Critical photoperiod and daylength threshold differences between northern and southern populations of the butterfly Limenitis archippus

r. Insect Pirysiol., 1975. I-01. 21. pp. 1159 to 1165.

Per,oamon Press. Printed in Grzat Britnin.

CRITICAL~PHOTOPERIOD ASD DXYLESGTH THRESHOLD DIFFEREXCES BETWEEX XORTHERN AXD SOUTHERS POPULATIONS OF THE BUTTERFLY L IMENI TIS A RCHIPP LiS J.IMES IV. HONG* AIVDAUSTIN P. PL.\T-rt Department

of Biological

Sciences,

University of &Iaryland Baltimore llaryland 21228, U.S.A.

County,

Catonsvilie,

(Received 20 LVooember 1974) Abstract--Laboratory studies show that different photoperiods induce diapause in northern (Vermont) and southern (.Maryland) larval strains of the butterfly Linrenitis archippus. The northern strain responds to 4 hr longer photoperiod thresholds and critical ranges than does the southern one. These responses are correlated with geographic differences in the ambient photoperiod of the two localities. In this facultative diapausing species, third instar larvae construct hibernacula within the basal portions of tubular leaves spun with silk, when daylength approaches either 13.5 hr (Vermont strain) or L3.0 hr (hIaryland strain). \Vhen reared in total darkness some larvae develop directly to fourth ins,:ar without diapause, although mortality is high. Among both strains different broods exhibit different incidences of diapause. Reciprocal inter-strain hybrids show intermediate diapause responses, suggesting that larval diapause is under the control of multiple genes. INTRODUCTION POP~L,W~OXS of insects having wide geographic distributions often exhibit genetically based adaptations to localized environmental and climatic conditions. Such local adaptations commonly occur among temperate winter diapausing species (DE WILDE, 1962; DANILEVSKY, 1965; BECK, 1968; D.INILEYSKY et al., 1970). In particular, local photoperiod conditions vary both seasonally and geographically. VJithin one species northern populations tend to enter diapause before southern ones. As a consequence, both (1) the critical photoperiod (that which induces diapause among 50 per cent of the individuals subjected to it), and (2) the daylength threshold (the longest photoperiod inducing diapause in some individuals) often exhibit increases from south to north in demes of a single species which differ latitudinally. Different photoperiod responses of this sort previously have been described in the European cornborer (Ostriniu m&M’s Hiibn.) by BECK (1963) and recently TXBER and T.AUBER (1972) demonstmted that latitudinal variation in photoperiod responses occurs also in the green lacewing, C’hryso~~a curneu Steph. The viceroy butterfly, Limenitis urchippus Cram., is a common species, well known for its mimetic association with the monarch, Danurls plexipptrs L. The viceroy is found throughout Xorth America from central Canada to AIexico, occurring as several Glen Burnie, * Present address : 7981 Crownsway, X\Iaqland 21601, U.S.A. + To whom reprint requests should be addressed.

phenotypically distinct sub-specific geographic Its geographic distribution extends from races. northern limits of about 53” latitude N to southern limits of approximately 22” latitude N (HOVANITZ, 1949; KLOTS, 1951; EHRLICH and EHRLICH, 1961). Unlike the monarch, the viceroy is non-migratory. L. urchippta is a polyvoltine species, being partly triple brooded over portions of its range (GREENFIELD and PLATT, 197-l). Half-grown viceroy larvae are capable of entering facultative diapause in the third instar, especially during the late summer and autumn. These larvae grow slowly, and upon reaching third instar each constructs a hibernaculum within the basal portion of a partly eaten leaf rolled into a tube and covered with silk. Larvae remain dormant within their hibernacula until the following spring (late March or early April), when those which survive the winter resume feeding activity and growth. Diapause is induced primarily by short-day photoperiod acting on the second and third instar larvae, as shown by reciprocal transfer experiments in which developing larvae were switched between shortand long-day conditions at various instars (CLARK and PLATT, 1969). These authors suggested that genetic differences in daylength thresholds exist between Vermont and New Jersey strains of viceroy larvae, based upon the responses of the two strains to three different photoperiod regimes. The purposes of this paper are twofold: (1) we demonstrate that northern (Vermont) and southern (ilraryland) strains of L. urchippus larvae differ in both their critical photoperiods and daylength

J.WFS \L-. Host

1160

threshold<, longer; and photoperiod sqgest that by multiple

ASD

those for the northern strain being 3 hr (2) we present preliminarv data on the responses of inter-s::ain _hybrids and lx\-al diapause is gcneticaliy controlled genes in this species.

T\vo strains of viceroy larvae were collected in hibernaccla from shrubs and saplings of willow (Saliv spp.) and poplar (Populz~ spp.). The northern strain was obtained in South Burlington (Chittenden Co.), Vt. (alt. 110 ft; lat. 44’3O’S) bet\veen 23 and 29 January 1972. The southern strain xvas collected from the University of hIar)lsnd Baltimore County (F.XI.B.C.) campus at Catonsville, IId. (alt. 250 ft; lat. 39’15’ X) beween 31 January and 7 February 1972. Alethods for maintaining the diapausing larvae in hibernacula and for rearing them were described by &AN and PLATT (1973). Adult butterflies reared from larvae emerging from the Table

1. AIortality

and incidence

so. of broods

hibernacula xvere hand-paired in the laboratory (PL.ATT, 1969). ?.Iated females were confined in perforated plastic bags placed over potted willolvs and were fed a dilute honey solution every 2 days, until oviposition :%-as completed. Experimental larvae lvere reared between 24 Alarch and 1 June 1972. Either eggs or recenrly hatched first instar lxx-ae xvere collected from the oviposition bags every 2 days and were placed in labelled Stvrofoam cups containing fresh willow leaves (10 .individuals per cup). Each cup was sprayed daily with a fine mist of water vapour, and then was covered lvith a small clear plastic bag secured lvith a rubber band. These cups were kept in transparent covered plastic shoe-boxes containing about j,, in. water, thus ensuring that the humidit! was high lvithin the rearing cups (to keep the foodplant lea\-es from wilting). The boxes containing experimental lanae were kept in photoperiod chambers. The temperature within the chambers was 25 ? 2”C, and high humidity (about 80-959,) was maintained Jvithin the rearing cups. The

of diapause among Vermont under various photoperiods

Larval mortality (first to third instar) Photoperiods (hr L : D)

ACSTIN P. PLATT

and XIavlnnd

Incidence of diapause

larval strains reared

Larvae showing Direct development

No.

16

1X0.

IO 10 17 8 14 32 8 2 2 6 2 0

45.4 20.8 23.6 10.7 12.6 25.6 12.3 11.8 11.1 31.6 54 0.0

0 35 36 52 72 38 16 0 0 0 0 0

0.0 92.1 65.5 77-6 74.2 40.9 28.1 0.0 0.0 o-o 0.0 0.0

12 3 19 15 25 55 41 15 16 13 35 35

100.0 7.9 34.5 224 25.8 59.1 71-9 100.0 100.0 100.0 100.0 100.0

111

17.2

249

46.7

284

53.3

15 7 13 30 34 31 8 0 11 6 8 27

65.2 17.1 19.1 33.0 31.5 31.0 16.0 0.0 204 17.6 16.7 55.1

0

31 4-k 54 39 6 0 0 0 0 0 0

0.0 91.2 80.0 88.5 52.7 8.7 0.0 0.0 0.0 0.0 0.0 0.0

190

25.1

17-I

34.0

0: 0

FO.

s’,

Vermont strain 0 8 12 12.5 13 33.5 14 l-l.5 15 15.5 16 20

: 24 : 16 : 12 : 11.5 : 11 : 10.5 : 10

1 3 2 3 6 6

: 9.5 :9 : 8.5 :8

3 3 3 4 2

: -I

Totals

Alaryland strain 0 : 24 8 : 16 12 : 12 12.5 : 11.5 13 : 11 13.5 : 10.5 1-F : 10 14.5 : 9.5 15 :9 15.5 : 8.5 16 :8 20 :4

2 5 3 3 5 5

5 1 3 1 3 -I

8 3 11 7 35 63 42 36 43 28 -CO 22 338

100.0 8-8 20.0 11.5 47.3 91.3 100.0 100.0 100.0 100.0 100.0 100.0 66.0

Photoperiod Table

and daylength threshold

2. Influence

of critical

differences

in northern

and southern

(Female\-,,. x >Iale& (Femalelr\rl. x SIalei-t.)

Totsls

Total So. of larvae 69 35

10-C

populations

range photoperiod on the per cent diapause in two reciprocal of inter-strain hybrid larvae Photophase

Crcsss type

butterfly

s

broods

(hr)

13

13.5

14

14.3

15

15.5

64.7 (17)

0.0 (16)

0.0 (12)

0.0 (10)

0.0 (8)

0.0 (6)

0.0 (1)

53.3 (13)

15.0 (10)

0.0 (9)

-

0.0

0.0

(1)

(1)

-

59.3

15.0

(30)

(26)

0.0 (21)

0.0 (10)

0.0 (9)

0.0 (7)

-

Note: l:he number of larvae (shown in oarentheses) individuals.

photoperiods used in the experiments varied from 8L : 16D to 20L, : 4D (Table 1). Sixteen broods of L. archippus were reared during the experiments, eight representing the northern \‘ermont strain, six the Maryland strain, and two others representing reciprocal inter-strain crosses (Tables 1, 2). The number of larvae within individual brood:: ranged from 1 to 223 (Appendis I). In all, 533 Vermont larvae, 512 lZInryland larvae, and 10-F inter-strain hybrid larvae were reared. All broods, except cne, represent F, larvae bred from are wild-collected individuals and, therefore, comparable to insects obtained from matings which might have occurred in the wild. However, Rtaryland brood 5 (73 larvae) is an F, cross, obtained from sibs of Maryland brood 3 (Appendix I). Some eggs wer’e kept in total darkness (OL : 24D). These were obse,ved briefly every second day under dim red light (670 nm), to which we assume the larvae are insensitive (DE WILDE, 1962; BECK, 1969). All experimenta: larvae were exposed to a single constant photopcriod regime from early first instar until they had either (1) developed to the fourth instar, or (2) had constructed hibernacula and entered diapause during the third instar. The larvae xvere observed, ti-reir cups cleaned, and fresh willow leaves were provided every second day during the experiments. \Ve considered that true diapause had begun when the larvae had completed and entered their hibernacula, and ceased feeding. The critical photoperiods of each strain and of the inter-strain hybrids were determined using the method of least s.quares to calculate the photophase at which 50 per (cent diapause occurs from the data given in Tables 1 and 2. For both strains the observed values within the critical photoperiod range were used as data points in these calculations. The critical photoperiod ranges used were the following: (1) Maryland strain, 12 to 14 hr; (2) \‘ermont strain, .l2.5 to 14.5 hr; and (3) inter-strain hybrids, 13 to 1-l hr.

1161

includes both diapausing and non-diapausing

Vermont

----’

*-‘-

strain

h!arylond strain (

Inter-strain

h‘jbrids

Fig. 1. Incidence of larval diapause vs. photophase in the I’ermont and Maryland strains of L. arciripp~s and in the inter-strain hybrids X joint regression analysis (hhTHER, 1960) of the effect of photoperiod on the per cent diapause within the Vermont and Maryland strains also was done using the diapause percentages over the critical photoperiod ranges (see above) for both strains. The results of this analysis are in Table 3. RESULTS Table 1 sholvs larval mortality and the incidence of diapause at different photoperiods for the X’ermont and hIaryland strains of L. nrchippus. Appendix I includes the raw data given by individual broods. The data for the two broods of inter-strain hybrids are given in TabIe 2. -4 summary of these findings is shown in Fig. 1. The day length threshold in the two strains differs by + hr: in the Maryland strain no diapause occurred when the photophase

J.WES

1161

IV. Host

AND ALXTIS

exceeded 14.0 hr, whereas, Vermont larvae exhibited 18 per cent diapause at l-1.0 hr, but no diapause at 14.5 hr or above. Least squares calculations based on the data over the critical ranges of the mo strains yielded critical photoperiods (diapause,,) of 12.9L : 1 l.lD (Marvland strain) and 134L : 10.6D (Vermont strain), respectively. For the inter-strain hybrids the daylength threshold was 14.0 hr photophase, and the calculated critical photoperiod was 13.1L : 10.9D, a value intermediate between those given above. Under photoperiods shorter than those of the critical range (i.e. 12L : 12D and 8L : 16D) both larval strains show a high incidence of diapause (between 66 and 91 per cent), as Table 1 and Fig. 1 shoxv. However, larvae placed in total darkness proceeded to develop directly without diapause, although the OL : 24D group exhibited considerably greater mortality (556 per cent) than was observed in the other groups. Table 3 includes the results of a joint regression analysis of the per cent larval diapause on photophase in the northern and southern strains of L. nrchippus over their respective critical ranges. Both strains show similar linear regression of the per cent diapause (Y) on hours of photophase (X), with 1v, = 13.5 hr (northern strain) and XUd = 13.0 hr (southern strain). The F ratio for the joint regression of the per cent diapause (both strains) on photophase is highly significant (I’< 0.01). However, the two strains are responding in similar ways to the variation in photoperiod,

Table

3.

diapause Sermont

Joint

regression

of the

per

Critical photoperiod

since neither the difference between the regressions, nor the mean incidence of diapause in the two strains exhibit significance (P> 0.25 in both cases). -Appendis I shoxvs that in both strains different broods displav different propensities toward dispause at any gi,-en photoperiod within or belo\v the respectit-e critical ranges.

DISCUSSION The diapause response cun’es of the northern and southern viceroy strains are similar to one another, except that the Iarval diapause responses are shifted by $ hr over the critical photoperiod range. These correlate well with the geographic responses differences in ambient photoperiod plotted for the two stock localities (Fig. 2): This graph (based on data obtained from DLXCOMBE, 1966) reveals that in northern Vermont the ambient dawn-dusk light cycle is about 4 hr longer from AIay to July (the time of greatest butterfly activity and reproduction) than it is in AIaryland. The two stock localities differ geographically by approsimately 5’ latitude, suggesting that temperate populations of L. archippus differ by about 6 min in their photoperiod responses for each degree of latitudinal displacement. This Q hr difference is found not only in (1) the critical photoperiod range of the two strains, but also in both (2) their daylength thresholds, and (3) their These findings calculated critical photoperiods. closely correspond to those of T.-\CBER and TALBER (1977) for reproductive diapause in C. carnea. Their

cent

on photophase over the critical ranges for the and Alaryland strains of L. orchippm. Based on data given in Table 1

Parameters

range

analysis

P. PL.%TT

Vermont strain

>Iaryland strain

12.5-14.5

12-14

w2

+6,0

---

V?m,or,t

-

L:oryiand

I

(hr)

&lean incidence of diapouse

(91)

Regression coefficients (:A) 5 S.E.

-40.3

t 3.0

-48~0t

10.1

d.f.

LIean square

F ratio

Joint regression

1

9727.0

61.4’”

Difference behveen regressions

1

73.9

0.3

Difference means

between

1

8.3

0.1

Deviation from regression (error)

6

158.3

.4nal~sis of variance:

** P
Fig. 2. Annual photoperiod changes beginning in 1Iay in northern Vermont and in SIaryland. Data obtained from DLSCONBE (1966) by plotting daylength for the 15th of each month using latitudes of 45”9. and iO’N.,

Photoperiod

Jnd daylength

threshold

ditferences

dara indicate that northern (S.Y.) and southern (.Ariz.) populatio,ls having a latitudinal difference of about 9”S’, differ in critical photoperiod by 1 hr. Our data confirm the earlier work of CL.IRK and PL.ITT (1969) by demons:rating more quantitatively that L. orchipp~~s is a long-day insect, and that facultatit-e diapause is initiated in response to shortday photoperiod. Extensive field and limited laboratory observations suggest that these criteria apply equally well to the other Sorth American species of the ge’lus Limenitis (see GREESFIELD and PL.\TT, 1951, for additional data). Some viceroy lanae develop directly to fourth instar when reared in total darkness, and do not exhibit diapause during third instar. However, if maintained in total darkness through the fifth instar they almost invariably die during the larval-pupal ecdysis. Nevert Teless, some of these larvae can complete normal metamorphosis if placed under a long-day light-dark regime. Certain broods in both strains exhibit higher incidences of diapause than do others at any given photoperiod within or below the critical range (.4ppendis I). These differences seem to be more variable in the Vermont strain than in the Maryland strain. They probably are important to the surl-ival of the species in nature, since they allow certain individuals to initiate diapause, while others still are undergoing direct development. As the seasons progress, northern populations would tend to initiate diapause earlier than southern populations, because of the more rigorous climatic conditions which they face. Thus, the higher critical photoperiod and daylength threshold characteristic of the Vermont insects ensures that diapause will be initiated in most individuals mainly during August and early September, whereas some Maryland larvae may continue direct development without diapause through September, and even into October, during favourable seasor s. TACBER and TAUBER (1972) have suggested that reproductive d&pause in C. cornea is under polygenic control. The di.5rent diapause responses of the geographic strains of L. arc/zippus, as well as both the inter-brood variability and the intermediate responses of inter-strain hybrids to photoperiod, all indicate that larval diapause in L. archippus like\vise is under the control of multiple genes.

in northern

and southern

butterfly

populAons

1163

CONCLUSIONS Northern (1-ermont) and southern (1Iaryland) strains of L. archippus larvae exhibit photoperiod responses rl;pical of long-day insects. Facultative diapause is induced at third instar by short-day (< 12 hr) conditions in 66 to 91 per cent of the individuals in both strains. Long-day conditions (> 14.5 hr) cause direct development to the imago, without diapause. The two strains exhibit similar diapause response curves, except that they are displaced by + hr, in relation toone an other: for the Vermont strain, the critical photoperiod range fell between 12.5 and l-C.5 hr, the daylength threshold was 14.5 hr, and the critical photoperiod was calculated to be 134L : 10.6D. For the &Iaryland strain, the critical photoperiod range xvas 12 to 14 hr, the daylength threshold was 14 hr, and the critical photoperiod was 12.9L : ll.lD. Two reciprocally crossed broods of inter-strain hybrids showed photoperiod responses intermediate between the above values. These differences in the photoperiod responses of the two strains are correlated with differences in the ambient photoperiods of the two geographic localities from which the strains were collected; summer dawn-dusk intervals are 4 hr longer in northern Vermont than they are in Maryland. Larvae reared under conditions of total darkness did not diapause at third instar, but proceeded to These apolyse to the fourth and fifth instars. also displayed excessive experimental groups mortality (only 20 of 45 larvae reared in total darkness survived to the fourth instar). In both strains, separate broods show different characteristic diapause frequencies which vary considerably under This inherent the same photoperiod rCgime. variability, together with the intermediate diapause responses of the inter-strain hybrids, suggests that larval diapause in L. archippus is under the control of multiple genes. .~ckno~cle~~ernetlts-ale thank PANELA C. PLATT for assistance in collecting the larval strains, J. WEISIASTER for helping rear the larvae, and P. KE.W for assistance in hand-pairing the bctterflies. S. J. BERRY of ‘IVesleynn University and F. E. HANSON of U.YL1.B.C. provided valuable comments on the manuscript. Finally, B. P. BRADLEY of U.1I.B.C. kindly assisted xvith the statistical

treatment

of the data.

18

16

167

4

5

6

533

‘I’ot;ds

32

223

167

7.1

15

512

2

3

-I-

s=

6

‘I‘ot;1ls

1.

-

-

100.0

-

(8)

(2) (61)

(3)

(1) (55)

(13) 50.0

(6) 100.0

(8) 0.0

(34)

(46) 76.9

(46) 66.7

(23) 87.5

(7) -

-

-

93.5

(I) 0.0

-

-

(67)

no.4

(1) 95.7

0.0

100.0

-

(1) 100.0

-

(55)

(13)

(2)

(38)

(27)

(20)

100.0

(12)

64.1 (39) 96.3

514 (35) 90.0

(3) 0.0

(1)

-

-

broods

-

-

12

in individual

0.0

(17)

-

-

(6)

-

-

94.1

(8) -

0.0

75.0

(1) -

percentages

0.0

01

Diapause

0.0

(28) 83.3

from

(1) (3) (3)

(42)

(5) 0.0 (5) 33.3 (19) 33.3

(69)

0.0

(1) 20.0

(12) 52.6

(74)

(29)

(55) 0.0

(29) 41.7

wild-collected

(36)

-

-

(36) -

0.0

(6) 0.0

0.0 (5) 7.3

36.5 (11) 65.5

(15)

X

(57)

(4) -

0.0

(93)

(6)

(30) -

(39) 39.3

(6)

(2) 20.0

46.7

0.0

0.0

individuals.

(44)

-

(3‘1) -

(4) 0.0 (13) X

(22)

2)

(15) 0.0

(17) 0.0

(40)

(1) 0.0

(27)

0.0

0.0 (l(J) 0.0

0.0

-

-_

(35)

-

(27) -.

_-

w

X

(35)

(9) X

0.0

(2‘1)

0.0

-

-

s

(11)

04

20

(12) oa

(1) 04

-

(13)

X

(7)

0.0

--

0.0

(16)

.--

(4) -

0.0

(1) -

(10) 50.0

04

(1) -

(6)

(4) -

04

0.0

(4) -

(7)

(X)

04

0.0

(7) 0.0

(6)

16

(25 + 2°C)

04

15.5

temper;lture

04

15

at room

30.0

(9) -

66.7

X’

14

14.5

photoperiods

100.0

(4)

2.5.0

(1::

13.5

different

per 24 hr day

under

Photophase

rewed

(1) 0.0

X

(97)

(24)

(20) 91.7

(38) 65.0

(6) 71.1

33.3

-

(5) -

(4) 1 (JO.0

75 .o

of I,. nrclrippw

The numbers in parentheses include both diapnusing and non-diapnusing individuals. ’ Lxwe rwred in total darkness were observed briefly under dim red light (see text). 2 In conditions represented by ‘S’, the eggsor larvae did not survive to third instar. J ‘I’llis is an I:, brood, the parents being sibs of brood 3. All other broods are I:, I:wvne reared

2

1

Maryland

98

8

strain:

1

3

13-l

32

2

67

Inrvne

No.

Vermont strain: I

Total No. of

Brood

Appendix

Photoperiod

and daylength threshold

differences

REFEREXCES BECK S. D. (1963) Diapause-physiology

and ecolog)- of photoperiodism. Bull. enz. Sot. _-Im. 9, S-16. BECK S. D. (1968) Insrc: Photoperiodism. Xcademic Press, Sex York. CLARK S. H. and F'LATT _I.P. (1969) Influence of photoperiod on development and larval diapause in the viceroy butterfly, Limenitis archippus. J. Insect Pitysiol 15, 1931-1957. D.UXLEYSKY .A. S. (1965) Photoperiodirm and Seasonal Development of I’nsects. Oliver SC Bosd, London. DANILEVSKY A. S., GOR~SHIS S. I., and TYSHCHEXO V. P. (1970) Biological rhythms in terrestrial arthropods. A. Rz. hi 15, 2oi-244. DLXCOSXBE R. L. (1966) The rlmericnn Ephemeris and Xat&nl Almanacfor theYear 1968. U.S. Government Printing office, 7Vashington, D.C. EHRLICH P. R. ant. EHRLICH A. H. (1961) How toKnow the Butterflies. Elrown, Dubuque,‘Iowa. GREEXFIELD J. C. .i~. and PL.XTT A. P. (1974) Report of the capture of an additional hybrid between Limenitir nrthemis astyarmx and L. nrchippus (S>-mphalidae). J. Iep. Sot. 28, 72-75.

in northern

and southern

butterfy

populations

1165

HOV.~ITZ IV. (1949) Increased variabi!in_ in populations following natural hvbridization. -In Gmetics, Paleontolory, and Erolution (Ed. by ]EPSOS G. L., 1I.\ix E., and SI.\IPSON G. G.), pp. 339-355. Princeton Universi? Press, Princeton, S. J. KE.LY P. J. and PL;\TT .A. P. (1973) llethods for estercaIlv sexing mature larvae and pupae of Limenitis (Nymphalidae). _Y. lep. Sot. 27, 122-129. KLOTS A. B. (1951) _4 Field Guide to the ButtqYies. Houghton 1Iifflin, Boston. ~I~THER K. (1960) Statisticnl Analysis in Biolofy. Butler & Tanner, London. PLATT A. P. (1968) A simple technique for hand-pairing Limenitis butterflies (Symphalidae). r. kp. Sot. 23, 109-112. T.~L-BER >I. J. and T.ICBER C. A. (1372) Geographic variation in critical photoperiod and in diapause intensit)- of Clrrysopn cclrnm (Seuroprera). _Y. Insect Phgsiol. IS, 25-29. DE )VILDE J. 1’. (1962) Photoperiodism in insects and mites. il. Rm. Ent. 7, l-26.