FORUM Genetic Differentiation of Loxoblemmus Appendicularis Complex (: ): Speciation Through Vicariant and Glaciation Events

1 2 1 1 2, 3 WEN-BIN YEH, YU-LIN CHANG, CHUN-HSIEN LIN, FU-SHENG WU, AND JENG-TZE YANG

Ann. Entomol. Soc. Am. 97(4): 613Ð623 (2004) ABSTRACT Taxonomic determinationbasedon morphology alone has failedto describethe evo- lutionary history of Loxoblemmus appendicularis Shiraki complex in Taiwan. Phylogenetic analysis using the 16S rDNA sequence reveals that three evolutionary lineages of L. appendicularis have been found to coincide with their area of geographical distribution: the Southern, Eastern, and Northern populations. Sequence distancewas equal between the Northern andSouthern andNorthern and Eastern populations (0.032), whereas between the Southern andEastern populations, the sequence distance was 0.026. Cross-breeding among these three populations has produced abnormal hybrids, suggesting that a possible postzygotic isolating mechanism exists. Biogeographical history suggests the speciation event in L. appendicularis began in the early Pleistocene (1.8 million yr ago [Mya]). Vicariant event createdby the rise of the Central Mountain Range Ͼ1 Mya ledto two separate Eastern andSouthern lineages. The following glacial event andformation of a landbridgebetween Taiwan andthe Chinese continent at the endof Pleistocene reintroduced L. appendicularis, currently known as the Northern population, to western andnorthern Taiwan. Results of sequence divergence, phylogenetic inferences, geographical distribution, and cross-breeding strongly show a current tax- onomic recognition of a single species with three parapatric cryptic species.

KEY WORDS Loxoblemmus, Gryllidae, speciation, vicariant, glaciations

PHYLOGEOGRAPHIC STUDIES HAVE GREATLY contributedto tral Mountain Range Ϸ2.5Ð1 Mya (Lin 1966, Huang et our understanding of evolutionary history (Avise al. 1997) in the formation of Ͼ100 peaks now above 2000). A clear success of phylogeographical study has 3,000 m in elevation, andthe ongoing tectonic collision been the improved descriptions of geographical dis- along the north-south axis of Taiwan has createddi- tribution, phylogenetic relationships, andgenetic dis- verse habitats for terrestrial organisms. In addition, it tance among evolutionary lineages (Bermingham and is believedthat Taiwan andthe Chinese continent Moritz 1998), especially with regards to the effect of have periodicallybeen connectedandseparatedbe- glaciations (Riddle and Honeycutt 1990) and moun- cause of sea level changes causedby glaciations during tain building (Trewick et al. 2000, Shaw and Lugo the Pleistocene Period. The periodic formation of a 2001). Taiwan is separatedfrom the Chinese continent landbridgeacross the Taiwan Strait has playedan by the shallow Taiwan Strait, which has interrupted important evolutionary role in TaiwanÕs biota. normal gene ßow in a species distributed on both sides The biota of Taiwan is rich in diversity, as has long of the Taiwan Strait. However, geographical history been notedby biologists, because of the extremely (Huang et al. 1997) andmammalian andreptilian fossil variable abiotic environments, such as mountain to- records (Shikama et al. 1975, Otsuka and Shikama pology, climate changes, and the multiple land-bridge 1978, Otsuka 1984) indicate that Taiwan was con- formations during the Pleistocene Ice Ages (Lue and nectedto the Chinese continent before the Pleisto- Chen 1997). The effects of the well-documented geo- cene Period. The present day conformation of Taiwan logical glaciations on the diverse biota of the island began Ϸ5 million yr ago (Mya) during the late Mio- have recently been the subject of an increasing num- cene (Teng 1987, 1990), causedby exhumation from ber of phylogeographic studies that focused on ge- the collision of the Eurasian andPhilippine plates netic patterns andprocesses of colonization andspe- (Teng 1990, Huang et al. 1997). The arc-continent ciation (Wang et al. 1999, 2000, Creer et al. 2001, collision resultedin a drasticexhumation of the Cen- Huang et al. 2001, Huang et al. 2002, Hwang et al. 2003). The diversiÞedspeciesandsubspecies of ter- 1 Department of Biology, Kaohsiung Medical University, Kaohsiung restrial in Taiwan might have been formedby 807, Taiwan. 2 Department of Entomology, National Chung-Hsiung University, peripatric speciation when geographic isolation oc- Taichung 402, Taiwan. curredbecause of the absence of a landbridgebe- 3 Corresponding author, e-mail: [email protected]. tween the islandof Taiwan andthe Chinese continent.

0013-8746/04/0613Ð0623$04.00/0 ᭧ 2004 Entomological Society of America 614 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4

Although the ecosystem in Taiwan supports diverse cesses of L. appendicularis by vicariant andglaciation communities of fauna andßora, the phylogeographic events are herein hypothesizedanddiscussed. impact on fauna has been poorly studied. Loxoblemmus The cricket genus Saussure (Saussure Materials and Methods 1877) has 46 species worldwide. Of these, 36 species are distributed throughout East Asia. Taxonomic rec- Collecting Materials. Forty-six individuals of L. ap- ognition of this genus has always met with some dif- pendicularis complex from various localities through- Þculties, because the descriptive characters of many out Taiwan (Fig. 1) were analyzedin this study. species overlap. Loxoblemmus appendicularis is small Twenty specimens of other Loxoblemmus species, L. (Ϸ1.5 cm) in body length and is one of the most equestris, and L. sylvestris, andthe three other genera, common local crickets in Taiwan. Fieldobservation Teleogryllus, Brachytrupes, and Velarifictorus, were andlaboratory observation have shown this species to usedas outgroups for comparisons. Collecting infor- have an annual life cycle without hibernatedeggs. The mation andsequence accession numbers (AY239043Ð numerous nymphs usually hatch in July andAugust AY239108) are shown in the Appendix. andmolt six times until adulthoodisreachedaround DNA Extraction, Amplification, and Direct Se- May to July. All stages live in moist or semimoist sites quencing. Live crickets were collected, Þxed, and pre- Ϫ Њ such as grasslands or in leaves under the trees. They servedin 95% ethanol at 20 C. The anterior leg was feedalmost entirely on the tiny roots of grasses or trees selectedfor homogenization by glass homogenizer in ␮ andoccasionally on the deadbodiesof other small 500 l digestion buffer containing 100 mM Tris-Cl (pH invertebrates or spiders. The taxonomic characters of 8.0), 10 mM EDTA, 100 mM NaCl, 0.5% SDS, 50 mM dithiothreitol, and 0.5 mg/ml proteinase K. The mix- L. appendicularis Shiraki Shiraki 1930 have been based Њ on a common andhighly conservedheadshape and ture was incubatedat 50 C overnight andextracted antennae forms (Gorochov 1985). Unfortunately, with phenol-chloroform (modiÞed from the process characters such as body size, head shade, front ridge, describedbyYeh et al. 1997). ExtractedcrudeDNA was dissolved in 50 ␮l TE buffer, andan aliquot of 10 antennae form, andveins in the tegmen are variable. ␮ Thus, the taxonomic determination of this species l crudeDNA was diluted10-foldandusedasa DNA complex has been ambiguous (Yang 1992). Prelimi- template in the following ampliÞcation reaction. Polymerase chain reaction (PCR) was employedto nary molecular evidence on L. appendicularis complex amplify a partial sequence of the mitochondrial 16S in Taiwan reveals three distinct clusters exist in the rDNA gene. The primers usedto amplify the region mitochondrial DNA sequence (Yeh et al. 1999). Thus, were 5Ј-GCCTGTTTATCAAAAACAT-3Ј (16SR21) these clusters couldbe usedto addressquestions con- and5 Ј-CCGGTCTGAACTCAGATCA-3Ј (16S22), cerning the patterns in L. appendicularis evolutionary which correspond, respectively, to nucleotides 13416Ð process. 13396 and12866Ð12884 of the 16S rDNA gene of Dro- In the past decade, molecular characters have been sophila yakuba (Clary andWolstenholme 1985). Am- usedto identifyinsect species. This has been espe- pliÞcation was carriedout for 35 cycles in a Þnal cially helpful in determining intraspeciÞc diversity volume of 100 ␮l containing 10 mM Tris-Cl (pH 9.0), (Mukha et al. 2000, Palmer et al. 2000) andin solving 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 0.1% Triton- the problem of sibling species (Kelley et al. 2000, Yeh X100,2UofSuperTaq polymerase (HT Biotechnol- et al. 2000, Parsons andShaw 2001), as well as that of ogy, LTD, Taiwan), 0.2 mM of each dNTP, 20 pmol of the species complex (Yeh et al. 1997, Guillet et al. each primer, and2 ␮l of DNA template. The reaction 2000). Molecular evidence is also the means of deter- was carriedout with the following temperature pro- mining whether or not introgression or hybridization Þle: denaturation for 50 s at 95ЊC, annealing for 1 min has occurred(Dowling andSecor 1997). Further- at 50ЊC, andextension for 1 min at 72 ЊC. AmpliÞed more, genetic analyses of natural species assemblages DNA fragment was excisedfrom agarose gel andex- render it increasingly likely that speciation occurs tractedfrom the gel with the Nucleotrap Kit (Mach- under sympatric conditions (Bush and Smith 1998, erey-Nagel, Germany) or puriÞeddirectlyfrom am- Hellberg 1998, Schliewen et al. 2001, Dawson et al. pliÞedproductusing the PCR puriÞcation Kit, 2002). Qiaquick (Qiagen, England). The resulting DNA We are interestedin genetic patterns andthe col- product was sequenced directly using radioisotope onization process of this species. This paper provides (Perkin Elmer using S35) or ßorescent dye of the the 16S ribosomal DNA sequence of mitochondrion, Cycling PCR Sequencing Kit for 29 cycles with the showing that what has been considered a single spe- following temperature proÞle: 40 s for denaturation at cies may actually be three differentiated geographical 95ЊC, annealing at 50ЊC, andextension at 72 ЊC (mod- populations (or species). Cross-breeding experiments iÞedfrom Yeh et al. 1997). Alternatively, some samples among these three populations have shown that a sequencedusing Taq dye terminator Cycle Sequenc- postzygotic isolating mechanism may be involvedin ing Kit (AppliedBiosystems) were analyzedwith the population differentiation. Speciation processes in ABI 377A sequencer. Three sequencing primers were this case may have been induced by the vicariant used, including 16SR21, 16S22, and 16S212 (5Ј-CAA- event of the Central Mountain Range longitudinally CATCGAGGTCGCAA-3Ј downstream). bisecting Taiwan and land-bridge formation during DNA Analysis. Initial alignment of the mitochon- the Ice Age. The colonization andevolutionary pro- drial sequences was conducted using the Pileup pro- July 2004 YEH ET AL.: SPECIATION EVENT OF L. appendicularis COMPLEX 615

Fig. 1. Illustration of collecting localities and individual specimens in Taiwan. N, S, and E, are Northern, Southern, and Eastern populations, respectively, of L. appendicularis. Members of the Eastern population are distributedina limitedarea andare not foundin other eastern areas. Symbols labeledon the map with F, E, ‚, ①, ②, and ③ correspondto L. apendicularis, L. equestris (L.e), L. sylvestris (L.s), Brachytrupes (Brach), Teoleogryllus (Teleo), and Velarifictorus (Velar), respectively. The Central Mountain Range is shown, with the light gray representing the altitudebetween 1,000 and2,000 m anddarkgray representing an altitude of Ͼ2,000 m. The dotted line shows the land-bridge border at the end of the Pleistocene (Boggs et al. 1979), and the hatched area is the rising seashore of the Hengchung peninsula during the Middle Pleistocene (Huang et al. 1997, Teng 1987). 616 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4

Table 1. Sequence distance based on proportion distance within and between species

La(S) La(E) La(N) Le Ls Velar Teleo Brach La(S) 0.007 Ϯ 0.002 La(E) 0.026 Ϯ 0.007 0.001 Ϯ 0.001 La(N) 0.032 Ϯ 0.006 0.032 Ϯ 0.008 0.006 Ϯ 0.002 Le 0.098 Ϯ 0.011 0.096 Ϯ 0.011 0.097 Ϯ 0.012 0.005 Ϯ 0.002 Ls 0.115 Ϯ 0.014 0.125 Ϯ 0.017 0.110 Ϯ 0.013 0.096 Ϯ 0.012 0.002 Ϯ 0.002 Velar 0.160 Ϯ 0.016 0.172 Ϯ 0.018 0.148 Ϯ 0.014 0.152 Ϯ 0.013 0.144 Ϯ 0.016 0.002 Ϯ 0.002 Teleo 0.156 Ϯ 0.017 0.165 Ϯ 0.019 0.154 Ϯ 0.016 0.155 Ϯ 0.015 0.145 Ϯ 0.014 0.122 Ϯ 0.013 0.013 Ϯ 0.005 Brach 0.150 Ϯ 0.015 0.157 Ϯ 0.015 0.145 Ϯ 0.012 0.135 Ϯ 0.015 0.143 Ϯ 0.017 0.145 Ϯ 0.011 0.137 Ϯ 0.015 0.002 Ϯ 0.002

Values in the Þrst three rows are that of L. appendicularis. Abbreviations are the same as those in Fig. 1. gram of the GCG software package andmanually another plastic container to avoidharming the newly reÞnedbasedon the secondarystructures of the 16S hatchednymphs andwere kept together until they rRNA sequences (Fang et al. 1993, Davis et al. 1994, died. Primary nymphs were reared collectively (Ϸ100 Kambhampati et al. 1996). The proportion of the nu- individuals) with the crushed food in 12-cm plastic jars cleotide composition of each taxon was calculated andmovedto an insect rearing cage (30 by 30 by 30 using the MEGA2.1 program (Kumar et al. 2001). cm, Bugdorm-1; Megaview, Taiwan) after the fourth Pairwise distance estimate was based on a model of the instar (Ϸ30 individuals in one rearing cage). The total proportion distance measure. Statistical analysis sys- number of eggs laid, of each instar, and of adult males tem (SAS Institute 2001) was usedto test the signif- and females was recorded. icant differences of nucleotide base composition among orthopteroidinsects. Results Both maximum parsimony (MP) andneighbor-join- ing (NJ) methods were used in phylogeny reconstruc- 16S rDNA Sequence Divergence Between Gryllid tion. MP was analyzedusing the PAUP 3.1 program Species. DNA sequences of 66 individuals of the gryllid (Swofford1993) with the multiple equally parsimony species were analyzed. A total of 559 bp was assayed, exhaustive search option, tree bisection-reconnec- including those for three Loxoblemmus species and tion, and 1,000 random addition sequences. MEGA2.1 three outgroup genera: Teleogryllus, Brachytrupes, and was usedto conductNJ analysis using the Kimura-2- Velarifictorus. Base compositions among the gryllid parameter distance estimate, because the substitution species were not signiÞcantly different. The average pattern between transversion andtransition was dif- base composition in G, A, T, andC was 21.9, 30.4, 35.9, ferent in this case. A bootstrap analysis of 1,000 rep- and11.8%, respectively. Sequence analysis of L. ap- lications was carriedout on the tree inferredfrom the pendicularis andthe other species of gryllidswas in- MP andNJ methods. vestigated, and the sequence distances are recorded in Rearing Method and Cross-Breeding Experiments. Table 1. Sequence distances among the three Lox- Three populations of L. appendicularis from Taichung oblemmus species were 9.5Ð11.1%. Comparison of Lox- (Northern population; collecting site referredto as oblemmus to other genera, such as Teleogryllus, N-A1 in Fig. 1; June 2000), Pingtung (Southern pop- Brachytrupes, and Velarifictorus, showedthat dis- ulation; collecting site referredto as S-Mancho in Fig. tances rangedfrom 12.2 to 15.3%. 1; April 2000), andTaitung (Eastern population; col- Plots of transversion (Tv) or transition (Ts) corre- lecting site referredto as E-F1 in Fig. 1; May 2000) sponded to a total substitution (Tv ϩ Ts) in two were usedin these experiments. Adultsandnymphs sequences that have shown differently evolved pat- were maintainedin the laboratory on commercial dog terns (Fig. 2). Three levels of the substitution pattern food, which was kept in plastic containers equipped depictedtheevolvedconstraint in the 16S rDNA gene. with sand1 cm thick on the bottom, andmoist cotton First, most of the transition substitutions were linear that linedthe water vial (Liu et al. 1999). They were andwere almost at zero of the tranversion within the kept under 14:10 h L:D cycles at room temperature species. Second, transversion or transition substitution (22Ð25ЊC). The crickets were fedwith fresh food,and event was, in the changing form, nearly linear among the uneaten foodwas removedat least two times per the species within genus Loxoblemmus. Third, in the week. Virgin adults were isolated before the Þnal molt saturation level among different genera, the transition andrearedseparately in a plastic vial ( Ϸ12 cm height substitution was similar to that in the secondlevel, and by 6 cm diameter). Adult males and females were aged transversion substitution retainedthe linear essence up to 3 wk to ensure they were sexually maturedfor of change. the cross-breeding experiments. All copulations, in- Patterns of Genetic Diversity Concordant to Geog- cluding the reciprocal sets, were maintained together raphy Among the Populations of L. appendicularis. in plastic containers (12 cm diameter and height) Sequence distances from 46 individuals of L. appen- providedwithmoistenedsandybottoms (which kept dicularis were clearly groupedinto three clusters. It is the female cricket reproductively active) and a small worth noting that the three clusters were distributed plastic foodcup. Adultcouples were transferredto consistently to the geographical areas. Individuals col- July 2004 YEH ET AL.: SPECIATION EVENT OF L. appendicularis COMPLEX 617

ets from the other two populations (Table 2). First, hatching proportion and progeny developing to adults were both nearly zero when the crossing parent is a male of the Northern population. Second, only a few the progeny developed to adults, in contrast to the S(() ϫ S(&) control set. When the crossing parent was a female of the Northern population, all of the progeny were female. Postzygotic isolation between Eastern andSouthern populations was also observed (Table 2). Although egg production and hatching pro- portion was high (Ͼ95%), the proportion of devel- opedadultswas low between Eastern andSouthern Fig. 2. Scatter plots of total substitution versus corre- crossing populations (10.8% in E[(] ϫ S[&] and sponding transistion (Ts, circles) or transversions (Tv, tri- 51.2% in S[(] ϫ E[&]) comparedwith the control set angles) of 16S rDNA sequences for all pairwise comparisons. ( ϫ & WS, distance within L. appendicularis complex or L. equestris; of S( ) S( ) (89.2%). Moreover, aberrations in the AS, distance among member species of Loxoblemmus; AG, sex ratio andthe emergent periodof adulthoodwere distance among compared genuses of Loxoblemmus, Te- also observedbetween the Southern (male) andEast- leogryllus, Brachytrupes, and Velarifictorus. ern (female) crosses. The sex ratio in the control set was Ϸ1:1, andthe emergent periodfor both male and female adulthoodwasaroundMayÐAugust. In the lectedfrom north of Chiayi county in west Taiwan hybrids of S(() ϫ E(&), however, the adult male constitute the Northern population (N), individuals emerged during FebruaryÐApril, which was distinctly collectedfrom Kaohsiung andPingtung counties con- different from adult female hybrids that emerged dur- stitute the Southern population (S), and individuals ing JuneÐSeptember. Alternatively, Þeldandlabora- collectedfrom Taitung county constitute the Eastern tory observations have shown that this species has an population (E; Fig. 1). The Northern population had annual life cycle without hibernatedeggs. The nu- an average distance of 3.2% from the Southern and merous nymphs usually hatch in July andAugust and Eastern populations, anda low 2.6% distanceexisted molt six times until adulthoodisreachedaroundMay, between the Southern andEastern populations. Sig- June, andJuly. niÞcant limitedsequence divergencewas obtained within each population; sequence variation for S, E, Discussion andN populations was an average of 0.007, 0.001, and 0.006, respectively (Table 1). Among 46 individuals of Base Compositional Bias and Substitution Pattern L. appendicularis, 21 haplotypes belonging to each in Transversion and Transition. It is well known that population were observed: 10 haplotypes from 18 in- insect mitochondrial DNA is rich in adenine and thy- dividuals in the Southern population, 2 haplotypes mine. Simon et al. (1994) inferredthat there might be from 8 individuals in the Eastern population, and 9 a trendof increasing A ϩ T bias in the more recently haplotypes from 20 individuals in the Northern pop- derived insect orders, but there has not been enough ulation (data not shown). The most common haplo- information to depict it. Yeh et al. (1997) have also type (N-L. app-A, B, D, E) was foundin eight indi- pointedout that the proportion of guanine in viduals in the Northern population. decreases from ancestral orders toward derived or- Phylogenetic Inference from 16S rDNA Sequence. ders. Nucleotide base composition of gryllid species is Three distinct lineages of L. appendicularis complex rich in adenine (A) and thymine (T), similar to the were foundby both distanceandparsimony analyses. previously studies of orthopteroid insects (Flook and The neighbor-joining tree under Kimura-2-parameter Rowell 1997a, b, Flook et al. 1999, 2000, Chapco et al. estimates (Fig. 3) showed that individuals of L. ap- 2001). However, different base composition can be pendicularis from the Northern, Southern, or Eastern area form a separate group. Bootstrap supportedthe branching of the three clusters. The maximum parsi- Table 2. Numbers of eggs produced and developed adults of mony trees revealeda similar topology. Heuristic hybrid progeny among the three populations search foundminimum length trees of 264 steps in 136 Hatching No. F1 Adult Parents No. eggs equivalent trees. The topology of the strict consensus proportion adults proportion (& produced parsimony tree (Fig. 4) approximatedthe neighbor- (%) (& (%) joining tree, andmost of the termini lineages of the ϫ trees were also in agreement. E S 787 97.8 6 79 10.8 E ϫ N 480 73.8 0 24 5.0 Cross-Breeding Among Three Populations. Results S ϫ N 412 92.7 0 72 17.5 from the cross-breeding experiments revealed that the S ϫ E 527 96.2 161 109 51.2 mating songs andbehaviors of these three populations N ϫ S 269 7.1 1 0 0.4 ϫ play no important role in interrupting their reproduc- N E 0 ÑÑÑÑ S ϫ S 462 97.6 226 186 89.2 tive communication. However, interesting observa- tions were made regarding hybrids produced when N, S, andE are the Northern, Southern, andEastern populations of crickets of the Northern population matedwith crick- L. appendicularis, respectively. 618 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4

Fig. 3. Phylogeny of three L. appendicularis complexÐrelatedspecies andtheir outgroups basedon 16S rDNA sequences, constructed by Kimura 2 parameter distance under NJ clustering methods. Bootstrap values are shown beneath the dendrogram. Specimens from the same species or population come from a robust cluster with a high bootstrap value. Each referencedoutgroup is from the same genus, or the other genus forms a robust cluster in itself. seen because the focus is at the base of guanine (G). sitions of guanine exist as the orthopteroidinsects are Base composition of the 3Ј portion of the 16S rDNA aligned. Guanine levels of Isoptera, Grylloblattaria, sequence of orthopteroidinsects obtainedfrom pub- andEnsifera are higher than in the other orthopteroid lishedpapers or retrievedfrom the GenBank (Table insects (Blattaria, Phasmida, and the Caelifera subor- 3) was calculated. The proportion of guanine in gryllid der in Orthoptera). Base composition bias may be species is nearly 21% on average, a value larger than causedby the function of the 16S rRNA. Stem-loop those of the other orthopteroids of Blattaria and Phas- forming in the tertiary structure is important for the mida and the suborder of Caelifera (Table 3). function of this gene. Guanine forms three hydrogen Variance analysis in the SAS program (SAS Institute bonds with cytosine, whereas two hydrogen bonds can 2001) shows that signiÞcantly different base compo- also form with uridine. Under the evolutionary con- July 2004 YEH ET AL.: SPECIATION EVENT OF L. appendicularis COMPLEX 619

Fig. 4. Cladogram of three L. appendicularis complexÐrelatedspecies andtheir outgroups basedon partial 16S rDNA sequences using heuristic search. Bootstrap values are shown beneath the cladogram. Specimens from the same species or population come from a robust cluster with a high bootstrap value. Strict consensus tree was shown from 130 trees. The tree lengths are 264 steps, andthe CI andRI values are 0.746 and0.945, respectively. straint of AT rich in mitochondrial genes, GU pairings Hewitt 1989). Cross-breeding experiments (Table 2) may be substitutedby AU pairing in the helix of the suggest that a hybridzone might exist in nature, al- 16S rRNA, reducing the guanine proportion in the though its range is uncertain. However, materials col- derived insect orders. lecteddidnotprovideinformation concerning hy- Hybrid Zones Among Three Populations. The col- brids of the Southern and Northern populations in lecteddata(Fig. 1) andphylogenetic results show that nature. Tsu-Feng-Gu in southeastern Taiwan is a geographic Hybrids reveal different adaptations for their sur- boundary between the Southern and Eastern popu- vival. In general, the predominance of hybrids more lations. In the contact zone, both genotypes of the S like one or the other parental species is typical of andE populations can be detectedhere,although hybrid zones in a wide variety of taxa (Goodman et al. most, three quarters of them, belong to the Southern 1999, Burke andArnold2001). However, increased population. One possible scenario is that the hybrid hybridgenotype, displacingthe parental genotype, zone in Tsu-Feng-Gu may have formedafter second- may come from the superior adaptation of some hy- ary contact between both populations, as generally brids(Burke andArnold2001) andthe probability of occurs in most studies of hybrid zones (Barton and introgression towardthe least abundantspecies (Ar- 620 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4

Table 3. Comparison of insect nucleotide compositions of the 3؅ portion of the mitochondrial 16S rDNA sequence of orthoptroid insects

Taxon N G% A% T% C% References Isoptera 7 20.1a 24.9 42.0 12.0 Kambhampati et al. 1996 Grylloblattaria 1 21.3a 28.3 38.8 11.6 Flook andRowell 1997b Orthoptera-Loxo. 66 21.9a 30.4 35.9 11.8 Current study Orthoptera-Gryllidae 62 20.6a 32.6 36.7 10.1 Huang et al. 2000, Jost et al.c Orthoptera- 51 20.1a 32.9 36.2 10.8 Chapco et al. 2001, Flook andRowell 1997b, Flook et al. 1999 Orthoptera-Caelifera 22 17.2b 33.2 39.0 10.7 Flook andRowell 1997a, 1997b; Flook et al. 2000; Flook et al. 1999; Chapco and Martelc Blattaria 34 17.5b 33.1 39.0 10.5 Kambhampati 1995 Phasmida 2 17.9b 28.4 44.2 9.6 Flook andRowell 1997b

Sequences of 16S rDNA of the orthopteroidinsects were downloadedfromthe publishedpapers or retrievedfrom GenBank andaligned to calculate their base composition. N: comparative number in that taxon. a or b : Variance analysis was performedon guanine distributedamongorthoptroidtaxa basedon the Duncan model.Different capital letters indicate the signiÞcant difference between them. c Retrievedfrom GenBank. noldet al. 1993, Burke andArnold2001). Different events eventually broke up the distribution range of levels of postzygotic isolation were involvedamong L. the ancestral cricket population, andeach group sub- appendicularis populations. Cross-breeding experi- sequently evolvedits own characteristics by either ment results for L. appendicularis suggest that hybrid- genetic drift or selection. In accordance with this ization capability is reciprocal between Southern and scenario, reptiles, salamanders, and freshwater Þsh Eastern populations. However, the introgression di- distributedineastern andwestern Taiwan have rection from Southern to Northern populations may evolved into different endemic species (Lue and Chen exist, as demonstrated by the asymmetrical crossing 1997). However, secondary contact may have oc- results between N(() ϫ S(&) andS( () ϫ N(&) curredwith the rising seashore of the Hengchung (Table 2). Although the control sets of E ϫ E andN ϫ peninsula during the Middle Pleistocene (Huang et al. N were not performed, only the control set of S ϫ S 1997), which permittedeasier communication be- will not bias the cross-breeding results. Two reasons tween the Southern andEastern populations. More- compelledus to use one control set only. First, labo- over, the existing Northern population wouldindicate ratory maintenance shows a similar life cycle among that yet another geographical event took place during three L. appendicularis populations (i.e., Northern, the Upper Pleistocene in Taiwan. Southern, andEastern), as describedabove.Second, Hewitt (1999, 2001) has arguedthat most major numerous nymphs were hatchedin the maintenance hybrid zones in Europe reßect divergence during the of the three populations we found. Multiple matings Quaternary Ice Ages, with periodic expansion and kept the female producing Ͼ300 eggs. To avoidwast- accumulating genetic differences. A similar biogeo- ing of feeding houses and materials, the control set of graphical event is also documented in South America only S ϫ S was used. (Bermingham and Avise 1986, Riddle and Honeycutt Hybrid Zones Indicated Secondary Contact Caused 1990). Four episodes of periodic glaciation in Taiwan by Vicariant and Glaciation Events. Geological evi- during the Pleistocene inßuenced many speciation dence indicates that the island of Taiwan has been events for TaiwanÕs fauna andßora (Lue andChen connectedandisolatedfrom the Chinese continent 1997) andmight also have providedtheopportunity more than once because of the interaction of arc- for L. appendicularis migration from China to Taiwan. continent collisions andsea level changes duringthe Thus, formation of landbridgesincreases the possi- late Pliocene. The mammalian fossil recordincludes bility of multiple L. appendicularis colonizations in elephants anddeer(Shikama et al. 1975, Otsuka and Taiwan. The earlier population (in the early Pleisto- Shikama 1978, Otsuka 1984) from the Lower Pleisto- cene, 1.8 Mya) might have withdrawn or survived only cene (1.8 Mya), indicating that Taiwan had been con- in the south, with the recently arrivedpopulation of L. nectedto the Chinese continent before the Pleisto- appendicularis from China during the last glaciation cene. Therefore, both the geological history andthe (20,000Ð110,000 yr ago) being distributed throughout fossil recordindicatethat L. appendicularis might have the north. originatedon the Chinese continent duringthe early Present day sequence divergences among these Pleistocene (1.8 Mya). The rise of the Central Moun- three cricket populations also consistently reßect tain Range has been estimatedto have occurredmore these two geographical events. Rate estimate of se- than 1 million yr ago (Lin 1966). The Central Moun- quence divergencewas calculatedbasedon the cali- tain Range runs longitudinally from north to south, bration time of the early Pleistocene (1.8 Mya) andthe with ridges above 3,000 m and declines abruptly to the rise of the Central Mountain Range (1.5Ð1 Mya) (Lin sea in southern Taiwan, andit may have actedas a 1966, Huang et al. 1997). Sequence divergence be- barrier to population dispersal or migration. Vicariant tween the original (Southern andEastern) andthe July 2004 YEH ET AL.: SPECIATION EVENT OF L. appendicularis COMPLEX 621 recent Northern populations is 0.032, whereas diver- sequence, gene organization, andgenetic code.J. Mol. gence is only 0.026 between the two vicariant popu- Evol. 22: 252Ð271. lations (Southern andEastern). The divergencerate Creer, S., A. Malhotra, R. S. Thorpe, and W.-H. Chou. 2001. between the original andrecently introducedpopu- Multiple causation of phylogeographical pattern as re- lations is 0.017/Mya (3.2/1.8), a value close to that of vealedby nestedcladeanalysis of the bamboo viper the two vicariant populations (S andE; 0.017Ð0.026/ (Trimeresurus stejnegeri) within Taiwan. Mol. Ecol. 10: 1967Ð1981. Mya), because the Central Mountain Range is steep Davis, R. E., T. J. Kelly, E. P. Master, B. S. Thyagaraja, C. A. enough to have separatedthese two populations be- Rote, and R. B. Imberski. 1994. Complete base sequence fore 1.5Ð1 Mya (2.6/1.5 and2.6/1). These divergence for the mitochondrial large subunit ribosomal RNA of the values are similar to the ribosomal DNA region of gypsy moth Lymantria dispar (L). Insect Mol. Biol. 3: Hawaiian Laupala crickets: 0.024/Mya (Fleischer et al. 219Ð228. 1998) or 0.02/Mya (Brower 1994). Dawson, M. N., K. D. Louie, M. Barlow, D. K. Jacobs, and The morphological variables within L. appendicu- C. C. Swift. 2002. Comparative phylogeography of sym- laris complex have been a source of confusion in patric sister species, Clevelandia ios and Eucyclogobius taxonomic classiÞcation andrevision (J. T. Y. and newberryi (Teleostei, Gobiidae), across the California Y. L. C., personal communication). Our results suggest Transition Zone. Mol. Ecol. 11: 1065Ð1075. Dowling, T., and C. Secor. 1997. The role of hybridization that the three populations of L. appendicularis do not andintrogression in the diversiÞcationof animals. Annu. represent a single panmictic evolutionary unit andare Rev. Ecol. Sys. 28: 593Ð619. sufÞciently distinct to be considered different species. Fang, Q., W. C. Black IV, H. D. Blocker, and R. F. Whitcomb. Biogeographical history suggests that the speciation 1993. A phylogeny of New World Deltocephalus-like leaf- event began during the early Pleistocene, and subse- hopper genera basedon mitochondrial16S ribosomal quent vicariant andglaciation events ledto these three DNA sequences. Mol. Phylogenet Evol. 2: 119Ð131. parapatric cryptic species. Fleischer, R. C., C. E. McIntosh, and C. L. Tarr. 1998. Evo- lution on a volcanic conveyor belt: using phylogeographic reconstructions andK-Ar-basedages of the Hawaiian islands to estimate molecular evolutionary rates. Mol. Acknowledgments Ecol. 7: 533Ð545. This work was supportedby the AcademiaSinica, Taipei, Flook, P. K., and C. H. Rowell. 1997a. 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Appendix: Collection information of crickets in this study

Abbrev. Accession no. Locality Latitude Longitude Species name Date S-ManCho AY230943 Manchou, Pingtung 231982 243632 L. appendicularis 6 March 2001 S-C28 AY239044 Tsu-Feng-Gu, Pingtung 232568 248293 L. appendicularis 7 February 1996 S-C29 AY239045 Tsu-Feng-Gu, Pingtung 232568 248335 L. appendicularis 7 February 1996 S-C32 AY239046 Tsu-Feng-Gu, Pingtung 232568 248335 L. appendicularis 7 February 1996 S-65 AY230947 Ken-Ting, Pingtung 230463 242997 L. appendicularis 11 May 2001 S-69 AY239048 Oulenpi, Pingtung 233664 242286 L. appendicularis 11 May 2001 S-89 AY239049 Tsai-Shan, Kaohsiung 172447 250549 L. appendicularis 10 June 2001 S-90 AY239050 Tsai-Shan, Kaohsiung 172475 250553 L. appendicularis 10 June 2001 S-91 AY239051 Tsai-Shan, Kaohsiung 172460 250555 L. appendicularis 10 June 2001 S-92 AY239052 Tsai-Shan, Kaohsiung 172455 250537 L. appendicularis 10 June 2001 S-93 AY239053 Tsai-Shan, Kaohsiung 172490 250562 L. appendicularis 10 June 2001 S-94 AY239054 Tsai-Shan, Kaohsiung 172451 250247 L. appendicularis 10 June 2001 S-110 AY239055 Fun-Shan, Pingtung 213987 246450 L. appendicularis 25 May 2001 S-112 AY239056 Fun-Shan, Pingtung 213547 246318 L. appendicularis 25 May 2001 S-117 AY239057 Fun-Shan, Pingtung 214764 246303 L. appendicularis 25 May 2001 S-120 AY239058 Hsiun-Liu, Pingtung 227929 245802 L. appendicularis 4 June 2001 S-121 AY239059 Hsiun-Liu, Pingtung 228056 245789 L. appendicularis 4 June 2001 S-122 AY239060 Hsiun-Liu, Pingtung 228213 245840 L. appendicularis 4 June 2001 E-F1 AY239061 Song-Ts-Jien, Taitung 247054 249658 L. appendicularis 18 January 1997 E-F1a AY239062 Song-Ts-Jien, Taitung 247054 249658 L. appendicularis 18 January 1997 E-C31 AY239063 Tsu-Feug-Gu, Pingtung 238568 248293 L. appendicularis 7 February 1996 E-C33 AY239064 Song-Ts-Jien, Taitung 247054 249658 L. appendicularis 2 April 1996 E-C34 AY239065 Song-Ts-Jien, Taitung 247054 249658 L. appendicularis 2 April 1996 E-C35 AY239066 Song-Ts-Jien, Taitung 247054 249658 L. appendicularis 2 April 1996 E-C37 AY239067 Song-Ts-Jien, Taitung 247054 249658 L. appendicularis 2 April 1996 E-C38 AY239068 Song-Ts-Jien, Taitung 247054 249658 L. appendicularis 2 April 1996 N-A1 AY239069 Ta-Keng, Taichung 218867 262576 L. appendicularis 18 July 1996 N-A2 AY239070 Ta-Keng, Taichung 218867 262576 L. appendicularis 18 July 1996 N-A3 AY239071 Ta-Keng, Taichung 218867 262576 L. appendicularis 18 July 1996 N-B1 AY239072 Shih-Gun, Taichung 225726 268643 L. appendicularis 28 September 1995 N-B2 AY239073 Shih-Gun, Taichung 225726 268643 L. appendicularis 28 September 1995 N-B3 AY239074 Shih-Gun, Taichung 225726 268643 L. appendicularis 28 September 1995 N-C1 AY239075 Heishun, Taichung 252347 266525 L. appendicularis 25 January 1996 N-C2a AY239076 Heishun, Taichung 252347 266525 L. appendicularis 25 January 1996 N-C2b AY239077 Heishun, Taichung 252347 266525 L. appendicularis 25 January 1996 N-D1 AY239078 Chunhsingleng, Taichung 227812 267843 L. appendicularis 28 September 1995 N-E1 AY239079 Hsiun-Dung, Taichung 226919 265224 L. appendicularis 16 February 1996 N-66 AY239080 Sammai, Chiayi 213470 258906 L. appendicularis 8 April 2001 N-67 AY239081 Wulai, Taipei 304911 275114 L. appendicularis 18 May 2001 N-98 AY239082 Tung-hsiao, Miaoli 217881 271473 L. appendicularis 3 June 2001 N-99 AY239083 Tung-hsiao, Miaoli 217881 271473 L. appendicularis 3 June 2001 N-105 AY239084 Tai-an, Miaoli 240945 269594 L. appendicularis 3 June 2001 N-106 AY239085 Tai-an, Miaoli 240945 269594 L. appendicularis 3 June 2001 N-107 AY239086 Tai-an, Miaoli 240945 269594 L. appendicularis 3 June 2001 N-108 AY239087 Tai-an, Miaoli 240945 269594 L. appendicularis 3 June 2001 N-113 AY239088 Tung-hsiao, Miaoli 217881 271473 L. appendicularis 3 June 2001 Le-A1a AY239089 Chung-Hsing village, Nantou 217306 265069 L. equestris 25 July 1996 Le-A1b AY239090 Chung-Hsing village, Nantou 217306 265069 L. equestris 25 July 1996 Le-B1a AY239091 Zey-ho, Hualien 265200 254243 L. equestris 2 April 1996 Le-B1b AY239092 Zey-ho, Hualien 265200 254243 L. equestris 2 April 1996 Le-C1 AY239093 Shan-Tseng-shi, Hualien 311267 266731 L. equestris 1 April 1996 Le-TWSK AY239094 Yuzin, Tainan 201762 257231 L. equestris 28 September 2000 Le-97 AY239095 Wufeng, Hsintsu 262005 272721 L. equestris 16 May 2001 Le-111 AY239096 Wufeng, Hsintsu 261687 272694 L. equestris 16 May 2001 Le-127 AY239097 Chipen, Taitung 264005 251669 L. equestris 10 October 2001 Le-128 AY239098 Chipen, Taitung 264005 251669 L. equestris 10 October 2001 Ls-70 AY239099 Wanlitong, Pingtung 218315 243325 L. sylvestris 11 May 2001 Ls-109 AY239100 Fun-Shan, Pingtung 213325 246314 L. sylvestris 25 May 2001 Ls-116 AY239101 Fun-Shan, Pingtung 213325 246310 L. sylvestris 25 May 2001 Ls-118 AY239102 Tsai-Shan, Kaohsiung 172475 250553 L. sylvestris 6 June 2001 Velar-A1 AY239103 Tachia, Miaoli 211278 269301 V. ornatus 3 April 1996 Velar-A2 AY239104 Tachia, Miaoli 211278 269301 V. ornatus 3 April 1996 Teleo-A1 AY239105 Minjien, Nantou 218211 263732 T. mitratus 25 July 1996 Teleo-B1 AY239106 Paoli forest, Pingtung 224850 244236 T. occipitalis 5 September 1996 Brach-A1 AY239107 Chung-Hsing village, Nantou 217306 265069 B. portentosus 31 January 1996 Brach-A2 AY239108 Chung-Hsing village, Nantou 217306 265069 B. portentosus 31 January 1996