageVI), and mature oocytes (stage VII). The granulosa cellsremaining after ovulation constitute the post-ovulatoryfollicles (POFs), which are cellular aggregates that are stainedby PAS. The granulosa cells undergo a degradation processuntil becoming indistinguishable from connective tissue.

The microscopic maturity scale used for ovaries wasmodified from Vazzoler (1996) and included seven stages:1) immature ovaries had mostly oogonia and primaryoocytes; 2) pre-mature ovaries were the same as immatures,but with cortical-alveoli oocytes; 3) ovaries in early vitello-genesis were similar to pre-matures, but with oocytes inearly vitellogenesis; 4) ovaries in total spawning hadabundant primary oocytes, few cortical-alveolli, atreticoocytes, and POFs, and rarely oogonias and late-vitelloge-netic oocytes; 5) ovaries in regeneration were similar toovaries in total spawning, but without POFs, nor late-vitellogenetic oocytes; 6) ovaries in late vitellogenesisshowed all kinds of oocytes, but late-vitellogenetic oneswere the most abundant, and never had POFs; 7) ovaries inpartial spawning were similar to ovaries in late-vitellogen-esis, except for having POFs.

Testes were classified microscopically using an adaptationof the scale of Blazer (2002): 1) pre-spermatogenic (only withspermatogonia); 2) early spermatogenic (mainly with sper-matogonia to spermatids, but with some spermatozoa); 3)mid-spermatogenic (with nearly equal proportions of sper-matocytes, spermatids, and spermatozoa); 4) late spermato-genic (with a high proportion of spermatogonia andspermatids in the cysts, and of spermatozoa in the lobularlumen and into the cysts); and 5) regressed (spermatogoniaand cells in resorption mostly or totally predominant in thecysts, but the lobular lumens could still contain spermato-zoa, even abundantly). The mean size at maturity wasdesignated as the length at which 50% of specimens weremature in a histogram of standard length (classes of 5 mmSL) versus percentage of mature specimens (Vazzoler, 1996).

Annual reproductive activity.—The monthly reproductiveactivity of adult specimens was determined using twoapproaches. The first was a series of ANCOVAs, one for eachdependent variable (gonad, liver, mesenteric fat, and eviscer-ated body weights) of each sex. Standard length was used as acovariate to control for variation due to body size. In femalesthe independent variable was month of capture, with samplesfrom June and July (both dry months), and from November2003 and November 2004 merged to increase the number ofobservations in the respective groups. For males, in order toreach the necessary sample size, two groups were formed bymerging samples from dry and rainy months. Assumptions ofnormality, linearity, homogeneity of variance–covariancematrices, homogeneity of regressions, and absence of outlierswere satisfied for all but one ANCOVA. The exception wasthat of male gonads, which showed heterogeneity of slopes.To overcome such heterogeneity we proceeded as recom-mended by Tabachnick and Fidell (2000), dividing standardlength in discrete groups and performing a two-way ANOVAusing the new variable as a random factor. For this analysisstandard length was divided in the two groups delimited bythe median. A square-root transformation was applied toweight-variables, to increase the normality, linearity, andhomocedasticity of groups.

We examined the degree of association between meanweights of gonads, liver, fat, and eviscerated body (as

Fig. 1. Monthly variation on adult female weight of: (A) gonads, (B) fatand liver, and (C) body. Values are marginal means adjusted for thecovariate (standard length), 6 SE. Sample size in (A). Shadow behindthe lines represent the rainfall pattern.

100 Copeia 2008, No. 1

obtained by ANOVA and ANCOVAs), and average rainfall.Additionally, we analyzed the possibility that correlationamong variables was lagged (e.g., the gonad weight could bemore correlated with the rains of one month before thanwith rains of same month). To do so, we calculated thePearson-correlations between each variable in month x withthe second variable at the same month (x), at one monthbefore (x21), at two months before (x22), at one monthlater (x+1), and at two months later (x+2). A degree offreedom (i.e., the unmatched observations at the tail of eachseries) was lost every time a month was lagged. To correctthe Type I error rate (a) due to the repeated use of same datafor various correlations, a false discovery rate control wasimplemented (Verhoeven et al., 2005).

The second method employed to characterize the repro-ductive activity was comparison of monthly proportions ofmaturity stages of adults using micro- and macroscopicobservations. Proportions were compared using contingencytables, computed with a standard spreadsheet. To fulfil therecommendation of having expected frequencies higherthan six on average (Zar, 1999), months with similarhydrological characteristics were merged when necessary.

RESULTS

Standard length was significantly greater (Mann–Whitney Ua0.05,2, n 5 153,50 5 790.5, P , 0.001) in females (mean 6 SD

5 59.44 6 6.54 mm) than males (37.63 6 2.32 mm).Females matured at a larger size than males (mean size atmaturity was 48 mm in females and 28 mm in males). Forfemales, standard length was significantly related withgonad, liver, and body weights, but not with fat weight(Table 1). Females showed a seasonal pattern of gonad, liver,fat, and body weights, as revealed by ANCOVAs (Table 1,Fig. 1). Lagged correlations, without nominal P-valuesadjusted for multiple testing, partially explained the sea-sonal patterns of variables (Table 2). However, after correct-ing the type-I error rate, only two correlations remainedsignificant. These two correlations revealed that fat andgonad weights were highest one and two months afterheaviest rains, respectively (Table 2).

The reproductive pattern obtained by macro- and micro-scopic observation of mature female gonads coincided withthe analysis of their weight. For the macroscopic-stageanalysis, the significant differences in the proportion ofreproductive stages (n 5 134, x2 5 68.14, df 5 10, P , 0.001)were due mainly, in order of importance, to the highproportion of non-reproductive females during April, Sep-tember, October, and March (all rainy months except thelatter), and the high proportion of reproductive females inJune–July, December and November (all dry months exceptthe latter; Fig. 2A). For the analysis of the proportion ofmicroscopic stages, months were pooled in four groups ofhigh and low rainfall (Fig. 2B), which showed a significant

Table 1. Results of ANCOVAs. * 5 P , 0.05, ** 5 P , 0.001. a Two-way t-test made with all males, using rainfall (high vs. low) as fixed effect and size as

random effect. Size codified in two groups, small and large, which were divided by the median (3.86 mm). b One way t-test made only with males with SL .

median. Partial g2 is shown to indicate the percentage of variance in the dependent variable that is explained by the independent variable, when their

relationship is significant (Tabachnick and Fidell, 2000).

DV

Significance of standardlength as covariant

Significance of grouping variable(month or groups of months)

F df F df Partial g2

Females Gonad 93.2** 1, 120 11.3** 10, 120 0.48Fat 0.009 1, 120 15.4** 10, 120 0.56

Liver 52.6** 1, 120 3.0* 10, 120 0.20Body 1120.9** 1, 120 8.1** 10. 120 0.47

Males Gonada — — 1.64 1, 59 —Gonadb — — 20.4** 1, 30 0.06

Fat 127.75** 1, 60 0.001 1, 60 —Liver 13.60** 1, 60 0.051 1, 60 —Body 1750.15** 1, 60 2.48 1, 60 —

Table 2. Correlation between Rain and Weights of Females, Lagging Them One or Two Months, and without Lagging. a 5 P , 0.05 without adjustment

nominal P-values for multiple testing; b 5 P , 0.05 after adjustment.

Fixed variable Lagged variable Month 22 Month 21 Same month Month +1 Month +2

Rainfall Gonad 0.35 20.77 a 20.53 0.45 0.89ab

Liver 0.01 20.45 0.18 0.53 0.42Fat 20.70a 20.25 0.49 0.81ab 0.20

Body 20.32 ,0.01 0.62a 0.75a 0.17Gonad Liver 20.22 0.39 0.48 20.31 20.46

Fat 0.44 0.76a 0.21 20.61 20.34Body 0.35 0.63a 0.06 20.63a 20.30

Liver Fat 0.11 0.22 0.39 20.12 20.31Body 20.01 0.59 0.40 20.21 0.17

Fat Body 0.19 0.35 0.75a 0.07 20.26

Torres-Mejia and Ramırez-Pinilla—Dry-season breeding of Creagrutus 101

variation in proportion of reproductive stages (n 5 134, x2 5

36.15, df 5 9, P , 0.001). This was mainly due to theproportion of females with ovaries in partial spawning andin regeneration.

For ANCOVAs of males, months were pooled in twogroups, dry and rainy months, to increase the number ofobservations per case. Five males were excluded due tomissing data. By using Mahalanobis distance with P , 0.001,two multivariate outliers were found and therefore deleted.The results for males showed that standard length wassignificantly related with each of the dependent variables(Table 1). The test for heterogeneity of slopes supported therelationship between the standard length and the weight ofgonads (ANOVA, F1,62 5 14.18, P , 0.001). In the case of

males, ANCOVAs showed that none of the dependentvariables varied significantly between rainy and dry months(Table 1, Fig. 2C). However, males larger than the medianshowed a very small but significant increase in gonad weightduring dry months, which explains the heterogeneity ofslopes obtained for this variable.

The analysis of microscopic observation of mature malesshowed that reproduction was concentrated during drymonths. For males, months were pooled in two groups ofhigh and low rainfall. Proportions of reproductive stagesvaried significantly between these two groups (n 5 70, x2 5

11.31, df 5 3, P , 0.025, Fig. 2D). The differences weremainly attributable to the high proportion of males in latespermatogenesis stage during dry months, and the lowproportion of males with testis in regression during drymonths.

DISCUSSION

Seasonality of reproduction.—Weight and microscopic obser-vation of gonads of both sexes, plus macroscopic observa-tion of ovaries, suggested that reproduction of C. guanes wasmore intense in dry months. Lagged correlations showedthat significant monthly variation of fat and gonad weightsis related to rainfall. One month after heavy rains fat weightincreased to its maximum. Two months after heavy rains,coinciding with dry seasons, gonads were heaviest. Thisindicates that gonad weight had an inverse relationshipwith recent rainfall. The lag between the acquisition of fatweight and the subsequent increase of gonad weight mayindicate that resources are being stored during rainy monthsto be invested later in reproduction. Other aspects of fat andgonad weight patterns, as well as the patterns of evisceratedbody and liver weight, were significantly correlated withunadjusted P-values, but not significant after adjusting formultiple testing. Nevertheless, these non-significant pat-terns were congruent with the hypothesis that rainfallpromotes resource acquisition, and breeding is concentratedon dry seasons. For example, the pattern of body weightswas similar to that showed by fat, with higher meansoccurring during and after the rainiest months, and lowermeans occurring after the months with heavy gonads.Further analyses, using longer time series, the monitoringof marked fishes, or a combination of both, may increase thepower to quantify the seasonal variation in body compart-ments associated with reproduction.

Liver weight showed a weak monthly variation (see lowpartial g2 values in Table 1), and was not associated with anyvariable in the lag analysis (Table 2). Since liver produces thevitellogenin that is sequestered by ovaries and becomes themain component of egg yolk (Mellinger, 2002), it wasunexpected that liver did not show the strong patternsimilar to those of fat and gonad weights. However,preliminary histological analysis revealed that hepatocytemorphology changes with reproductive stage (pers. obs.),indicating a massive involvement of liver in seasonalreproductive activity. The seasonal changes of liver weightmay have been obscured by other processes that this organperforms.

Two months depart from the pattern of reproductionconcentrated on dry months: March, a dry month withreduced reproduction, and November, a rainy month with ahigh proportion of vitellogenic females. In March, repro-duction may be prevented by resource depletion. This idea issupported by the low weight of fat observed during this dry

Fig. 2. Seasonal pattern of reproduction of both sexes. (A) Macro- and(B) microscopic reproductive stages of adult females. (C) Gonad, fat,liver, and body weights, and (D) microscopic reproductive stages ofadult males. ** 5 P , 0.001. 1 All testes. 2 Small-male testes. 3 Large-male testes.

102 Copeia 2008, No. 1

period. An alternative explanation could be that fishesfollow an environmental cue that indicates upcomingrainfall, assuming that rainy-season reproduction is disad-vantageous for juveniles (see hypotheses below). Thereforereproduction during March may be avoided by femalesbecause at that time young would not have time to growenough to survive the coming high water period. Thesituation in November is opposite to that of March. DuringNovember, females with ovaries in late vitellogenesisaccounted for 60–80% of all mature females, but no femalewith POFs was captured. This could indicate that vitello-genic eggs were retained, perhaps delaying reproductionuntil the proper environmental cues or conditions occurred.Moreover, during November females were also storing fat,which was spent during the subsequent months. Thisevidence indicates that November was a month of prepara-tion for breeding. Numerous reproductive patterns withpeaks of gonad weight before the spawning season has beenreported by Munro (1990). This frequent delay mayconfound the inference of reproductive patterns by exclu-sively inspecting the gonad weights, a common practice inichthyology.

Comparison with other fishes.—From the 67 species ofCreagrutus, the phenology of only three species has beenstudied: C. bolivari (Ortaz, 1997), C. brevipinnnis (Roman-Valencia, 1998), and C. melasma (Winemiller, 1989). Theformer two species were sampled in mountain rivers, andthe latter in a lowland stream. These analyses concludedthat reproductive season was extended, including both dryand rainy months. However, since these studies were basedon the analysis of gonad weights, the apparent reproductionduring rainy months may correspond in fact to a monthprevious to spawning, similar to what we observed in oursamples from November. The latter scenario may be whatoccurred in studies of C. bolivari (Ortaz, 1997) and C.brevipinnis (Roman-Valencia, 1998). The species of Creagru-tus that inhabit mountain rivers seem to reproduce duringdry months. Further analyses of reproductive patterns ofCreagrutus inhabiting lowlands are needed.

In general, the reproduction of many tropical freshwaterfish species has been studied (Munro, 1990), most of thembreeding during wet season in the tropical areas, or duringspring and summer in subtropical regions. However, thereare some species that breed during dry months, few of themin lowland streams (Payne, 1975; Kramer, 1978; De Silva etal., 1985), and most of them corresponding to studies madeon mountain rivers (Sazima, 1980; Flecker et al., 1991;Harikumar et al., 1994; Wang et al., 1995; Pusey et al., 2002).The intensity and predictability of flow regimes may lead tolife history adaptation of freshwater organisms (Lytle andPoff, 2004). With evidence from invertebrates, plants, andfishes, dry season reproduction has been proposed to be anadaptation to floodings (Lytle and Poff, 2004). The gener-ality of this pattern and its adaptive significance deservesmore investigation.

Factors favoring dry-season reproduction.—Several factorshave been proposed for explaining the advantage ofreproduction during dry season in tropical stream fishes.Kramer (1978) invoked biotic factors, such as food availabil-ity, interspecific competition, and avoidance of interspecificbreeding. An alternative explanation, defined as the ‘‘lowflow recruitment’’ hypothesis (Humphries et al., 1999), gives

importance to both biotic and abiotic factors. It states thatduring dry seasons tropical rivers have a higher concentra-tion of larval food (zooplankton and algae) and a morestable habitat (no flash floods), both factors favoringsurvival of offspring. Neither Kramer’s nor Humphries’hypothesis has been tested.

In the specific case of the Creagrutus population studiedhere, circumstantial evidence weakens the support for thehypothesis that reproduction is synchronized with foodabundance for adults. The observation of females with highfat weight soon after rains suggests that reproduction duringdry season is not synchronized with food availability foradults of C. guanes. Another hypothesis, that reproductionduring dry season prevents juveniles from being washed outby heavy rains, is doubtful for C. guanes. Like other tropicalfishes that inhabit mountain rivers (Vari and Harold, 2001),this species prefers backwaters and embayments, where flowspeed is null even during the strongest floods, and it isunlikely that juveniles can be washed out in thoseconditions. Additional studies on the habitat use by C.guanes along its ontogenetic development would help toclarify this question.

The unusual peak of reproductive activity during the dry-season described here is another example that the patternobserved in other more studied habitats, such as floodplainrivers, should not be considered predominant in tropicalfreshwaters fishes (Kramer, 1978; Ortaz, 1997). Moreover,the wide-encompassing hypotheses of Kramer (1978),Humphries et al., (1999), and Lytle and Poff (2004) couldbe tested, by observation of natural systems or by experi-mental approaches (Brown and Shine, 2006). Currentobservational data is imbalanced. Thus future researchshould focus on poorly studied habitats, such as low-orderstreams. A greater understanding of the reproductive timingof tropical freshwater fishes is needed given the increasinganthropogenic alteration on rivers and climate.

MATERIAL EXAMINED

Creagrutus guanes. All catalog numbers have the prefix UIS-T.For dates and sites of collections see Materials and Methodssection. UIS-T 24, 27, 29, 43–44, 48, 49–52, 54, 56, 59, 61,64–55, 67, 69, 74, 79, 81, 92, 94–95, 102, 107, 117, 120–121,126–128, 132–136, 139–141, 143–144, 146–148, 150, 152–156, 160, 166–168, 172–174, 178, 181–182, 189, 234–235,252–253, 256, 258, 260–262, 264, 267–269, 276, 279, 286,291–292, 294, 296–298, 1003, 1010, 1021, 1023, 1038, 1041,1048–1050, 1052, 1054–1056, 1058, 1060, 1071, 1092, 1126,1130, 1133–1134, 1137–1138, 1141, 1144, 1149, 1156–1157,1161–1164, 1167, 1175–1176, 1188, 1191, 1206, 1219, 1222,1231 ,1251–1252, 1255, 1260–1261, 1263–1267, 1271–1273,1275–1277, 1278–1280, 1284, 1289, 1292–1293, 1295–1296,1306–1307, 1314–1315, 1333, 1335–1336, 1341, 1344,1346–1347, 1410–1412, 1441, 1530–1531, 1533, 1535–1536, 1540–1547–1548, 1551, 1553, 1555, 1557–1558,1560–1563, 1568, 1570–1572, 1574–1575, 1592–1597.

ACKNOWLEDGMENTS

G. Galvis, J. Mojica, J. Burns, and F. Rangel made significantrecommendations about the project. R. Gavilan and Escuelade Biologıa–UIS facilitated equipment. F. Rangel, P. Rueda,M. Valderrama, J. Santander, N. Pinto, O. Rojas, M. Rojas, M.Myyo, A. Motta, and M. Quijano assisted with field work. D.Kramer, M. Alkins-Koo, and D. Reznick made important

Torres-Mejia and Ramırez-Pinilla—Dry-season breeding of Creagrutus 103

suggestions to the manuscript. J. Rotemberry and V. Serranogave statistical advice. Thanks to the members of the LBRV–UIS lab and the UCR Reznick’s lab. From the former, N.Pinto gave opportune assistance. From the latter, A. Bannetinspected English and J. Arendt helped with figures.INCODER issued the permission to capture the specimens.This study was supported by LBRV–UIS. L. Mejia, P. Torres,and G. Serpa.

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