The relationship between genome size, development rate, and body

size in copepods

Grace A. Wyngaard1,*, Ellen M. Rasch2, Nicole M. Manning1, Kathryn Gasser1 &Rickie Domangue31Department of Biology, James Madison University, Harrisonburg, VA 22807, USA2Department of Anatomy and Cell Biology, James H. Quillen College of Medicine, East Tennessee

State University, Johnson City, TN 37614, USA3Department of Mathematics, James Madison University, Harrisonburg, VA 22807, USA(*Author for correspondence: E-mail: wyngaaga@jmu.edu)

Received 10 December 2003; in revised form 4 May 2004; accepted 11 May 2004

Key words: genome size evolution, nucleotype, developmental rate, body size, copepod

Abstract

Freshwater cyclopoid copepods exhibit at least a fivefold range in somatic genome size and a mechanism,chromatin diminution, which could account for much of this interspecific variation. These attributessuggest that copepods are well suited to studies of genome size evolution. We tested the nucleotypichypothesis of genome size evolution, which poses that variation in genome size is adaptive due to the ‘bulk’effects of both coding and noncoding DNA on cell size and division rates, and their correlates.We found a significant inverse correlation between genome size and developmental (growth) rate in fivefreshwater cyclopoid species at three temperatures. That is, species with smaller genomes developed faster.Species with smaller genomes had significantly smaller bodies at 22 �C, but not at cooler and warmertemperatures. Species with smaller genomes developed faster at all three temperatures, but had smallerbodies only at 22 �C. We propose a model of life history evolution that adds genome size and cell cycledynamics to the suite of characters on which selection may act to mold life histories and to influence thedistribution of traits among different habitats.

Introduction

The prevailing explanation of the C-value enigma(Gregory, 2001a) is that variation in genome sizeamong organisms of comparable complexity isnonadaptive and provides no understanding of themechanisms underlying life history variation (John& Miklos, 1988). This view ascribes the terms‘junk’ and ‘parasitic’ DNA to heterochromaticDNA that accounts for much of the variation ingenome size (Ohno, 1972; Doolittle & Sapienza,1980; Orgel & Crick, 1980).

A contrasting explanation considers genomesize itself to be a phenotypic character subject to

selection. As evidence accumulates supportingfunctional roles of heterochromatin, the merits ofincorporating genome size evolution into modelsof life history theory become more appreciated(McLaren et al., 1966, 1988, 1989; Cavalier-Smith,1978, 1980; Escribano et al., 1992; Beaton &Cavalier-Smith, 1999; Gregory & Hebert, 1999).To paraphrase Cavalier-Smith (1985a) the nullhypothesis to be tested when challenging the pre-vailing explanation is one in which the bulk of thenoncoding DNA that comprises genome size is‘junk DNA’ that is insignificant in an evolutionarysense. The alternative hypothesis that we test inthe present study is that genome size, a large

Hydrobiologia (2005) 532: 123–137 � Springer 2005

portion of which must be heterochromatic, non-coding DNA, covaries with fitness traits in cyclo-poid copepods. This hypothesis most closelyresembles the nucleotypic hypothesis proposed byBennett (1971, 1972) and later discussed byGregory (2001a). McLaren and colleagues haveargued persuasively that genome size is animportant determinant of developmental rate andbody size in marine calanoid copepods, throughits influence on cell size and cell division rates.Here we extend some of their ideas and ap-proaches to a copepod taxon that possesses adocumented mechanism that expands the range ofgenome size. Some cyclopoid species dramaticallyreduce the size of their somatic nuclear genomesby 35–99% during embryonic chromatin diminu-tion (Grishanin et al., 1994; Wyngaard & Rasch,2000). Our goal is to explore how large scale fea-tures of the genome, which operate at levels belowthe whole organism, may either constrain evolu-tion or enhance evolutionary change, and socontribute to the life history variation we observeat the organismal level.

In the present study we explore the relationshipbetween genome size and developmental rate andbody size in five species of warm adapted, fresh-water species that span the smallest to largest ge-nomes known in cyclopoids. The cyclopoidcopepods seem to be especially well suited for thisgoal because they (1) possess an obvious mecha-nism, chromatin diminution, for expanding thetotal range of interspecific genome sizes, (2) mayhave constancy in cell number among closely re-lated species so that genome size would influencebody size via cell size, (3) have determinate growthwhich facilitates ease in measuring nuclear DNAcontents in adults and may exert definitive effectson adult body size, and (4) exhibit a considerablerange of developmental rates and body sizes,which contribute substantially to fitness.

We predict that species with smaller genomeswill develop faster. This exploratory study wasconducted at 15, 22 and 30 �C to determine if fu-ture surveys would need to incorporate the effectsof temperature in the experimental design. Wepredict that relationships between genome size anddevelopment rate may differ among the threetemperatures, if genotype by environment inter-actions occur such as those typically reported forphysiological traits (Wyngaard, 1986).

Because development rate is often intertwinedwith body size and some marine calanoid copepodspecies have similar numbers of total nuclei perindividual organism that could enhance the effectsof genome size (McLaren & Margoliese, 1983), weexamined the relationship between genome sizeand body size. We predict that species with smallergenomes will have smaller bodies at maturity.

We contribute data on variation in genome si-zes and life histories to a small body of relatedpublished literature, with the hope of increasingthe awareness of holistic approaches that shownew promise in connecting organismal traits togenomic and ecological functions (Sterner & Elser,2002). Finally, we propose a model that linksvariation in genome size, cell cycle dynamics, andzooplankton life history traits to the types ofhabitat that favor particular combinations of thetraits. This model explores how variation at ahierarchy of levels from the nucleotype to theorganism, may constrain or enhance evolutionarychange in zooplankton life histories.

Materials and methods

Species sources and identifications

Species were chosen because they spanned therange of known adult somatic nuclear DNA con-tents in freshwater cyclopoid copepods. All sam-ples were from warm temperate waters. Thecollecting locale and species identification for theViet Nam population of Thermocyclops crassus aregiven in Wyngaard & Rasch (2000), for the Vir-ginia population of Mesocyclops edax in Dobrzy-kowski & Wyngaard (1993), and for the Louisianapopulations of Megacyclops latipes, Mesocyclopslongisetus, and Mesocyclops pehpeiensis in Dor-ward & Wyngaard (1997). In previous studies(Dorward & Wyngaard, 1997; Wyngaard & Ras-ch, 2000), we used the name Mesocyclops ruttnerito refer to the M. pehpeinensis population. In thepresent study we follow Guo (2000) who synony-mized M. ruttneri with M. pehpeinensis.

Laboratory culture – All species were fedCryptomonas ozolini Skujja (UTEX-LB2194) andcultured in defined media according to Wyngaardand Chinnappa (1982) except that newly hatchedArtemia salina nauplii were substituted for cala-

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noid copepodid prey. Copepods were reared in15 ml capacity Petri dishes at 15, 22, and 30 �Cand L:D 14:10 in a Conviron CMP 3244 envi-ronmental chamber that varied less than ±0.5 �C.Media and algae were replenished every secondday at 15 and 22 �C and every day at 30 �C. FiveArtemia nauplii were fed to copepodid III–VItwice daily, except for the small bodied T. crassus.

Specimen preparation and Feulgen–DNA cytopho-tometry

Squashes of adult females for determination ofnuclear DNA content were prepared according toDorward & Wyngaard (1997). Measurements ofstaining intensity and optical density were madeusing a Vickers M85 scanning and integratingmicrodensitometer. This method allows identifi-cation and selection for measurement of hetero-geneous cell types of individual nuclei that aresparsely dispersed in squash preparations of cy-clopoid copepods that exhibit both chromatindiminution and endoreduplication. Specific detailsfor fixation, tissue handling and staining protocolsfor the Feulgen–DNA reaction are given elsewhere(Rasch & Wyngaard, 1995, 2001). Simultaneouslystained chicken red blood cells (2.5 pg DNA pernucleus) and trout red blood cells (5.0 pg DNAper nucleus) were used as internal reference stan-dards to estimate actual genome size in pg DNAfor copepod nuclei (Rasch, 1985, 2003). With theexception of Mesocyclops pehpeiensis, for whichonly a limited cell population was available, thenumber of somatic nuclei measured for each spe-cies ranged from 375 to 2800. Coefficients of var-iation were less than 2% for repeated scans onindividual copepod nuclei.

Species that exhibit embryonic chromatindiminution characteristically have sperm withDNA levels that are significantly higher thanwould be predicted from the DNA content ofdiploid or 2C somatic nuclei of the same species(Beermann, 1977; Rasch & Wyngaard, 1995).Thus, sperm DNA levels cannot be used as a baseline to estimate C-values for other types of cellsduring postembryonic development. For this rea-son we report genome size as the diploid or 2Cnuclear DNA content found in adult somatic tis-sues. Although adult copepods have ceasedgrowing and lack mitoses, occasionally cells occur

with twice the DNA content of typical adult so-matic cells, as also reported by Escribano et al.(1992).

Development time and body size measurements

Times of hatching of embryos into nauplii wereeither observed and known exactly or recorded tooccur within a 2 h interval. Times of molting fromCV to adult (for determination of maturation rate)were observed at no longer than 5 h intervals at 15and 22 �C and 3 h periods at 30 �C. Sample sizesof body size measurements were occasionallysmaller than those for maturation time when onlya portion of the adults were preserved for mea-surement. Body size was computed as the cube ofadult female cephalothorax length, as measured at40· or 100· using a compound microscope. Thismeasure assumes a similar tear-drop shape amongthese species, but is preferable to using bodyweight which can reflect rapidly fluctuating lipidstores.

Statistical analyses

Development rate (reciprocal of development time)was used in the analysis to normalize the slightlypositively skewed distributions of developmenttime and to reduce the heterogeneity of variancesamong species. Heterogeneity of variances indevelopment rate among species was found only incomparisons with M. latipes. The ordered hetero-geneity test (OH) is a more powerful test than anondirectional test of heterogeneity such as the F-test in the one-way analysis of variance (ANOVA)(Rice & Gaines, 1994a) and was used to test H0

that l1 ¼ l2 ¼ l3 ¼ l4 ¼ l5 versus the ordered Ha

that l1 ‡ l2 ‡ l3 ‡ l4 ‡ l5, with at least one strictinequality. The l1, l2, l3, l4, l5, denote, respec-tively, the true mean development rates of thespecies T. crassus, M. pehpeiensis, M. longisetus,M. edax and M. latipes. This a priori hypothesisspecifies that species with smaller genomes developas fast as or faster than species with larger ge-nomes. The test statistic is the product rsPc wherers is Spearman’s rank correlation between the ob-served ranking of the sample means of develop-ment rates and the ranking prescribed on thealternative hypothesis (Rice & Gaines, 1994b). Thevalue Pc is the complement of the p-value based on

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the F-ratio in the usual one-way ANOVA fordevelopment rate. The OH tests reject the nullhypothesis for large values of rsPc. The p-value forthe OH test statistic rsPc was estimated usingTable 1 of critical values from Rice & Gaines(1994b). For example, at p ¼ 0.05, the critical va-lue for rsPc, when there are five groups, is 0.509and thus p is a value <0.05 if the value of rsPc islarger than 0.509. The OH test was also used totest the null hypothesis that mean body size is thesame for the different species against the orderedalternative that species with smaller genome sizeshave less than or equal body sizes as species withlarger genomes.

To examine pairwise differences in develop-ment rate between species, as well as pairwisedifferences in body size, at a particular tempera-ture, Scheffe’s multiple comparison procedure wasused. Additionally, the degree of association be-tween body size and development rate was com-puted using product moment correlations basedon the mean values of traits for each of the fivespecies at each of the three temperatures. Onlyspecimens for which measurements of both devel-opment rate and body size were available were

used in computation of these product momentcorrelations. All statistics were computed usingSPSS (2000).

The particular species were chosen because theyspanned the range of genome sizes known forwarm water cyclopoids. To the extent that mea-sures of any statistical association can be attrib-uted to common ancestry, rather than toindependent changes such as occur during parallelor convergent evolution, the strength of the sta-tistical relationships we report may be inflated.This concern is more consequential in comparisonsamong distantly related taxa than in the presumedclosely related taxa studied here (Rylov, 1948;Ueda & Reid, 2003).

Genome size

Genome size varied from 0.84 to 4.11 pg per nu-cleus among the five species studied here. Differ-ences in genome size between males and females ofa single species were negligible, as were differencesamong the several specimens sampled for a givenspecies (Table 1).

Table 1. Estimated genome size (pg DNA per diploid, 2C nucleus) for adult tissues from five species of cyclopoid copepods

Species Sex N DNA pg per nucleus n Genome

Mean ± SE size (nps)

Megacyclops latipes

Female 5 4.11 0.052 275 3.75 · 109

Male 3 3.93 0.041 150 3.59 · 109

Mesocyclops edax*

Female 38 3.02 0.021 2800 2.76 · 109

Male 34 2.97 0.005 2051 2.67 · 109

Mesocyclops longisetus*

Female 10 1.79 0.012 510 1.63 · 109

Male 5 1.78 0.094 228 1.62 · 109

Mesocyclops pehpeiensis

Female 2 1.34 0.156 70 1.22 · 109

Male 2 1.56 0.027 100 1.42 · 109

Thermocyclops crassus

Female 8 0.84 0.054 580 0.76 · 108

N designates the number of specimens examined, n denotes number of individual nuclei measured. nps is the estimated number of

nucleotide pairs based on the conversion factors to convert quantities of DNA expressed in pg to number of nucleotide pairs or to

number of kilobases given by Rasch et al. (1971). Species which exhibit chromatin diminution are marked by an asterisk after the

species names. All data, except for nps, are after Wyngaard & Rasch (2000).

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Genome size and developmental rate

Among the five species tested, only M. latipesshowed nonnormal distributions of developmentrate, with a positive skew at 15 �C and a negativeskew at 22 and 30 �C. Species with smaller ge-nomes developed as fast or faster than species withlarger genomes at all three temperatures (15 �C,p < 0.01; 22 �C, p < 0.001; 30 �C, p < 0.001;OH) (Fig. 1, Table 2). Scheffe’s procedure wasperformed to examine how mean developmentrates differed among species within each tempera-ture and if significance in the OH tests reflectsinequalities among means. The mean developmenttimes on which the rates were based and theScheffe’s procedure computed are given in Table 3.

Temperature effects

At 30 �C (Fig. 1, Table 3), the developmentaltime from nauplius I to adult female was shortestfor T. crassus and M. pehpeiensis. The variancesaround the very similar mean development timesof these two species were small, so the Scheffe’smultiple comparison test detected a statisticallysignificant difference (p ¼ 0.015) (Table 3). The

other three species (M. longisetus, M. edax, M.latipes) each developed at times that were signifi-cantly longer and different from each other, withM. latipes developing the slowest. Thus the sig-nificant result in the OH test reflects an equalitybetween T. crassus and M. pehpeiensis and differ-ences between all other comparisons.

At 22 �C (Table 3), T. crassus matured fasterthan M. pehpeiensis. M. longisetus, M. edax, andM. latipes matured at successively slower rates. Allpairwise differences in species were significant(p < 0.001), except for M. edax and M. latipes(p ¼ 0.422, Scheffe’s multiple comparison test),and thus the results of the Scheffe’s procedure areconsistent with the results of the OH test results.

At 15 �C (Table 3), M. pehpeiensis matured thefastest and M. latipes matured the slowest.M. longisetus and M. edax developed at the samerate (Scheffe’s procedure, p ¼ 0.983). Similar tothe relative developmental rates observed at 30 �C,M. pehpeiensis matured faster than T. crassus(Scheffe’s procedure, p < 0.001). Thus the signif-icance in the OH reflects equality or faster devel-opment times between smaller genome sizescompared to larger genomes, with the exception ofT. crassus and M. pehpeiensis which are in reverse

Figure 1. Plot of developmental rate of five cyclopoid species under the three experimental temperatures. Development time (days) is

transformed to a rate function using 1/development time in order to satisfy the assumption of a normal distribution in the statistical

analyses.

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Table 2. Directed test of relationship between (A) genome size and development rate and (B) genome size and body sizea

Temperature (�C) Pc rs Pc rs Critical value p-Value

A. Development rate

15 1.0 0.8 0.8 0.740 <0.01

22 1.0 1.0 1.0 0.740 <0.001

30 1.0 0.9 0.9 0.740 <0.001

B. Body size

15 1.0 0.7 0.7 0.740 <0.05

22 1.0 0.9 0.9 0.895 <0.001

30 1.0 0.7 0.7 0.509 <0.05

aA tests the alternative hypothesis that smaller genomes develop as fast or faster than larger genomes: Thermocyclops

crassus ‡ Mesocyclopspehpeiensis ‡ Mesocyclops longisetus ‡ Mesocyclops edax ‡ Megacyclops latipes. B tests the alternative

hypothesis that species with smaller genomes mature as small or smaller than species mature as small or smaller than species with

larger genomes: Thermocyclops crassus £ Mesocyclops pehpeiensis £ Mesocyclops longisetus £ Mesocyclops edax £ Megacyclops

latipes. The OHs tests were computed for each temperature separately. P = p value, Pc = (1 ) P), rs = Spearman’s rank correlation

coefficient, and Pc rs = OH test statistic after Rice & Gaines (1994b). Critical values of 0.509, 0.740, and 0.895 correspond to 0.05,

0.01, and 0.001, respectively.

Table 3. Mean female development time (days) and body sizes (lm3 · 106) of five cyclopoid copepod species reared at 15, 22, and

30 �C

Species 15 �C 22 �C 30 �C

A. Development time

T. crassus 29.9 (0.74) 9.7 (0.33) 5.5 (0.28)

n = 14 n = 16 n = 12

M. pehpeiensis 22.2 (0.92) 11.1 (0.46) 5.4 (0.12)

n = 10 n = 19 n = 29

M. longisetus 40.2 (4.9) 13.9 (1.47) 7.6 (0.65)

n = 18 n = 24 n = 16

M. edax 39.6 (3.65) 17.2 (0.68) 10.7 (0.44)

n = 10 n = 12 n = 6

M. latipes 72.4 (12.8) 18.9 (2.20) 16.9 (2.68)

n = 13 n = 8 n = 10

B. Body size

T. crassus 0.1548 (0.0176) 0.1570 (0.0085) 0.1673 (0.0321)

n = 13 n = 16 n = 11

M. pehpeiensis 0.5672 (0.0325) 0.4626 (0.0357) 0.3329 (0.0304)

n = 7 n = 18 n = 29

M. longisetus 0.6819 (0.0442) 0.7057 (1.0856) 0.5159 (0.0327)

n = 14 n = 24 n = 16

M. edax 0.5192 (0.0291) 0.5879 (0.0427) 0.2343 (0.0262)

n = 3 n = 7 n = 6

M. latipes 0.9905 (0.1814) 0.8556 (2.0890) 0.6230 (2.0508)

n = 12 n = 6 n = 7

Standard deviations are in parentheses. Number of adult females measured is indicated by n. Means of development time were

transformed as described in Methods for the OH tests and Scheffe’s tests.

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order of the prediction of more rapid maturationof species with smaller genomes.

Genome size and body size

All species showed normal distributions of bodysize at all temperatures. Species with smaller ge-nomes matured at a size as small or smaller thanspecies with larger genomes at all three tempera-tures (15 �C, p < 0.05; 22 �C, p < 0.001; 30 �C,p < 0.05; OH) (Fig. 2, Table 2). As with devel-opment rate, a Scheffe’s procedure was performedto examine how mean body sizes differed amongspecies within each temperature. The means onwhich these tests were based are shown in Table 3.

Temperature effects

At 30 �C (Table 2), the adult female body size wassmallest for T. crassus and largest for M. latipes.The Scheffe’s multiple comparison test detected astatistically significant difference (p £ 0.007) in allpairwise comparisons, although M. edax, M. peh-peiensis, and M. longisetus are not in the order thatwould be predicted by a perfect ranked correlationbetween genome sizes and body size (Table 3).

At 22 �C (Table 3), the adult female body sizewas smallest for T. crassus and largest forM. latipes. All pairwise differences in species weresignificant (p < 0.001), but the body sizes ofM. longisetus and M. edax were reversed based onthe prediction that species with smaller genomeshave smaller body sizes.

At 15 �C T. crassus and M. latipes were sig-nificantly different from each other and from theother three species (p < 0.001) (Table 3). How-ever, no statistically significant differences in adultbody size were detected among M. edax, M. peh-peiensis, and M. longisetus. This is consistent withthe OH and reveals that equalities, rather thandifferences, were detected among three species.

Developmental rate and body size

Mean developmental rate was negatively corre-lated with mean body size at 22 �C (r ¼ )0.814,p ¼ 0.047, Fig. 2), but not at 15 �C (r ¼ )0.693,p ¼ 0.351) and 30 �C (r ¼ )0.583, p ¼ 0.341)(Fig. 3). These statistical results are nearly indis-tinguishable from results computed on pairs ofdevelopment rate and body size for individuals.

Figure 2. Plot of body size of five cyclopoid species under the three experimental temperatures.

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Discussion

Genome size

Somatic nuclear DNA contents of the five fresh-water cyclopinid species varied from 0.84 to4.11 pg per nucleus (Table 1). This variationencompasses the published values for somatic nu-clear DNA contents for freshwater cyclopoids,except for Cyclops kolensis and Cyclops strenuuswhich contain 0.28 and 0.36 pg, respectively(Grishanin et al., 1994; Gregory, 2001b).

Intraspecific variation in genome size was neg-ligible for all of the cyclopoid species analyzed here(Table 1). Using methods of DNA–Feulgen cyto-

photometry essentially similar to those employedby McLaren et al. (1988, 1989), Rasch & Wyng-aard (1995) searched extensively for intraspecificvariation in genome size both within and amongpopulations of Mesocyclops edax from NovaScotia, Virginia and Florida. They found no evi-dence in support of levels of intraspecific variationcomparable to those reported for the marine cal-anoids C. glacialis, Pseudocalanus acuspes, andPseudocalanus elongatus (Escribano et al., 1992).Taking into account the practical limits of DNA–Feulgen cytophotometry (0.01 pg DNA, Rasch,1985) and the consistent constancy of genome sizesfound here for multiple specimens of both maleand female adults of each of the species analyzed,

Figure 3. Development rate (day)1) and body volume (cephalothorax length3) in T. crassus (0.84 pg), M. pehpeiensis (1.34 pg), M.

longisetus (1.79 pg), M. edax (3.0 pg), and M. latipes (4.11 pg) reared at 15, 22, and 33 �C.

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adherence to the hypothesis of DNA constancyremains a valid assumption for these cyclopoidcopepods.

Genome size and development rate

The five cyclopinid species studied here have di-verged in both somatic genome size and develop-ment rate. Genome size correlates negatively withdevelopment rate in females, and does so in aconsistent fashion over a range of temperaturesencountered in nature (Fig. 1). These inverserelationships were observed over only a fivefoldrange in genome size and could be even strongerover the broader range of genome sizes reportedfor calanoid, cyclopoid, and harpacticoid cope-pods.

McLaren et al. (1988, 1989) provided the firstcorrelations between somatic genome size anddevelopment rates in copepods. They reported thatgenome sizes of marine calanoid Calanus andPseudocalanus embryos and larvae were negativelycorrelated with development rate. Similar negativecorrelations between genome size and moltingrates of late stage juveniles were found in C. gla-cialis (Escribano et al., 1992). The present studyreveals similar relationships between genome sizeand the entire postembryonic development fromhatching into a nauplius I larvae to maturation atcopepodid CVI among freshwater cyclopoid spe-cies. A companion study of 14 warm water cy-clopoid species (including four species used in thisstudy) found that genome size correlates nega-tively with embryonic development rate as well(Edgerton et al., in preparation). All of thesestudies reasoned that multicellular copepods fol-low much the same rules as the single celledorganisms for which many of the strong correla-tions between C value and cell size and cell divi-sion rates duration are reported (Bennett, 1971,1972). Measurements of cell cycle durations areknown for only nine cyclopoid species (Beermann,1977; Leech &Wyngaard, 1996), and most of thesemeasurements are of prediminuted embryos. Ef-forts to relate cell cycle durations to selection forfast or slow development, as Strathmann et al.(2002) have done for marine planktonic larvae,would enable a finer dissection of the interrela-tionships among genome size, cell division rates,and development rates.

Genome size and body size

The relationship between genome size and bodysize was weaker and more variable than that be-tween genome size and development rate (Fig. 2,Table 2). Positive correlations between genomesize and body size have been reported in studieswith larger sample sizes of marine calanoids(McLaren et al., 1988, 1989), freshwater calanoids(Gregory et al., 2000), and freshwater cyclopoids,including four of the species in this paper (Wyng-aard & Rasch, 2000). McLaren & Margoliese(1983) found similar total numbers of nuclei inwhole organisms in closely related species of themarine calanoid Pseudocalanus and argued thatlarger nuclear DNA contents were associated withlarger cells, and hence larger bodies. Thus, inspecies that exhibit eutely, the effect of cell size onadult body size may be enhanced.

The freshwater cyclopoids have not beenexamined for eutely and absence of this phenom-enon could explain the weak relationship betweengenome size and body size in the present study.Whether or not eutely is present in cyclopoids, thecovariance of body size in relationships betweengenome size and development rate should beconsidered, at least in experimental design.

More comparisons at the interpopulational le-vel are needed before we can discern the variabilityin maturation time and body size associated with aparticular genome size, and when assuming gen-ome size is constant within a (cyclopoid) species.McLaren’s reports in intraspecific variation innuclear DNA contents could explain how some ofthe genetic differentiation among populations inlife history traits is accomplished.

Body size and developmental rate

Smaller bodied species developed faster than largerbodied species at 22 �C, but not at 15 and 30 �C.The faster development of smaller bodied forms at22 �C is consistent with most studies (Allan &Goulden, 1980), but exceptions exist (Wyngaard,1986). The common assumption is that ecophysi-ological and energetic demands prevent maximi-zation of both development rate and body size inthe same individual and that this tradeoff shouldbe considered, at least statistically if not by design,in studies of development rate. The evolution of

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lifestyles that demand either fast or slow develop-ment or small or large body sizes may be availableonly to those lineages that possess small and largegenome sizes, respectively, or mechanisms to alterthem. In this regard, genome size may operate as aconstraint on the evolution of life history traitsthat are genetically correlated with it. If closelyrelated taxa, such as the species examined in thepresent study, diverge in both genome size and lifehistory traits, this is evidence supporting corre-lated evolution. In the instances where a particularenvironment might select for negative correlations,such as rapid developmental rate and large bodysizes, it becomes difficult to dissect the relation-ships among these three characters.

Temperature effects

The present study suggests that relationships areweak between genome size and body size, andbetween developmental rate and body size at ex-treme temperatures compared to relationships at22 �C. This genotype–environment interactionsuggests that a decoupling between developmentrate and body size occurs at extreme temperatures.At 22 and 30 �C, M. edax was smaller bodied thanpredicted by genome size alone. At 15 �C, body

sizes of M. pehpeiensis, M. longisetus, and M. edaxwere not different from one another and weresmaller than would be predicted by temperaturealone, based on the common observationthat body size increases inversely with tempera-ture. M. edax showed dramatic plasticity in bodysize related to temperature in the present study,which mirrors variation reported in Florida andMichigan populations (Wyngaard, 1986). M. edaxhas the largest latitudinal range of the five speciesin this study and as such may exhibit more pop-ulation differentiation than the other species in thisstudy. Some of these genotype–environmentinteractions may be ascribed to differences insensitivity to extreme temperatures, and reflectlocal adaptation.

Hierarchical model of genome size and life historyvariation

Ecophysiological traits define the patterns of re-source allocation to growth, reproduction, de-fense, and stress tolerance in the organism. Duringdevelopment, a cascade of links from the molecu-lar and biochemical levels to the physiological levelinteract to produce the final performance of theindividual. Figure 4 explains how interspecific

Figure 4. The nucleotype is one of a hierarchy of biological levels, each of which shows adaptive variation. Pre-existing interspecfic

variation in size of the nucleotype (measured in picograms or number of base pairs in the somatic nucleus) of copepods is expanded

when some species reduce somatic nuclear DNA contents via chromatic diminution or increase nuclear and/or germline DNA contents

via endoreduplication. (A) Smaller nuclear DNA contents are associated with smaller cells, and require less time to replicate and to

complete the cell cycle. These effects result in more rapid development to maturity and smaller sizes at maturity, particularly in species

where eutely occurs. Ephemeral habitats and those that inflict intense mortality on copepods will favor these traits. (B) Larger nuclear

DNA contents are associated with larger cells and require more time to replicate and to complete the cell cycle. Larger cells may

produce more protein transcripts and carry more lipid stores, which are particularly important for early stage nauplius larvae. These

effects result in slowed development to maturity and larger sizes at maturity, particularly in species where eutely occurs. This suite of

traits will be favored in habitats with low level or poor quality food and/or weak predation.

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variation in nuclear DNA contents of copepodsmay be translated into variation at the organismallevel, and thus contribute to understanding howselection at the nucleotypic level may play a role indetermining the distribution life history traits inparticular types of habitats. To construct thisconceptual model, we drew from data in thepresent study and literature on genome sizes andlife history traits in copepods, dynamics of cell sizeand cell division rates, and the selective forces thatshape fitness traits in zooplankton.

The nucleotype

Somatic cell genome sizes of adult copepods varyover 100-fold at the interpecific level among cy-clopoids, calanoids, and harpacticoids, with themost comprehensive listing given by Gregory(http://www.genomesize.com.). Freshwater cyclo-poids have values from 0.28 to 4.1 pg DNA persomatic (or 2C) nucleus (Grishanin et al., 1994;Wyngaard & Rasch, 2000) while marine calanoidshave values from 4.3 to 24.9 pg DNA per somatic(or 2C) cell (McLaren et al., 1988; McLaren et al.,1989) and freshwater calanoids vary from 1.33 to5.71 pg DNA per haploid C-value (Gregory et al.,2000). Estimates of somatic cell genome size areknown for only a single species of the estuarinecalanoids (0.65 pg DNA per nucleus for Euryte-mora affinis), for the marine cyclopoid (0.5 pgDNA per nucleus for Diothona culvata), and forthe marine harpacticoid (0.5 pg DNA per nucleusfor Tigriopus californicus ) (Rasch & Wyngaard,2000; Rasch et al., 2004). These data suggest atrend of calanoids possessing larger genomes thancyclopoids, but begs the question of whether theselineages are constrained to have relatively largeand small genomes, respectively.

The most dramatic mechanism for stabilizingsomatic nuclear DNA contents in freshwater cy-clopoid copepods is chromatin diminution, whichremoves 33–90% of the germline-limited DNAfrom somatic progenitor cells during earlyembryogenesis in certain, but not all, species (re-viewed by Wyngaard, 2000; Wyngaard & Rasch,2000). Chromatin diminution reduces the DNAcontent of M. edax embryos from 25–30 pg perpre-diminuted germ cell nucleus to 3.0 pg DNAper somatic cell genome and reduces the DNAcontent of M. longisetus embryos from about 12–

15 pg per pre-diminuted germ cell nucleus to 1.7–1.8 pg per somatic cell genome (Rasch & Wyng-aard, 1995, 1996). Little is known about theprevalence of chromatin diminution in the marineand estuarine calanoid copepods. Chromatindiminution occurs in the marine calanoid Pseudo-calanus sp. (Robins & McLaren, 1982), later pre-sumed to be P. acuspes (Nanton, 1993; McLaren,pers comm.). Eurytemora herdmani excises DNAjust prior to fusion of the pronuclei that forms theembryo (Robins & McLaren, 1982), an intriguingphenomenon that may be related to the chromatindiminution observed in cyclopoids. The estuarinecalanoid Eurytemora affinis lacks chromatin dim-inution (Rasch et al., submitted). Chromatindiminution appears to be absent in the few harp-acticoid species thus far examined (Beermann,1959; Wyngaard & Rasch, 2000).

Mechanisms for increasing genome sizes in-clude polyploidy and endoreduplication, whichentails a partial or complete replication of thegenome without an ensuing mitosis (Fig. 4). Rasch& Wyngaard (2001) suggested that endoredupli-cation occurs in the germ line nuclei of Mesocy-clops edax and other species that exhibit chromatindiminution (Rasch & Wyngaard, 1996). Theintriguing quantum jumps in genome size (ca.4.193 pg DNA per nucleus) among 13 species ofCalanus and Pseudocalanus, which are not attrib-utable to polyploidy (McLaren et al., 1989), mightalso result from endoreduplication and explain therelatively large genome sizes in these two genera.Lastly, the small accretions and deletions over timethat contribute variations in genome size amongclosely related taxa presumably operate as they arehypothesized to do in most multicellular organ-isms (Graur & Li, 2000). These broad categories ofmechanisms that alter DNA contents providevariation at the nucleotypic level that undoubtedlyhave consequences for the dynamics of cell cycleregulation.

The cell cycle

The significant correlations between DNA contentand mitotic division rate in single celled species arewell established (reviewed by Gregory, 2001a).Genome size correlates positively with cell volumeand negatively with division rate (Mirsky & Ris,1951; Cavalier-Smith, 1985b, c). New models have

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been proposed to explain how changes in genomesize may be associated with critical checkpoints inthe cell cycle (Gregory, 2001a). Multicellularorganisms which have different types of cells,sometimes with different amounts of DNA, con-ceivably could complicate the comparisons ofDNA content with cell division rates and cell size.However, it seems reasonable to assume that thesecellular processes are acting in a similar fashion inmany of the different types of cells. The premise isthat smaller somatic genomes possess more rapidmitotic division rates and shorter cell cycles, whichconfer faster organismal growth rates, and earlierages at first reproduction at the organismal level(Fig. 4A). White & McLaren (2000) report a po-sitive correlation between rDNA iteration andcopepod development rate. Positive correlationsbetween rDNA copy number and genome sizewere found in a study of several copepods species(Wyngaard et al., 1995), as well as a broad rangeof plants and animals (Prokopowich et al., 2002).Wyngaard & Gregory (2001) proposed that exci-sion of heterochromatin during chromatin dimi-nution may eliminate the late ORI’s (origins ofreplication) that lie within the heterochromatinand convert those that flank the heterochromatinto early ORIs, thus providing a mechanism thatspeeds up the cell cycle. In this model, we extrap-olate from the division rates and sizes of cells tothe development rates and sizes of the organism.The effect of cell size on adult body size would beenhanced in organisms that possess eutely. Similarnucleus numbers among adults has been reportedfor marine calanoids (McLaren & Margoliese,1983), but remains unexamined for other cope-pods. The relationships we report may be of sig-nificance whether genome size is under directselection, or whether variation in genome size hasco-opted the adaptive significance conferred toother traits that are under selection.

Developmental rate, body size and habitat

Rapid development enhances the likelihood ofcontributing to the gene pool of the next genera-tion when predation is intense or the environmentis ephemeral (Fig. 4). Habitats with intense pre-dation, especially by visually feeding predators,select for smaller bodied forms (Brooks & Dodson,1965). The likelihood of rapid development is en-

hanced further where food resources are high, butthis may lead also to large body sizes. Slowerdevelopment rates may be favored in water bodieswith poor quality or low levels of resources. Largebodied forms may have higher survival whereinvertebrate predation is more intense than verte-brate predation (Zaret, 1980).

Thermocyclops crassus is a planktonic species(Ueda & Reid, 2003) that seems to prefer eutro-phic ponds and lakes. Its small body size likelyreduces its vulnerability to fish predation.M. pehpeiensis inhabits eutrophic ponds and lakes,as well as ephemeral marshes, ditches, and creeks,two attributes that would appear to select for ra-pid maturation (Ueda & Reid, 2003). M. longisetusinhabits ponds, pools, littoral zones of lakes, la-goons and subterranean waters and appears torequire permanent water bodies (Ueda & Reid,2003). This relatively large bodied species mayreduce its vulnerability to fish predation byinhabiting shallow areas with vegetation. All threespecies encounter predation in environmentswhere vertical migration is not likely to be effectiveagainst visually feeding predators, and rapiddevelopment rates are advantageous. All threespecies have rapid development rates relative toM. edax and M. latipes.

M. edax has the broadest latitudinal distribu-tion of the five species in this study and is notablefor its interpopulation variability in life histories(Wyngaard, 1986). Its restriction to permanentwater bodies ensures that it encounters vertebratepredation, but its diel vertical migrations and tol-erance of low oxygen conditions in the hypoli-minion, enable it to ameloriate intense predation(Zaret, 1980). Body size in M. edax may be tiedmore to resource level than to predation (Wyng-aard, 1986), as its body size reflects that whichwould be predicted by food level rather than pre-dation intensity. Development rate and body sizeare inextricably intertwined with resource avail-ability and predation intensity, as resource leveland size of fish stocks often are correlated witheach other in nature.

Megacyclops latipes commonly inhabitsephemeral environments, and thus is less likely tosuffer intense fish predation (Marten et al., 1989;Bruno et al., 2001). This may explain the mainte-nance of its large body size. The study populationwas collected from ditches in Louisiana where

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food often consists of numerous insect larvae prey,but probably relatively low amounts of phyto-plankton suitable for younger developmental in-stars of copepods (Marten et al., 2000). Such aselective regime, if typical of what this speciesencounters, would favor diverting more energyfrom growth rate into maturing at a large bodysize and producing large embryos with larger re-serves than in species experiencing high food andhigh predation. Embryonic size of M. latipes ismore than twice that of the other four species inthis study (Edgerton et al., in preparation). Selec-tion may be more intense on the various compo-nents, such as egg production, that contribute toreproductive output after adulthood is reached(Twombly et al., 1998). Early naupliar develop-ment relies on lipid reserves provided to the em-bryo, whereas copepodid development is morestrongly dependent upon food resources in thewater (Hart et al., 1995). Selection for larger em-bryos, perhaps at the expense of clutch size, wouldbe predicted to occur in low food environments.Relative to freshwaters, much of the marine envi-ronment can be characterized as low food (Allan,1976) and perhaps this explains why we observethe largest genome sizes among the marine cope-pods.

Additionally, genetic variability in timing ofreproduction may be characteristic of copepodsthat exhibit a bet-hedging strategy to maximizefitness in ephemeral environments (Hairston,1987). M. latipes exhibited the largest coefficient ofvariation in development time of the five species.

Integrative approaches in the future

Our data, albeit on a small number of species,support the view that further exploration of theadaptive variation of genome size in copepods iswarranted. We are currently expanding this pre-liminary survey to include a broader taxonomicsampling. As phylogenies of the copepods becomeavailable and attention is focused on mechanismssuch as chromatin diminution and DNA endore-duplication, our abilities to infer something of thenature of nucleotypic evolution will be enhanced.We may achieve some explanatory perspective forthe constraints that limit and mechanisms thataccelerate life history evolution to account for theamazing diversity seen in these copepods. Studies

of the adaptive significance of genome size in thecopepods offer the potential to link micro- andmacroevolutionary processes and to consider how‘bulk’ DNA may act in concert with coding genesto unravel the multiple layers of patterns thatinteract to produce the amazing diversity in lifehistory seen in these forms in nature.

Acknowledgements

We are grateful to G. Marten and M. Nuygen forcollecting and sharing their laboratory cultures ofM. pehpeiensis, M. longisetus and M. latipes and toM. Holynska for T. crassus. E. Colliver assisted inrearing M. longisetus. Janet Reid identified allspecies except M. edax. The manuscript benefitedconsiderably from comments by T.R. Gregory,S.I. Dodson, D. Leech, and I.A. McLaren. Thetechnical expertise of Mrs. D.H. Lee and B.A.Connelly is gratefully acknowledged. JMU pro-vided facilities and support. These studies weresupported in part by the James H. Quillen Collegeof Medicine, East Tennessee State University,Johnson City. This work was also supported byNSF 93-06607 to GAW and DEB 0080921 toEMR.

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