BIOLOGICAL CONSERVATION
Biological Conservation 118 (2004) 341–351 www.elsevier.com/locate/biocon
Mitochondrial DNA sequence, morphology and ecology yield contrasting conservation implications for two threatened buckmoths (Hemileuca: Saturniidae)
Daniel Rubinoff a,*, Felix A.H. Sperling b a Department of Plant and Environmental Protection Sciences, University of HawaiÕi, 310 Gilmore Hall, 3050 Maile Way, Honolulu, HI, 96822 USA b Department of Biological Sciences, University of Alberta, CW405A Biological Sciences Ctr., Edmonton, Alta., Canada T6G 2E9
Received 9 April 2003; received in revised form 17 September 2003; accepted 22 September 2003
Abstract
Taxa of conservation interest are frequently identified using morphological or ecological characters. These characters are as- sumed to represent evolutionary importance, population structure and/or phylogenetic relationships in such organisms. We tested this assumption using two species complexes of the moth genus Hemileuca (Saturniidae). Both have populations threatened by habitat loss and need conservation protection. Legislation protects one taxon with apparent ecological differences. We sequenced 624 base pairs of mtDNA from the COI gene for geographically distant populations of the Hemileuca maia species complex and the H. electra species complex. Resultant phylogenies contradict prior assumptions about relationships in both species complexes. The legislatively protected Bog Buckmoth is paraphyletic with widespread H. maia, and its use of a novel hostplant seems to be a local adaptation. Divergent morphology and hostplant use among H. electra subspecies are associated with modest genetic divergence (0.48%). However, a group of unrecognized populations that are morphologically similar and geographically close to H. electra electra have mtDNA that is divergent by an average of 4.1%. There is disagreement regarding prioritization of ecological divergence over neutral genetic distance in conservation. We place ecological variation in a phylogenetic context and recommend that ex- ploration of genetic relationships be undertaken when populations are threatened. Adaptive ecological variation should be evaluated in a phylogenetic context to understand its conservation importance. This study illustrates the importance both of phylogenetic context and the use of independent characters in assessing biodiversity for conservation prioritization. Ó 2003 Elsevier Ltd. All rights reserved.
Keywords: Systematics; Insect conservation; Conservation prioritization; Cryptic species; Genetic distance; Adaptive ecological variation
1. Introduction between very similar populations (Daugherty et al., 1990; Baker et al., 1995; Burbrink et al., 2000; Dawood Identification of unique populations or species that and Channing, 2000; Lee, 2000). As a result, conserva- are considered to represent independent, historically tion attention will be focused on what are actually isolated, evolutionary lineages is often based solely on widespread, relatively undifferentiated populations, morphological or ecological traits (Moritz, 1994). These while much older lineages are left unrecognized and traits become the basis for assigning conservation value unprotected (May, 1990). Systematists generally agree to unique populations. However, phylogenetic rela- that conservation efforts should be focused on phylo- tionships established by morphological or ecological genetically distinct taxa, since such ‘‘long branches’’ characters may fail to recognize cryptic species, or ex- represent more evolutionary time than the ‘‘bush phy- aggerate what are actually modest genetic differences logenies’’ of recently diverged lineages (May, 1990; Vane-Wright et al., 1991; Faith, 1994; Crozier, 1997; * Corresponding author. Moritz, 2002 but see Erwin, 1991; Crandall et al., 2000). E-mail addresses: rubinoff@hawaii.edu (D. Rubinoff), A lack of systematic knowledge for potentially [email protected] (F.A.H. Sperling). threatened taxa thus precludes effective conservation
0006-3207/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocon.2003.09.013 342 D. Rubinoff, F.A.H. Sperling / Biological Conservation 118 (2004) 341–351 prioritization (Daugherty et al., 1990). In such situa- not restricted to Menyanthes and are commonly found tions, molecular systematics can play a crucial role in the on other genera like Salix and Betula, especially in the identification and conservation of threatened taxa later larval instars. Furthermore, polyphagy is well es- (Haig, 1998; Soltis and Gitzendanner, 1999). tablished in some populations of H. maia and H. ne- The North American moth genus Hemileuca Walker vadensis (Scholtens and Wagner, 1994; Martinat et al., (Saturniidae) contains approximately 20 species, in- 1997; Pryor, 1998). Legge et al. (1996) and Tuskes et al. cluding two taxa that are threatened: the Bog Buck- (1996) assert that the Bog Buckmoth is an independent moth, which is a member of the maia species complex evolutionary lineage, having arisen out of populations that has no formal scientific name (Tuskes et al., 1996) of the western species H. nevadensis that became isolated and Hemileuca electra electra Wright, one of four sub- during interglacial periods. However, there are few species of the H. electra complex. Both species com- ecological or morphological differences between the Bog plexes have been the subjects of intensive morphological Buckmoth and parapatric populations of H. maia to the and ecological investigations to determine systematic east and south and H. nevadensis to the west (Fig. 1), relationships (Scholtens and Wagner, 1994, 1997; Tus- leading to suggestions that the maia complex may ac- kes and McElfresh, 1995; Legge et al., 1996; Tuskes tually represent one variable species using many habitats et al., 1996). Even so only limited, and inconclusive, and hostplants (Scholtens and Wagner, 1997; Kruse, genetic data have been gathered on the Bog Buckmoth 1998). (Legge et al., 1996) and nothing is known of the genetic The H. electra species complex contains four mor- relationships among the four electra subspecies. The phologically distinct subspecies that range across parts electra and maia species complexes represent a rich of southwestern California (electra), southeastern Cali- model for examining how well systematic relationships fornia and southern Nevada (mojavensis Tuskes and based on morphological and ecological characters pre- McElfresh), northern Arizona (clio Barnes and dict genetic relationships between taxa, especially those McDunnough), and Baja Norte, Mexico (rubra McEl- pertinent to conservation prioritization. fresh and Tuskes) (Table 1 and Fig. 2). The nominate The Bog Buckmoth (Hemileuca sp., Tuskes et al., subspecies, H. electra electra, is found predominantly in 1996) is a legislatively protected taxon restricted to a few coastal sage scrub, a habitat which has been greatly re- bogs in the Great Lakes region of North America, al- duced by urban development (Westman, 1981). Never- though debate has arisen over the evolutionary origins theless, no conservation status has been suggested for and phylogenetic distinctness of the Bog Buckmoth H. electra electra, even though Tuskes and McElfresh (Legge et al., 1996; Scholtens and Wagner, 1997; Kruse, (1995) raised concern about the future of the taxon, and 1998). It is differentiated from its putative sister species, Rubinoff (2001) found that it was not adequately pro- H. maia (Drury) and H. nevadensis Stretch, solely by its tected by current reserve designs. This inaction is based occurrence in bog habitats and its ability to feed on bog on morphological and ecological characters that suggest buckbean, Menyanthes trifoliata (Menyanthaceae) as a that the H. electra electra phenotype is a coastal form larva (Tuskes et al., 1996). Yet Bog Buckmoth larvae are that blends eastward into adjacent Colorado Desert
Fig. 1. Range map for the Hemileuca maia complex (Tuskes et al., 1996). Collection localities are denoted by black circles. D. Rubinoff, F.A.H. Sperling / Biological Conservation 118 (2004) 341–351 343
Table 1 Populations of Hemileuca sampled (names from Tuskes et al., 1996) Taxon Collection locality Number of individuals sequenced Maia complex H. maia NY: Suffolk Co. 2 H. maia FL: Clay Co. 1 H. maia LA: Livingston Parrish 2 BBMa;b NY: Oswego Co. 4 BBMa WI: Marquette Co. 2 BBMa WI: Ozaukee Co. 1 H. nevadensis CA: Merced Co. 1 H. nevadensis NV: Lyon Co. 2 H. nevadensis WI: Douglas Co. 1 Electra complex H. electra electra CA: San Diego Co. 6 H. electra electra CA: Riverside Co. 1 H. electra clio AZ: Gila Co. 2 H. electra rubra MX: Baja Calif., Catavina 2 H. electra mojavensis CA: San Bernardino Co. 2 Colorado Desert popsa CA: Imperial Co. 5 Colorado Desert popsa CA: Riverside Co. 2 Colorado Desert popsa CA: San Diego Co. 1 Outgroup taxa H. eglanterina CA: Mono Co. 1 H. griffini AZ: Coconino Co. 1 H. burnsi CA: San Bernardino Co. 1 H. neumogeni UT: Washington Co. 1 H. juno AZ: Cochise Co. 1 H. oliviae NM: Lincoln Co. 1 a Undescribed taxa, BBM is the Bog Buckmoth. b Taxon with legislative protection.
Fig. 2. Range map for the Hemileuca electra complex (Tuskes and McElfresh, 1995). Collection localities are denoted by black circles. 344 D. Rubinoff, F.A.H. Sperling / Biological Conservation 118 (2004) 341–351 populations that are not currently threatened (Tuskes We obtained samples of all four H. electra subspecies and McElfresh, 1995). These Colorado Desert popula- and the unnamed Colorado Desert populations (Table 1 tions are insufficiently differentiated morphologically or and Fig. 2). In order to examine genetic relationships ecologically to merit taxonomic recognition and are across as much of their distributions as possible, we thought to represent an introgression zone between at took multiple samples from the range of H. electra least three other electra subspecies (Tuskes and McEl- electra in San Diego and Riverside Counties, California, fresh, 1995; Tuskes et al., 1996). to compare with Colorado Desert populations from San An independent phylogenetic hypothesis based on Diego, Imperial and Riverside Counties, California. For molecular data for these species complexes presents an all subspecies except mojavensis, we sequenced individ- opportunity to compare the reliability of morphological uals collected from more than one location (Table 1). To and ecological traits to recover underlying systematic give a broader phylogenetic comparison across Hemil- relationships, even for species complexes within the same euca and to provide outgroups for both species groups, genus. While it is common for genetic divergences to be the following congeneric taxa were included in the concealed by a lack of morphological and ecological analyses: H. eglanterina (Boisduval), H. neumogeni Ed- differences (Good and Wake, 1992; Baker et al., 1995; wards, H. juno Packard, H. griffini Tuskes, and H. Johnson and Jordan, 2000; Wake and Jockusch, 2000), oliviae Cockerell. Specimens were either frozen at )70 this study examines the correlation in two separate, °C or air-dried prior to DNA extraction. but congeneric, species complexes between mtDNA and ecological (H. maia complex) or morphological 2.2. DNA selection, extraction, amplification, and se- (H. electra complex) divergence. We ask first whether di- quencing vergences in morphological or ecological traits are corre- lated with genetic distance in other gene systems, Rubinoff and Sperling (2002) found very low levels of specifically mitochondrial DNA sequences? The answer to divergence between species in both the electra and maia this question is relevant beyond systematics because al- complexes for the nuclear gene Elongation Factor 1 al- location of conservation resources is often based solely on pha but a different pattern emerged from relatively the presence of ecological and morphological characters. faster-evolving mtDNA data suggesting that a more We then ask whether the levels of conservation protection thorough mtDNA-based investigation would be infor- and taxonomic recognition afforded populations in the H. mative. We used mtDNA as a marker because, in the- maia and H. electra species complexes (and based on the ory, it has one quarter the effective population size of existence of apparent morphological or ecological diploid nuclear genes, making it more susceptible to differentiation) are commensurate with phylogenetic dis- genetic bottlenecks. Therefore, at recent internodes, tance as indicated by mtDNA? And finally, how does mtDNA is more likely to reflect genetic differentiation valuation of ecological and morphological divergence in a than nuclear genes (Moore, 1995), at least when dis- phylogenetic context affect conservation prioritization? persal is not strongly female-biased. Furthermore, be- cause females are the heterogametic sex in Lepidoptera, the inviability of interspecific hybrid females expected 2. Methods under HaldaneÕs Rule makes mtDNA less vulnerable to the introgression that might lead to a disagreement be- 2.1. Field sampling tween species trees and gene trees (Sperling, 1993, 1994). DNA was extracted from specimens following Qiagen Hemileuca maia, the Bog Buckmoth, and H. nevad- (1999) QIAampâ DNA mini kit extraction protocols. We ensis populations were sampled from geographically used PCR amplification with two primers designed for distant points within each taxonÕs range (Table 1 and this study: ÔHemiÕ C1-J-2375 (50-GTAGGAATAGA- Fig. 1) in order to maximize genetic diversity. Bog TATTGATACC/TCGAGC-30)andÔLeucaÕ TL2-N-3020 Buckmoth populations feeding on Menyanthes trifolium (50-GGTTTAAATCCATTACATATAATCTGCC-30). were sampled from two separate bog populations in The thermal profile (94 °C for 30 s; 53 °C for 30 s; and 72 Oswego County, New York, and from disparate popu- °C for 1 min) was repeated 35 times. These primers en- lations in Marquette and Ozaukee Counties, Wisconsin. compass a 624 base pair region in Cytochrome Oxidase I This sampling regime includes specimens from two of and the beginning of tRNA regions of the mitochondrial the three populations identified as Bog Buckmoth (no genome. Purified PCR product was sequenced in both samples were obtained from the third population near directions using fluorescent terminators in an ABI 377 Ottawa, Ontario, Canada). Additionally, it allows us to automated sequencer. Sequences are available from compare Bog Buckmoth genotypes to some of the geo- GenBank (Accession Nos. AF387983–AF388024). Ad- graphically closest and most distant populations of the ditionally, sequence from one specimen of H. electra sister taxa H. maia (an oak-feeder) and H. nevadensis (a electra published by Caterino et al. (2001) was added to willow-feeder) (Table 1 and Fig. 1). the analysis (GenBank AF170856). D. Rubinoff, F.A.H. Sperling / Biological Conservation 118 (2004) 341–351 345
2.3. Phylogenetic analysis the maximum parsimony search maintained the mono- phyly of both the electra and maia species complexes. Sequences were hand aligned using Sequence Navi- The consensus tree also supported some individual gator. We sequenced 16 individuals representing 10 sample level resolution in individual trees. Relationships geographically separated populations of the maia com- between the previously named taxa, particularly the Bog plex, and 21 individuals representing 13 geographically Buckmoth and H. maia, were represented as paraphy- separated populations of the electra complex. We ana- letic in all trees and did not change between them, al- lyzed both species complexes and six outgroup taxa to- though precise relationships between individual samples gether to avoid a priori assumptions about relationships varied slightly. Maximum likelihood scoring of the 288 within and between the populations or taxa. Addition- trees given by the parsimony analysis (see Section 2) ally, combined analysis facilitated direct comparison of gave 194 trees with a best score of 2571.585. For those divergence levels between the two species complexes. No 194 trees the estimated Ti/Tv is 2.498 (j ¼ 5:774), esti- a priori outgroups were designated, since the two species mated proportion of invariable sites ¼ 0.4917, and the groups serve as functional outgroups for each other. estimated gamma shape parameter ¼ 0.8879. Modeltest We began with a heuristic search under a maximum software suggested a TVM + I + G model with a gamma parsimony model (all defaults) in PAUP* 4.0b4a shape parameter ¼ 0.7546 (see Modeltest http://in- (Swofford, 2000). No outgroup status was assigned a bio.byu.edu/Faculty/kac/crandall_lab/modeltest.htm for priori to any of the taxa in the analysis. The trees given details). The resulting tree had a likelihood score of by the maximum parsimony search were then assigned 2559.525 and was identical to that produced by the likelihood scores with a maximum likelihood model in other ML method. The maximum likelihood tree re- which all defaults were used except estimated Ti/Tv rate, sulting from the analysis using parameter values given estimated proportion of invariable sites, and a gamma by the highest scoring maximum parsimony trees had a distribution for variable sites with an estimated param- score of 2571.58. The only variation between the MP eter shape based on the data. We then performed a and ML trees concerned relationships between individ- maximum likelihood analysis using estimated parameter ual samples and not between named taxa. The ML tree values because there is good evidence that more complex (Fig. 3) had the same basic branching pattern as the models produce phylogenies that are better supported maximum parsimony trees and had the exact same to- by the data (Huelsenbeck, 1995; Wilgenbusch and De- pology as one of them. The Colorado Desert samples of Queiroz, 2000). We also estimated a maximum likeli- electra were always monophyletic, including popula- hood model using Modeltest software (Posada and tions sampled from localities across the region. Boot- Crandall, 1998) for comparative purposes with the strap and decay index branch support estimates aforementioned method. Parsimony-based bootstrap supported the monophyly of both the maia (DI ¼ 14, values (all defaults, 500 replicates) were obtained using BS ¼ 100) and electra (DI ¼ 13, BS ¼ 100) species com- PAUP* 4.0b4a. Decay indices were calculated using plexes, but resolution within the species complexes TreeRot (Sorenson, 1999). The number of synonymous varied (Fig. 3). and non-synonymous sequence differences were calcu- lated using PAUP* 4.0b4a, MacClade 3.06 (Maddison 3.1. Maia species complex and Maddison, 1996), and Sequence Alignment (Ram- baut, 1995). Because the Kishino–Hasegawa test Neither the strict consensus parsimony tree nor the (Kishino and Hasegawa, 1989) can be overly conserva- maximum likelihood tree supported the monophyly of tive in assessing the significance of likelihood differences maia or the Bog Buckmoth. The nevadensis sample from among topologies (Goldman et al., 2000), we used the northwestern Wisconsin (Douglas Co.) is in the middle Shimodaira–Hasegawa test (Shimodaira and Hasegawa, of the Bog Buckmoth populations from Wisconsin and 1999), as implemented in PAUP*4.0b4a (1000 replicates New York. While equally parsimonious trees disagreed with full optimization), for this purpose. Mean pairwise concerning the exact relationship of particular individ- distances between clades were calculated using uals in the Bog Buckmoth and maia complex, these in- PAUP*4.0b4a. dividuals were always paraphyletic with respect to geography and prior taxonomic designations (Fig. 3). An individual from the Wisconsin Bog Buckmoth pop- 3. Results ulation in Marquette County had a haplotype identical to that of an individual from the Bog Buckmoth pop- Maximum parsimony analysis gave 288 most parsi- ulation in Oswego County, New York. Another haplo- monious trees of 344 steps. All trees had the following type from the same Marquette Co. population was more statistics: CI ¼ 0.67, RI ¼ 0.90, RC ¼ 0.60, and when similar to maia from Louisiana, Florida or New York uninformative characters are excluded CI ¼ 0.58, than to the other Ozaukee haplotype. Populations of RC ¼ 0.52. The strict consensus of all 288 trees given by nevadensis from Nevada and California always formed a 346 D. Rubinoff, F.A.H. Sperling / Biological Conservation 118 (2004) 341–351
Fig. 3. Hemileuca phylogeny given by maximum likelihood search. Decay index values are above branches, bootstrap values (500 replications) below if >50%. BBM is Bog Buckmoth. Numbers of specimens with similar haplotypes from the same locality are shown, if >1, in brackets behind species name. In Hemileuca electra complex, Colorado Desert populations have wing morphology that is intermediate between H. electra electra and H. electra mojavensis (Tuskes and McElfresh, 1995). monophyletic sister group to the Midwestern and east- maia complex was 1.9%, between nevadensis from Ne- ern maia, the Bog Buckmoth, and the Wisconsin ne- vada and maia from Clay Co., Florida. Because the Bog vadensis samples. The Shimodaira–Hasegawa test Buckmoth is not monophyletic, there was no meaningful indicated that a topology in which the Bog Buckmoth divergence within the taxon. and nevadensis were constrained to be monophyletic (one theory of the Bog Buckmoth origination; Legge 3.2. Electra species complex et al., 1996; Tuskes et al., 1996; Scholtens and Wagner, 1997) was significantly (P < 0:05) less likely than the All samples of H. electra form a clade in all maximum best ML topology (Table 2). The maximum level of se- parsimony trees. There are two synonymous base pair quence divergence between any two individuals in the substitutions supporting monophyly in clio, one sup- D. Rubinoff, F.A.H. Sperling / Biological Conservation 118 (2004) 341–351 347
Table 2 Comparison of maximum likelihood phylogeny with competing hypotheses Phylogeny ln L Difference in ln L from P value best phylogeny Original phylogeny (Fig. 3) 2700.36354 Best na electra electra and Colorado Desert 2723.29597 22.93243 0.033 electra populations constrained to be monophyletic The Bog Buckmoth and nevadensis constrained 2722.27668 21.91314 0.047 to be monophyletic Results based on Shimodaira and Hasegawa (1999) test using bootstrap (1000 replications) with full optimization. porting electra, one supporting rubra and none sup- Buckmoth, maia, nevadensis split is the salient point of porting mojavensis. In contrast the Colorado Desert this data. This evidence of very recent contact between populations of electra, which apparently bear interme- maia and the Bog Buckmoth shown by our mtDNA diate morphological characters (Tuskes and McElfresh, sequence is confirmed by independently inherited nu- 1995) always sorted as monophyletic and as a sister clear gene sequences (Rubinoff and Sperling, 2002) and group to the rest of the electra clade. The Shimodaira– therefore is not an artifact of a single-gene lineage Hasegawa test indicated that the likelihood of a topol- sorting differentially. Low-level divergence and para- ogy for which the Colorado Desert and electra electra phyly of mtDNA between the Midwestern and eastern taxa were constrained to be monophyletic was signifi- Bog Buckmoth populations suggest recent contact, and cantly lower (P < 0:04) than the likelihood of the best proximity of the taxa makes continued gene flow a ML tree (Table 2). There were nine synonymous chan- possibility. Future research using population-level ges supporting the Colorado Desert population branch. markers such as microsatellites or amplified fragment The mean level of sequence divergence between the rest length polymorphisms (AFLPs) may be useful in un- of the electra complex and the Colorado Desert popu- derstanding very recent levels of isolation between lations was 4.1%. The maximum divergence within the populations in the Bog Buckmoth/maia complex. Colorado Desert clade was 1.9% between San Diego and While use of Menyanthes by the Bog Buckmoth Imperial County populations. populations is a unique trait, local specialization to habitat and different hostplants are common conspecific phenomena in the Saturniidae (Tuskes et al., 1996) and 4. Discussion may evolve quickly under selective pressure. In the case of the Bog Buckmoth, which is listed as endangered in Systematic treatments of the maia and electra species New York State, hostplant use and bog habitat are cited complexes have previously been based on morphology as justification for the taxonÕs recognition and protected and ecological traits. However, our molecular data status. Conservation action may be warranted to save suggest that it may be necessary to make changes in the unusual populations, but such action may be at the conservation status, management, and taxonomy of expense of other, more threatened or distinctive, both species complexes. complexes. Several authors have hypothesized that Bog Buck- The H. electra complex provides a comparison to the moth populations differentiated following postglacial or H. maia complex, not only in terms of genetic diversity interglacial colonization, possibly by H. nevadensis but also in its need for conservation. In this complex, populations spreading from the west (Legge et al., 1996; morphology, divergent hostplant use, and pheromone Tuskes et al., 1996; Scholtens and Wagner, 1997). variation have been used to assess levels of phylogenetic However, the Bog Buckmoth is not genetically distinct divergence. Our results indicate that these characters from widespread H. maia, despite some ecological dif- conflict with DNA-based markers. Described H. electra ferentiation, and the likelihood values for such a Bog subspecies have easily visible morphological traits that Buckmoth–nevadensis association are significantly lower distinguish them (Fig. 3, Tuskes and McElfresh, 1995). than the Bog Buckmoth–maia paraphyly we found One subspecies, H. electra rubra, has been considered (Table 2). The lack of mtDNA monophyly and the very for species status. In addition to morphological differ- low rates of nucleotide substitution within and between ences, it is the only member of the complex to use H. maia and the Bog Buckmoth populations contradict hostplants in families other than Polygonaceae, and the assumption that the Bog Buckmoth populations are rubra/electra hybridization experiments have resulted in isolated from surrounding maia populations. Although sterile offspring (Tuskes and McElfresh, 1995). mtDNA, especially at low divergence levels, may sort However, rubra/electra hybrid inviability may not be paraphyletically with respect to species lineages (Talbot phylogenetically important. In a genus of parapatric and Shields, 1996; Avise, 2000), the recency of the Bog water strider bugs (Heteroptera), hybridization 348 D. Rubinoff, F.A.H. Sperling / Biological Conservation 118 (2004) 341–351 compatibility was not correlated with genetic distance moth, the lack of divergence (neither nuclear nor (Sperling et al., 1997). In contrast, compatibility and mtDNA), has conservation relevance. Preservation of distance are correlated in a genus of tortricid moths historically isolated populations should take precedence (Landry et al., 1999). In the electra complex, the most over saving geographic variants (Vane-Wright et al., ecologically and morphologically divergent populations 1991; Moritz, 1994, 2002). Further work is needed to (rubra and clio subspecies) are not the most genetically determine whether the Bog Buckmoth populations rep- divergent. Morphological variation, hostplant use resent anything other than another hostplant and hab- (Tuskes and McElfresh, 1995), and pheromone varia- itat on the periphery of the range for an already tion (McElfresh and Millar, 1999) were not correlated adaptable species. It is possible that, with additional with the patterns of molecular divergence shown by sampling, a monophyletic DNA-based difference be- mtDNA. These molecular differences suggest long-term tween the Bog Buckmoth and H. maia might be found. isolation of the Colorado Desert populations from the But the low mtDNA divergence levels and lack of rest of the electra complex. The ÔintermediateÕ morpho- monophyly in H. maia, the Bog Buckmoth, and H. ne- logical characters of the Colorado Desert populations vadensis populations from Wisconsin to Louisiana to noted by Tuskes and McElfresh may, in fact, represent Florida to New York casts doubt on the isolation of the ancestral form from which the four subspecies have these taxa. Perhaps previously contiguous (and currently diverged (Fig. 3). There are only six substitutions (all proximate) habitat promoted gene flow across these synonymous) between the nevadensis populations from wide-ranging populations and slowed divergence. Nevada and California and the rest of the maia complex; For H. electra electra the situation is more compli- there are nine between the Colorado Desert populations cated. While geographically close to H. electra popula- and the closest electra subspecies. Even within the Col- tions from the Colorado Desert, H. electra electra has orado Desert populations there is more divergence than consistent and much greater genetic divergence, indi- within the maia complex; seven substitutions separate cating isolation and an independent evolutionary tra- the Riverside and eight separate San Diego County jectory, at least for mtDNA. These results concur with populations from those in Imperial County. Thus both others suggesting that the relatively old mountains of their divergence from other populations and the extent southern California have facilitated a pattern of speci- of variation within the Colorado Desert populations ation events across several unrelated taxa (Wake, 1997; show evidence of isolation over relatively long periods of Riddle et al., 2000; Wake and Jockusch, 2000; Jockusch time compared to the Bog Buckmoth. and Wake, 2002). H. electra electra populations are an The issue of ‘‘species’’-level recognition is conten- average of 4.1% diverged from adjacent Colorado Des- tious, especially in a conservation context (Phillips et al., ert populations to the east, but only 1.7% from the 1996; Sites and Crandall, 1997). Sites and Crandall have geographically closest subspecies to the northeast in the argued that information from multiple genes must be Mojave Desert or to the south in Baja California, incorporated to establish species status for conservation. Mexico. Therefore, because the Colorado Desert pop- However, because there are so many incompatible spe- ulations do not represent an avenue of genetic exchange cies concepts, firmly delineating a ‘‘species’’ is often a for the rest of the H. electra complex, the conservation difficult process. While we agree that species boundaries implications for H. electra are clearer. H. electra popu- are not necessarily discerned by single-gene phylogenies, lations in Mexico are connected with populations in populations of conservation importance can be assessed. southern California, the Mojave desert and Arizona Individual genes represent evolutionary lineages, and only through a narrow band of sage scrub habitat that mtDNA is one of the best molecular markers for eval- has been subjected to tremendous development pressure uating the short internodal distances typical of species (Westman, 1981; Rubinoff, 2001). By comparison to the and populations in conservation biology (Moritz, 1994; Bog Buckmoth (which has a range peripheral to that of Moore, 1995). Additional independent markers assess- maia) these coastal, connective, H. electra electra ing the underlying phylogenetic pattern are desirable populations are smaller, more threatened, and may (Johnson and Jordan, 2000) and we have morphologi- be worthy of conservation protection to preserve cal, ecological and behavioral characters (from Tuskes historical genetic contact between the four subspecies of and McElfresh, 1995) to compare to mtDNA. H. electra. MtDNA divergences in H. electra, especially at the Characters such as morphology, hostplant use, and levels present between populations from the Colorado pheromone chemistry are under strong local selection Desert and the four described subspecies, indicate an (Tuskes and McElfresh, 1995; Tuskes et al., 1996; independent evolutionary trajectory for at least part of McElfresh and Millar, 1999) and are not reliably cor- the genome. Differences between species concepts do not related with the Hemileuca phylogeny (Rubinoff and preclude the conservation of what are, at a minimum, Sperling, 2002). Sole use of such fast evolving characters greatly divergent DNA lineages. We feel that divergence to set conservation priorities can lead to misappropria- in the electra complex, or in the case of the Bog Buck- tion of conservation resources and, in the electra species D. Rubinoff, F.A.H. Sperling / Biological Conservation 118 (2004) 341–351 349 complex, a failure to recognize the most evolutionarily have a genetic component (Saville and Belote, 1993); independent lineages. It is possible that this problem pathogen susceptibility in Saturniidae varies widely be- occurs in many other taxa where genetic data are lacking tween congeneric taxa depending on the species and and conservation action, or inaction, is based solely on climate (Tuskes et al., 1996). Perhaps the level of isola- characters subject to rapid change. tion indicated by the mtDNA divergence between Col- Crandall et al. (2000) argue that, for conservation orado Desert and coastal populations reflects such purposes, preservation of populations with ecological differing thermal or disease-related adaptations, not differentiation should take precedence over those with readily obvious from a superficial ecological review, but deeper genetic divergences because recent ecological crucial to the speciesÕ survival in those regions. differentiation is evidence of higher evolutionary activity The use of neutral genetic markers to prioritize taxa which will be important to species survival (i.e., greater for conservation is not primarily focused on saving adaptive significance). The Bog Buckmoth is one of the neutral variation for its own sake (Crandall et al., 2000). examples (from Legge et al., 1996) they use to illustrate Rather, neutral variation is an empirical measure of this point; an ecological difference, that, despite a lack of isolation between taxa; isolated lineages, when exposed genetic differentiation, should be prioritized in a con- to selection under different conditions, are likely to servation context. In contrast, we agree with MoritzÕs evolve ecological specialization (Avise, 2000). Because (2002) emphasis on the irreplaceable nature of histori- we do not know enough about how and which genetic cally isolated lineages. We argue that the ÔsignificanceÕ of differences translate into ÔsignificantÕ, or evolutionarily ecological differentiation must be examined in the con- important ecological differences essential to the long- text of the taxa in question. There is no correlation be- term survival of a species, it is unwise to assign greater tween hostplant use (or specificity) and phylogeny importance to those traits that are simply the most ev- (evolutionary distance) in Hemileuca (Rubinoff and ident. We must preserve ÔunobservedÕ features as well Sperling, 2002). Furthermore, at least one-third of the (Faith, 2002). Prioritizing ÔbushÕ phylogenies due to taxa in Hemileuca exhibit local hostplant preferences what may turn out to be ultimately minor, rapidly (Tuskes et al., 1996). Other species of Saturniid moth evolving, and possibly superficial ecological differentia- frequently have local host specialization greater than tion can only be avoided by evaluation in a broader that found in the Bog Buckmoth, to the extent that taxonomic context. This is especially important when populations of some species in adjacent mountain ran- prioritization is at the expense of genetic differentiation ges specialize on completely unrelated hostplants and demonstrating a longer evolutionary history and the are unable to feed on the adjacent populationÕs preferred unknown ecological variation that additional time may host (Tuskes et al., 1996). In the context of all Hemil- have brought. euca, the Bog BuckmothÕs acquisition of novel host- The need for good taxonomy in effective conservation plants is not an unusual circumstance (i.e., ‘‘functional has been recognized (Daugherty et al., 1990), but the diversity’’ that can be regenerated sensu Moritz, 2002). importance of molecular methods in taxonomy is still Perhaps the Bog Buckmoth has been intensely studied being elucidated. Molecular methods frequently have and received more conservation attention than any revealed morphologically cryptic species relationships. other Hemileuca because maia/Bog Buckmoth are one of The discovery of cryptic species is not limited to insects only two species in the genus (which has about 20 spe- and presents a challenge to conservation (Baker et al., cies in the US) occurring in the eastern third of the 1995; Burbrink et al., 2000; Lee, 2000; Johnson and country, where the density of interested biologists is Jordan, 2000). We recommend that when populations of highest. Decisions about which ecological characters are a taxon appear to be threatened it is desirable to add a most important in the evolution of a species or lineage molecular component to conservation planning to pre- should not be addressed without detailed information vent the loss of cryptic, independent, evolutionary lin- (phylogenetic context) about the characters to be eages. Molecular systematic methods should take a compared. larger role in evaluating levels of conservation protec- A further difficulty with using ecological novelty to tion, both for listed taxa and for unprotected, but determine conservation priority is that not all ecological threatened populations. Adaptive variation must be variation is as obvious as hostplant use, and often very evaluated in a phylogenetic context in order to under- little is known about the adaptive significance or im- stand its long-term conservation importance. Obviously, portance of most ecological traits which Crandall et al. there are many cases in which morphology and ecolog- (2000) propose be used for conservation prioritization. ical characters accurately reflect relatedness. However, While the Colorado Desert and coastal populations of in both species complexes in Hemileuca in which there H. electra do not exhibit hostplant differentiation, the are populations of conservation concern, primary reli- former live in the hottest, driest part of the electra ance on morphology and ecology may have been phy- complexÕs range and the later in the coolest and most logenetically misleading. This leads us to speculate that humid. It is known that heat tolerance in insects can conservation actions, or the lack thereof, based on 350 D. Rubinoff, F.A.H. Sperling / Biological Conservation 118 (2004) 341–351 phylogenies lacking a molecular component may not be Crozier, R.H., 1997. Preserving the information content of species: saving an appropriate measure of genetic diversity. genetic diversity, phylogeny, and conservation worth. Annual Therefore, in the development of management plans, we Review of Ecology and Systematics 28, 243–268. 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Goldman, N., Anderson, J.P., Rodrigo, A.G., 2000. Likelihood-based We thank S. McElfresh and J. Millar, University of tests of topologies in phylogenetics. Systematic Biology 49, 652– California, Riverside for the use of pheromone lures and 670. S. McElfresh for specimen samples. E. Stanton supplied Good, D.A., Wake, D.B., 1992. Geographic variation and speciation samples of the Bog Buckmoth from New York and in the Torrent salamanders of the genusRhyacotriton (Caudata : Wisconsin that were invaluable; J. Kruse also donated Rhyacotritonidae). In: University of California Publications in Zoology, vol. 126. University of California Press, Berkeley, CA. samples from Wisconsin and A. Cognato donated Haig, S.M., 1998. Molecular contributions to conservation. Ecology samples from New York. C. Conlan donated samples of 79, 413–425. the electra complex. We are grateful to S. Weber of Huelsenbeck, J.P., 1995. Performance of phylogenetic methods in Cabrillo National Monument for a research and col- simulation. Systematic Biology 44, 17–48. lecting permit. M. Caterino provided helpful feedback. Jockusch, E.L., Wake, D.B., 2002. Falling apart and merging: diversification of slender salamanders (Plethodontidae: Batracho- J. Herbeck, J. Powell, C. Schmidt, D.Wake, D. Wall, seps) in the American West. Biological Journal of the Linnean and S. Welter, University of California, Berkeley, pro- Society 76, 361–391. vided comments that greatly improved the quality of Johnson, J.B., Jordan, S., 2000. Phylogenetic divergence in leatherside this paper. C. Villet, American Museum of Natural chub (Gila copei) inferred from mitochondrial cytochrome b History and K Will, U.C. Berkeley kindly drew the sequences. Molecular Ecology 9, 1029–1035. Kishino, H., Hasegawa, M., 1989. Evaluation of the maximum moths in Fig. 3. D. Rubinoff received support at U.C. likelihood estimate of the evolutionary tree topologies from DNA Berkeley from the Achievement Rewards for College sequence data, and the branching order in Homindoidea. Journal Scholars Foundation, the Margaret C. Walker fund, a of Molecular Evolution 29, 170–179. Harvey I. Magy fellowship, the Usinger Memorial Fund Kruse, J.J., 1998. New Wisconsin records for a Hemileuca (Lepidop- for systematics, a Keen Fellowship, and an Agriculture tera: Saturniidae) using Menyanthes trifoliata (Solanales: Meny- anthaceae) and Betula Pumila (Betula ceae). Great Lakes Experiment Station grant to F. Sperling. In addition, F. Entomologist 31, 109–112. Sperling gratefully acknowledges research grants from Landry, B., Powell, J.A., Sperling, F.A.H., 1999. Systematics of the NSF-PEET and NSERC. 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