Natal origins of migratory monarch butterflies at wintering colonies in Mexico: New isotopic evidence

We measured δD and δ¹³C values of 597 monarch butterflies collected from natural mortality at 13 wintering colonies (44–50 per colony) during February of 1997 (Table 1). These were individuals that migrated from North America in late summer and fall of 1996, and so could be related directly back to our 1996 breeding range isotopic study (15) (Fig. 2). Fifty male and 50 females were taken at random from each colony and stored in paper envelopes. Monarch wing membranes were separated from the abdomen, placed in glass vials, solvent-cleaned, air-dried, and stored.

Because a portion of the total hydrogen of monarch wing membrane (largely keratin) is available for isotopic exchange with ambient water vapor, it was necessary to quantify and eliminate the effect of this uncontrolled, temperature-dependent variable. Unfortunately, complete elimination of exchangeable hydrogen (i.e., hydrogen involved in O—H bonds) by chemical techniques such as nitration is not possible for complex organic matter (10). Hydrogen–isotope exchange between wing membrane and water vapor was first quantified by equilibrating samples with steam having a wide range of hydrogen-isotopic values (−135 to +525‰) at constant temperature (130 ± 0.1°C) and then measuring the total hydrogen δD values (10, 15). Hydrogen in monarch wing membranes available for isotopic exchange at this temperature was determined to be 19.5 ± 0.7% (r² = 0.99, P < 0.001, n = 27). In all our samples, potential variability resulting from uncontrolled hydrogen-isotopic exchange was eliminated by controlled equilibration of all wing membrane samples with steam (δD = −135‰) at 130 ± 0.1°C for 2 hr. Sample reproducibility of repeated equilibrated samples was better than ±2‰ for δD. Thus, total hydrogen-isotopic results for equilibrated samples could be compared reliably among samples and sites. After steam equilibration in Vycor break-seal tubes, all water vapor was cryogenically removed, and samples were sealed under vacuum, combusted at 850°C in the presence of cupric oxide, and followed by cryogenic separation of CO₂ from H₂O. Waters of combustion were reduced to H₂ gas on hot zinc (15). Stable-isotope analyses were performed on a Micromass Optima dual-inlet isotope-ratio mass spectrometer. Stable-carbon isotope analyses are reported in parts per thousand (‰) deviation from the Pee Dee belemnite (PDB) standard, with a sample reproducibility of better than ±0.1‰. Stable-hydrogen isotope results are reported in parts per thousand deviation from the SMOW standard and normalized on the Vienna Standard Mean Ocean Water/Standard Light Antarctic Precipitation (VSMOW/SLAP) scale, with a sample reproducibility of better than ±2.0‰.

No effect of sex on the distribution of stable isotopes in monarch butterflies was observed (MANOVA F_2,557 = 1.6, P = 0.2), and populations from all colonies showed normal distributions for both δ¹³C and δD (Kolmogorov–Smirnov, P < 0.01 in all cases). In general, δD and δ¹³C values of monarch wings overlapped considerably among wintering colonies (Table 1, MANOVA F_24,1114 = 1.5, P = 0.05), but some difference in the distribution of δD values among sites (ANOVA F_12,558 = 2.2, P = 0.01) was apparent (Table 1). The Barranca Hondon and La Gota de Agua monarch colonies were more depleted in δD values and did not overlap at the 95% confidence interval with the more enriched δD distributions of Llano el Amparo, Altamirano, Las Palomas, and La Mesa, suggesting more northern natal origins of monarchs at these two colonies.

We inferred natal origins of wintering monarchs from Mexico by comparing δD and δ¹³C data of wintering individuals with isotopic values found in monarchs field raised at natal sites throughout their breeding range (Fig. 2). Our δD and δ¹³C data show that 95% of wintering monarchs originated from throughout the known breeding range (Fig. 3). However, 50% of wintering monarchs originated from a fairly restricted geographic part of the breeding range, including the states of Kansas, Nebraska, Iowa, Missouri, Wisconsin, Illinois, Michigan, Indiana, and Ohio. This corresponds to an area of intense corn, soybean, and dairy production in the midwestern United States. Possibly, larger numbers of monarchs are produced in such areas of cultivation since milkweed host plants are persistent there despite pesticide and weed control measures. Proportionately fewer monarchs originated from the most southern or northern reaches of the breeding range (Fig. 3). Fewer wintering monarchs from the extremes of the breeding range were expected since relatively fewer individuals are produced there. Lack of representation from southern parts of the breeding range also is a result of the lack of larval host plant availability in the southern United States in late summer (11).

Our isotopic evidence shows that the 13 discrete monarch wintering colonies in Mexico generally are well mixed and thus demonstrates the existence of a panmictic model of monarch wintering colony composition. With the exception of the two sites noted, our results further suggest that the loss of a single wintering roost site is unlikely to affect one part of the breeding population in eastern North America over another. However, because the primary geographic production area for monarchs is centered in the American Midwest, that area should be focal for conservation efforts in North America. The combination of δD and δ¹³C measurements of tissues of breeding and wintering populations of monarchs measured in a single year represents a new and powerful tool for understanding the ecology of this species and avoids interannual isotopic variability (12). Furthermore, our approach can be readily applied to other migratory organisms in North America and likely elsewhere and possibly refined through the assay of isotopes of other elements (13, 14).

This paper was submitted directly (Track II) to the Proceedings Office.

We thank Constantino Orduña Trejo of the Mexican National Institute of Forestry and Agriculture (INIFAP) for coordinating field sampling in Mexico, as well as the numerous staff members at the Mexican monarch reserves for assistance in the field. We thank the INIFAP and Mexican National Institute of Ecology for permission to sample at all wintering colonies. S. Polischuk, B. Boldt-Leppin, and R. George assisted with lab analyses. This research was funded by Environment Canada and by grants from the Canada–Mexico International Partnerships Program and the Manitoba Model Forest Project.