Part I. Elucidating origins of the Cycad Blue
I- 4. Discussion
Molecular systematics and phylogeography of Cycad Blue
Mitochondrial DNA sequences often represent little variation in widely distributed species. However, in two other recently investigated cases of polyommatine lycaenids feeding on “weedy” species, mtDNA provides good resolution on genetic structure and systematic validity. The species Lampides boeticus, one of the most widely distributed Old World butterflies, has never been considered to geographical differentiation (Lohman 2008). However, phylogeographic analyses show this butterfly forms three distinct groups based on mitochondrial COI and cytB genes.
Yago et al. (2008) address taxonomic problems in the genus Zizina, small butterflies often difficult to identify by wing pattern. The mitochondrial ND5 gene in combination with male genitalic morphology allows reliable identification of Zizina taxa. As in the above widely distributed Asian butterflies, most sampled populations of Ch. pandava also showed little genetic variation (only 29 haplotypes were found in 810 specimens).
However, in this case genetic data supports the validity of existing subspecies. Ch. p. pandava is the most widely distributed subspecies.
This subspecies could in fact represent the source population where it feeds on several native Cycas populations that are distributed in south of mainland China and Indochina (Jones, 1993). Ch. p. pandava exhibits one to four step mutations to Ch. p. peripatria endemic in Taiwan, and nine step mutations to Ch. p. vapanda endemic in the Philippines. Based on the mitochondrial COII gene, Ch. p. pandava also possesses 21 haplotypes, considerably more than Ch. p. peripatria (five haplotypes) and Ch. p. vapanda (two haplotypes). Although Ch. p. lanka was not surveyed in this study, this subspecies may be more closely related to Ch.
p. pandava than to Ch. p. peripatria or to Ch. p. vapanda, based on its
geographic proximity. These molecular data in combination with significant morphological differences (Hsu, 1989) both supports the subspecies validity of Ch. p. peripatria.Low genetic variation and the star-like haplotype network of the Cycad Blue (Figure 3) is consistent with a population bottleneck after rapid range expansion, as also in the example of the highly invasive Horse Chestnut leaf miner moth Cameraria ohridella in Europe (Valade et al., 2009). At the same time, Cycad Blue hostplants have suffered severe reduction through habitat destruction and collecting for the horticultural trade and for subsequent planting in urban and suburban areas (Donaldson, 2003). On the one hand, the gene flow of Cycad Blue may nevertheless have been limited due to CITES restrictions forbidding transport of wild cycads without permits, in accord with COII gene data that show a localized, highly endemic distribution of haplotypes (Figure 2;
Table 5). On the other hand, at present, the planted range of wild cycads is much vaster than native range in Asia, representing many opportunities
for the Cycad Blue to increase its population size and range. A strongly analogous situation is found in another cycad feeding lycaenid that was formerly of conservation concern, the local race of the Atala Hairstreak butterfly (Eumaeus atala) in southeastern USA. This butterfly, represent in the Caribbean, became extinct between 1937 and 1959 in Florida (Landolt, 1984). However, after initial reestablishment in greenhouses, the Atala Hairstreak is now commonly found in southeast Florida feeding on the genera Zamia and Cycas wherever they are planted horticulturally (Hall and Butler, 1995).
The origin of Taiwanese populations
The results support the hypothesis that Taiwanese outbreak populations, especially in the western part of CMR, were mostly caused by range expansion of the native population. The hypothesis that outbreak populations were delivered by means of direct introduction along with alien cycads was rejected because the major haplotypes (haplotypes A, B, and C) represented the dominant populations which were only found in Taiwan. Western populations (haplotype C) showed a different dominant haplotype from eastern populations (haplotype A, B), supporting a hypothesis of longer coexistence of alien and native populations in Taiwan. However, the haplotype network suggests that haplotype C was derived from haplotype A, occurring only in eastern part of Taiwan (Figure 3). In this case, the outbreak populations of western populations in Taiwan would have been maintained entirely by horticultural cycads.
The Cycad Blue has thus expanded its range through a single rather than through multiple colonization events, to become widespread around the
whole island. There is no evidence that the striking biogeographic difference in haplotypes divided by the CMR (C compared with A and B) is related to a now extinct population of Cycas (such as Cy. taitungensis) native to western Taiwan, i.e. that the pattern is explained by divergence in allopatry. Taiwanese herbarium records also show that few horticultural cycads were planted in Taipei and southwestern Taiwan before 1950 (plant records at website: http://taif.tfri.gov.tw/taif_en/), and there are no records of Ch. pandava prior to 1976 in Taiwan (Hsu, 1987).
Before recent anthropogenically induced outbreaks, Taiwanese populations must have been founded from neighboring regions, considering also that Taiwan is a relatively young island, formed c. 9 Ma (Sibuet and Hsu, 2004). Hsu (1987) has pointed out that the source of the Taiwanese population is likely to have been either mainland China or the Philippines archipelago. According to the haplotype joining network, Taiwanese populations are more closely related to populations from mainland China than those in the Philippine archipelagos which lack a direct connection in the network. Moreover, populations in Taiwan could indeed represent a relatively old colonization. Multiple lines of evidence suggest that southeastern Taiwan constituted a Pleistocene refuge (see examples in Cheng et al., 2005 and Lee et al., 2006). The native hostplant,
Cy. taitungensis has high genetic variance indicating a large population
during interglacial stages (Huang et al., 2001), and thus sufficient resources for local survival of Ch. pandava over this time. Although Taiwanese populations show only one to four-step mutations from other regions (Figure 3), the significant genetic structure among the three subspecies indicates a long period of isolation because none of Taiwaneseendemic haplotypes (A-D, and F) was found in other native regions of the Cycad Blue.
Origins of the introduced populations in other geographical regions
Many native species have been threatened or even extinguished when introduced species successfully establish populations in their native habitats. Therefore, an understanding of the origin, biology, and ecology of alien species could help to focus conservation efforts for native species.So far, Chilades pandava has already been introduced to many parts of the Old World as far apart as Korea (Takeuchi, 2006), Japan (Mitsuhashi, 1992; Takegami, 2001; Hirai, 2009), Hong Kong in 1978 (Bascombe et al., 1999), Pacific islands including Guam in 2005 (Calonje, 2007; Moore, 2008), the neighboring island of Rota in 1996 (Moore, 2008), Saipan in 1996 (Schreiner and Nafus, 1997; Moore et al., 2005) and in the western Indian Ocean in Réunion since 2000 (Martiré and Rochat, 2008;
Guillermet, 2009), Mauritius since 2000 (Williams, 2006; Williams, 2007), Madagascar since 2006 (DCL, pers. comm.), and in Miami (SHY, pers. comm.). In some places, the native Cycas plants, Cy. micronesica (Guam) and Cy. thouarsii (Madagascar), are threatened by the Cycad Blue (Table 8). The molecular data could provide enough information to quickly ascertain the origin of the haplotypes of different subspecies or populations, and thus provide a phytosanitary monitoring tool. For example, the most likely origin of the Guam populations is from the Philippine archipelago and that of the Korean populations, from Taiwan because the haplotypes found were shared with these regions (Figure 2 or Table 5). Tracing the source of Cycad Blue populations is more important
in Japan because haplotypes characteristic of two subspecies were detected, Ch. p. peripatria found in Okinawa (No. 13) and Ch. p.
pandava found in Honshū (No. 14-15). Besides, the increasing frequency
and wide, rapid range expansion of Ch. pandava in Japan (Mitsuhashi, 1992; Hirai, 2009) increases the urgency to protect the last native region of Cy. revoluta, in the Ryukyu Islands of southwestern Japan (Wang et al., 1996). Nevertheless, COII sequence data was unable to trace the origin of Madagascan populations because haplotype O was found in many parts of the present range of Ch. p. pandava. Therefore, to improve the identification of the origin of introduced populations, more native populations should be surveyed and more sensitive genetic methods such as microsatellites (e. g. Habel et al., 2008) should be developed to more finely discriminate the origins of Ch. pandava outbreaks.The role of the CMR on genetic structure of Ch. pandava
The Central Mountain Range (CMR) of Taiwan at over 3000 meters elevation provides a primary north-south barrier considered to divide native populations of many species (examples in Peng, 2006; Wang et al., 2007). Such a dominant biographic barrier that clearly structures populations of many species is exceptional for such a small island. The maximal elevation recorded for Ch. pandava so far is about 700m (Hsu, 1989). As expected, CMR also serves as an effective barrier to divide the eastern and western populations of this Cycad Blue. The scattered nature of larval hostplants is commonly a significant factor in the population structure of tropical insect herbivores (Ehrlich, 1984). However, haplotype distribution and population dynamics of this butterfly are
clearly influenced by the CMR (Table 4; Table 7). This barrier may also be reflected in differences in emergence times in eastern and western populations: Ch. pandava in Yilan (Figure 2, No. 6) emerges in September or later whilst populations in Hualian (No. 7) emerge in March (LWW, pers. obs.).
Indirect effects of cycad cultivation on native Cycas species
Introduced plants have not only the potential to enlarge the distribution of native insects, but also to increase their biomass (Tallamy, 2004) and thus herbivore pressure on native plants. In Taiwan, the additional Cycad Blue food resource (Cy. revoluta) appears indeed to have augmented the population size of Ch. pandava, as apparent in increased levels of plant attack in southern monitoring sites (Lan, 1999;
Wu et al., unpublished data). Although the monitoring and genetic data show that western Taiwanese populations seldom disperse to the eastern side, the extra food resource provided by the introduced Sago Palm that are planted abundantly in eastern Taiwan may still greatly increase the overall population size of the Cycad Blue, threatening the survival of the rare native Cy. taitungensis. Adding greatly to this threat, another harmful pest, the scale insect Aulacaspis yasumatsui, has been introduced to Taiwan with horticultural Sago Palm since 2000 (Germain and Hodges, 2007). This scale continuously sucks nutrients from the leaves, stem and primary root until the host dies (Weissling et al., 1999). It is reported that the cycad scale causes high cycad mortality in Guam (Moore et al., 2005) and in Florida (Howard et al., 1999). While the monitoring of native Cy.
taitungensis initially showed no significant mortality from heavy attacks
by Ch. pandava (only 4 of 162 cycads observed died between 2000 to 2004, Wu et al. unpublished data), the increased level of herbivory over many years combined with the presence of the new cycad pest A.