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Biological Control 45 (2008) 308–318 www.elsevier.com/locate/ybcon
A molecular phylogeny of the genus Lygodium (Schizaeaceae) with special reference to the biological control and host range testing of Lygodium microphyllum
Paul T. Madeira *, Robert W. Pemberton, Ted D. Center
United States Department of Agriculture, Agricultural Research Service, Invasive Plant Research Laboratory, 3225 College Avenue, Ft. Lauderdale, FL 33314-7700, USA
Received 28 June 2007; accepted 5 February 2008 Available online 14 February 2008
Abstract
Lygodium microphyllum, first naturalized in the 1960s, has aggressively invaded forest-dominated wetlands in southern and central Florida. The indeterminate growth of this invasive climbing fern creates thick rachis mats which climb over shrubs and trees smothering the underlying growth and carrying ground fires into the forest canopy. Foreign surveys for natural enemies identified 20 species of insects and two species of mites. Host range testing of three insect species and one mite included five Lygodium species (L. palmatum, L. volubile, L. cubense, L. venustum, and L. oligostachyum) native to the United States and the West Indies. A molecular phylogeny of the genus was conducted using the trnL intron and the trnL-F intergenic spacer of chloroplast DNA to determine the relationship of L. microphyllum to other Lygodium species. Three major clades appeared, one with L. palmatum and L. articulatum (the most basal), a second with L. reticulatum and L. microphyllum, and a third comprising the other species examined. Lygodium microphyllum appeared at the end of a long branch approximately equidistant from all species of interest preventing the correlation of genetic distance and host range behavior. However, inspection of the results of host range experiments showed a relationship between the presence of a related biotype or species of the insect on a related alternate host and the ability of that insect to develop on that alternate host. Ancestral host usage and ecological fitting are examined as possible explanations for the acceptance of L. palmatum by the tested musotimine insects. Published by Elsevier Inc.
Keywords: Lygodium microphyllum; Lygodium palmatum; Neomusotima conspurcatalis; Austromusotima camptozonale; Floracarus perrepae; Neostromboc- eros albicomus; Ancestral host usage; Biocontrol; Host range; Host shift; Molecular phylogeny
1. Introduction ‘‘stems” (actually single compound fronds) exhibit indeter- minate growth creating thick rachis mats over the tops of Lygodium microphyllum (Cav.) R. Br. (Lygodiaceae), shrubs and trees. These smother the underlying vegetation first recognized as naturalized in southern Florida in 1965 reducing native plant cover (Brandt and Black, 2001)and (Beckner, 1968), has aggressively invaded forest-dominated plant diversity (Nauman and Austin, 1978; Pemberton wetlands (Pemberton and Ferriter, 1998). These include and Ferriter, 1998), thereby rare native flora (Craddock bald cypress (Taxodium distichum [L.] Richard) stands, Burks, 1996). The rachis mats alter the normal course of Everglades tree islands, mesic pine flatwoods, saw grass fires by carrying flames into the canopy thus destroying (Cladium jamaicense Crantz) marshes, wet prairies, and trees that would survive ground fires. Burning fronds also mangrove communities (Pemberton et al., 2002). Its carry fire over extended distances as wind-borne pieces (Roberts, 1998; Schmidz et al., 1997). For these reasons the Florida Exotic Plant Pest Council has included * Corresponding author. L. microphyllum in the most invasive category, Category E-mail address: [email protected] (P.T. Madeira). I label (FLEPPC, 2005).
1049-9644/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.biocontrol.2008.02.004 P.T. Madeira et al. / Biological Control 45 (2008) 308–318 309
The cost of chemical treatment for L. microphyllum is actually be polyphyletic or paraphyletic. Instead clades high (Pemberton et al., 2002) and, although effective, re- derived from molecular phylogenetic analysis are more growth and non-target damage usually occur (Ferriter, strictly monophyletic and therefore more useful for the 2001). Given L. microphyllum’s rapid spread, natural area structuring of host range plant lists. Similarly, Pemberton managers have turned to biological control for a cost-effec- et al. (2002) noted that the subgenera of Lygodium have tive, long-term solution. Starting in 1997 scientists from the been established using morphology. They proposed a study USDA-ARS Invasive Plant Research Laboratory and the ‘‘to look for congruency in placement of the species in sub- USDA-ARS Australian Biological Control Laboratory in genera with molecular-based phylogenies”. co-operation with CSIRO Entomology began exploratory This research, essentially a molecular taxonomic study surveys in Asia and Australia for natural enemies of of the genus Lygodium, was initiated as a direct result of L. microphyllum (Goolsby et al., 2003). Surveys in its native that proposal. The regions chosen for sequencing are two range identified 20 species of herbivorous insects and two non-coding regions of chloroplast DNA, the trnL intron species of mites. The pyralid moth Neomusotima conspurca- and the trnL-F intergenic spacer (Taberlet et al., 1991). talis Warren and the eriophyid mite Floracarus perrepae Goolsby et al. (2006), looking to find the origin of the inva- Knihinicki and Boczek are the most widely distributed. sive Florida population, previously used these chloroplast These, along with another pyralid Austromusotima campto- DNA sequences to identify the geographic haplotypes of zonale Hampson, formerly Cataclysta camptozonale (Yen L. microphyllum. Non-coding regions contain greater vari- et al., 2004), were selected for further work. ation than coding regions and are therefore very useful for Host range testing generally followed the principles for studies within a genus and often even within species. The selecting test plants formalized by Wapshere (1974) under Lygodium species identified for host range testing (the ‘‘spe- the name of the centrifugal-phylogenetic method (CPM) cies of interest” defined earlier) along with any other spe- where the candidate agent is subjected to a sequence of test cies available from other sources were used for plants, beginning with (and emphasizing) those most clo- sequencing. Secondarily, the study compared the molecular sely related to the target weed and proceeding (with phylogeny with results from the host range testing to deter- decreasing emphasis) to successively more distant taxa. mine whether the results of the former are predictive of the Increasingly it is considered important to assess the risk latter. to related indigenous flora, especially rare and endangered species. A ‘‘safeguard” component is designed to identify 2. Materials and methods additional plants for testing based on unrelated plants that share secondary chemicals with the target weed which 2.1. Plant material and DNA extraction could serve as token feeding stimulants for the candidate insects. Some plants of economic importance are usually This study analyzed 38 samples of plant material col- tested; as well as plants identified in the host plant usage lected for 14 Lygodium (ingroup) species and 2 Anemia patterns for the insect taxon involved. This model has (outgroup) species. Sample collection information, sample served biological control well for 30 years and is widely identification numbers, and GenBank accession numbers used by government regulatory authorities. are presented in Table 1. Samples were collected in the field Pemberton et al. (2002) discussed Lygodium species of and placed directly into air-tight tubes containing silica gel. interest (and therefore those to be used for host range test- Upon arrival at the molecular laboratory they were placed ing) taking into account taxonomic relatedness to L. micro- on fresh desiccant inside 3 dram shell vials. Table 1 pre- phyllum, geographic proximity to insect release sites in sents the collections by species, sample identification num- Florida, plant biology, and their status (native/invasive). ber, GenBank accession number, collection site, country, Within the genus Lygodium they identified five native spe- collection date, latitude, longitude, and collector. Total cies of interest: Lygodium palmatum (Bernh.) Sw., a tem- DNA from up to 20 mg dry weight of sample was extracted perate native with a range from Appalachia north to using the Qiagen DNeasy Plant Mini kitÒ. Purified DNA New England; Lygodium volubile Sw., which occurs in was quantitated using a microplate spectrophotometer. Cuba and other parts of the West Indies; Lygodium cubense Kunth., endemic to Cuba; Lygodium venustum Sw., found 2.2. Amplification and sequencing of PCR products in the West Indies; and Lygodium oligostachyum (Willd.) Desv., endemic to Hispanola. The taxonomic relatedness PCR products for sequencing of the trnL intron and the of Lygodium japonicum (Thunberg ex. Murray) Swartz, trnL-F intergenic spacer were generated using the ‘‘c” another invasive species in the southeastern U.S., was also (CGA AAT CGG TAG ACG CTA CG) and ‘‘f” (ATT of interest. TGA ACT GGT GAC ACG AG) universal primers Briese (2003) pointed out that the CPM usage of formal located on the trnL (UAA) 50 exon and the trnF (GAA) taxonomic classification, while intended to reflect phylog- gene, respectively (Taberlet et al., 1991). Reaction tubes eny, could better be described as ‘‘taxonomic circumspec- were added to a preheated (94 °C) block, incubated at tion” because these rankings are not always informative 94 °C for 3 min, and then cycled 35 times (denaturation: of the true evolutionary history of the group, which may 94 °C, 1 min; annealing: 55 °C, 1 min; extension: 72 °C, 310
Table 1 Lygodium sample collection and identification information Species Sample ID GenBank accession # Collection site Country collected Date collected Latitude Longitude Collector A. adiantifolium L120A DQ845241 Loxahatchee Wildlife Refuge, Florida USA 6/7/2001 26.5oN 80.3oW L. Kasarjian A. phyllitidis L125A DQ845242 Not specified COSTA RICA Sent 6/6/2001 16oN84oW UC Berkeley Botanical Garden L. articulatum L97A DQ845214 Coromandel Peninsula NEW ZEALAND 5/26/2001 37o070S 175o520E E. Cameron L. articulatum L98A DQ845215 West Auckland NEW ZEALAND 5/19/2001 36o580S 174o310E E. Cameron L. circinnatum L130A DQ845228 Botanical Garden, Bogor, Java INDONESIA Sent 6/6/2001 6.6oS 106.8oE UC Berkeley Botanical Garden L. cubense L160B DQ845219 Contact authors CUBA L. flexuosum L22A DQ845236 Klong Pla Kang Waterfall, Rayong THAILAND 5/13/1999 12.8oN 101.6oN A. Wright L. flexuosum L80A DQ845237 Huai Nam Rin, Chiang Mai THAILAND 8/30/1998 19o05.220N99o27.370E W. Somprasong L. flexuosum L180A DQ845235 Pang Mo, Chiang Mai, Chiang Dao THAILAND 11/27/1998 19o29.860N99o02.6370E A. Wright L. flexuosum L82AS DQ845238 Litchfield, Northern Territory AUSTRALIA Sent 7/6/2001 13.2oS 130.7oE J. Goolsby and A. Wright 308–318 (2008) 45 Control Biological / al. et Madeira P.T. L. flexuosum L82C DQ845239 Huai Nam Rin, Chiang Mai THAILAND 8/30/1998 19o05.220N99o27.370E W. Somprasong L. japonicum L51B DQ845232 Lamaroo Beach, Darwin AUSTRALIA 8/4/1999 12o28.050S 130o50.530E T. Wright and Jeloak L. japonicum L56A DQ845233 Ugata, Mie JAPAN 10/11/1997 34.2o0N 136oE R. Pemberton L. japonicum L63B DQ845234 Hillsborough Co., Florida USA 7/26/2000 27.9oN 82.4oN L. Kasarjian L. japonicum L57B DQ845231 Silver Bay, Lan Tao Island, CHINA 10/19/1997 22.3oN 114.2oN R. Pemberton Hong Kong L. lanceolatum L135A DQ845225 Near Andranovory MADAGASCAR 8/24/1967 23.1oS 44.2oE Fischer Forest Preserve L. microphyllum L10A DQ845212 Moe Mpe Hia Village GHANA 5/11/1999 5oN2oW R. Pemberton L. microphyllum L13A DQ845213 Kisi Bay Reserve, Kwazulu-Natal S. AFRICA 9/18/1998 26o5701900S32o4904800E G. Cook, Ward and Glen L. microphyllum L16A DQ845211 Daintree Ferry, Queensland AUSTRALIA 1/14/1999 16.3oS 145.2oE J. Makinson and K.Galway L. microphyllum L19C DQ845210 Morang District NEPAL 6/16/1998 26.8oN 87.3oE M. Rayamajhi L. microphyllum L26A DQ845209 Shing Mun Reservoir, New Territories CHINA 10/17/1997 22.4oN 114.1oE R. Pemberton L. microphyllum L42A DQ845205 Tirunelvelli, Tamil Nadu INDIA 5/1/2002 8o32.5070N77o21.3340E J. Goolsby L. microphyllum L44A DQ845206 Iron Range, Cape York, QN AUSTRALIA 6/25/2002 12.7oS 143.2oE J. Goolsby and M. Purcell L. microphyllum L101B DQ845207 Hobe Sound, Florida USA 6/28/2000 27.0oN 80.1oE L. Kasarjian L. microphyllum L105A DQ845208 Hillsborough County, Florida USA 4/30/2001 28.1oN 82.3oE L. Kasarjian L. oligostachyum L95A DQ845220 Cordillera Central, south of DOMINICAN R. 2/23/2001 18o350N70o130E R. Pemberton Loma de Casabe L. oligostachyum L96A DQ845221 Santa Domingo DOMINICAN R. 2/27/2001 18o280N69o540W R. Pemberton L. palmatum 150A DQ845229 McMinnville, Tennessee USA 7/22/2001 35.7oN 85.8oW S. Brother L. palmatum 150C DQ845230 McMinnville, Tennessee USA 7/22/2001 35.7oN 85.8oW S. Brother L. polystachyum L81A DQ845240 Huai Nam Rin, Chiang Mai THAILAND 8/30/1998 19o05.220N99o27.370E A. Wright L. reticulatum L140A DQ845217 Mount Dzumac NEW CALEDONIA 5/25/2001 22o05.290S 166o26.810E A. Wright L. reticulatum L141A DQ845216 Tully, Queensland AUSTRALIA Sent 7/6/2001 17.9oS 145.0oE J. Goolsby and A. Wright L. reticulatum L143A DQ845218 Road to Aerodrome, Isle de Pin NEW CALEDONIA 9/13/2001 22o37.370S 167o26.570E J. Goolsby and R Pemberton L. smithianum L8A DQ845222 Near Ayi Sakro village GHANA 5/12/1999 5oN 2.3oW R. Pemberton L. venustum L90A DQ845226 Rio Higuero, Aeropuerto DOMINICAN R. 2/22/2001 18oN69oW R. Pemberton del Mismo Novembre L. venustum L91A DQ845227 Sierra Prieta DOMINICAN R. 2/22/2001 18o190N69o580W R. Pemberton L. volubile L70A DQ845223 Iguazu National Park ARGENTINA 9/2/2000 25.6oS 54.6oW R. Pemberton L. volubile L71A DQ845224 Iguazu National Park ARGENTINA 8/28/2000 25.6oS 54.6oW R. Pemberton P.T. Madeira et al. / Biological Control 45 (2008) 308–318 311
2 min) followed by a final extension (72 °C, 10 min). Reac- The bootstrap procedure re-samples the original data, with tion mixtures contained 1 reaction buffer (10 mM KCl, replacement, to create a series of bootstrap datasets the 10 mM (NH4)2SO4, 20 mM Tris–HCl, 2 mM MgSO4,1% same size as the original data. The bootstrap value of a Triton X-100), 0.5 mM Betaine, 0.2 mM dNTPs, 0.5 lM node is then defined as the percentage of times that a node each primer, and 0.04 U/ll Taq polymerase (New England is present in the trees constructed from the new data sets. BiolabsÒ). PCR products were cleaned for sequencing Consensus trees (50% majority rule) were also examined. using the Zymo Clean and Concentrate KitÒ. Sequencing Maximum likelihood analysis was also carried out using was performed using the same external primers used for PAUP. Initially, MODELTEST 3.06 (Posada and Crand- the PCR products. Internal primers used include (Taberlet all, 1998), as implemented in the Windows interface et al., 1991) primers ‘‘d” (GGG GAT AGA GGG ACT MTgui, was run to find the optimal evolutionary model TGA AC) and ‘‘e” (GGT TCA AGT CCC TCT ATC parameters. The model chosen using Akaike weighting CC). The sequencing reaction was performed using the (10245) was K81uf+G (Kimura 3-parameters with unequal Amersham/GE DYEnamicTM ET terminator kit. Sequenc- base frequencies) with base frequencies A = 0.3498, ing was performed on an ABI 310 sequencer using POP-6 C = 0.1671, G = 0.1839, T = 0.2992, Rmat (1.0000 2.2543 capillaries. 0.3867 0.3867 2.2543), and a gamma distribution shape parameter of 2.2198. Base frequencies, rates of nucleotide 2.3. Alignment parameters substitution, and the gamma parameter were then entered into PAUP to obtain the optimal maximum likelihood tree. Trace files were compiled and viewed using Sequencer A bootstrap analysis was conducted using 1000 replicates. 4.1.4 (Gene Codes). The DCA (Divide and Conquer) algo- rithm (Stoyle, 1998) was chosen for the alignment. It pro- 3. Results duces simultaneous multiple sequence alignments by recursively cutting the sequences under consideration into 3.1. Parsimony analysis sets of smaller subsequences which are then aligned using the MSA algorithm (Carrillo and Lipman, 1988). Hickson The parsimony analysis produced 60 best equally parsi- et al. (2000) reviewed five global multiple sequence align- monious trees with a length of 958 (where the sum of the ment programs (Clustal W, Divide and Conquer, Malign, minimum possible weights was 819 and the maximum PileUp, and TreeAlign) and found the DCA program pro- was 1803). These 60 trees each produced a consistency duced the highest relative score, among the shortest align- index (CI) = 0.855, a retention index (RI) = 0.859, and a ment lengths and number of gaps, and among the most rescaled consistency index (RC) = 0.734. Results of the consistent alignment scores and alignment lengths when analysis are presented in Fig. 1. External branches are gap costs were varied. Various gap opening and extension labeled with the species and sample identification numbers. costs were examined from GO = 1, GE = 0.5 to GO = 16, Sample identification numbers may be cross-referenced GE = 8 using Neighbor Joining trees. Only a minor branch with the sample collection information and GenBank shift in the resulting tree was produced over this range [at accession numbers in Table 1. Where multiple sample ID the highest gap extension (GE = 8) costs]. The settings cho- numbers appear together they are identical sequences. sen (Costs: Tv = 2, Ti = 1, GO = 4, GE = 2, L = 20, k =0, The upper numbers at each node represent consensus num- W = 0) produced one of the most common alignment bers for the 60 most parsimonious trees. The lower num- lengths in the gap cost analysis. bers at each node represent the bootstrap percentages for 1000 replicates. Differences between the 60 equally parsi- 2.4. Phylogenetic analysis monious trees occurred ‘‘within” L. microphyllum and L. japonicum samples, and in the placement of the root rel- The alignment for the chloroplast sequences was 537 ative to L. palmatum and Lygodium articulatum A. Rich. bases in length for the trnL intron and 470 bases in length The bootstrap analysis shows some weakness in the place- for the partial trnL-F spacer. The trnL gene was excluded ment of (1) L. cubense and L. oligostachyum relative to from analysis. There were 350 characters which were vari- Lygodium smithianum Pr., (2) of L. venustum relative to able but parsimony uninformative and 261 characters Lygodium polystachyum Wall. ex Moore, (3) the rooting which were parsimony informative. Analysis was con- of Lygodium circinnatum (Burm. f.) Sw., and (4) the mono- ducted using PAUP (Swofford, 2002). A partition homoge- phyletic nature of L. palmatum and L. articulatum. neity test was conducted using the PAUP ‘‘homopart” command to compare the trnL intron and trnL-F spacer. 3.2. Maximum likelihood analysis There was no significant difference between the sequences so they were analyzed together. Parsimony analysis was The maximum likelihood analysis produced a single best conducted using a parsimony branch-and-bound analysis. tree presented in Fig. 2A. Branches are identified by sample Gaps were treated as ‘‘missing”. Bootstrap analysis, a type identification numbers and species. The same pattern of of statistical analysis to test the reliability of branches in clusters appears as in the parsimony analysis but with the the evolutionary tree, was conducted using 1000 replicates. advantage of displaying a distance metric (sequence diver- 312 P.T. Madeira et al. / Biological Control 45 (2008) 308–318 gence). Three major clades appear, one with weak sup- (Collinson, 1996; Halle, 1940; Manchester and Zavada, port containing L. palmatum and Lygodium articulatum 1987; Rozefelds et al., 1992) and appear in North America, [labeled a], a second with Lygodium reticulatum Schkuhr Europe, Northern Asia, Chile, Australia, and New and L. microphyllum [b], and a third with the other species Zealand. examined [c]. Percentage values from 1000 bootstrap Wikstru¨m et al. (2002) provide a molecular phylogenetic replicates appear along the branch lengths. Most relevant study of the Schizaeaceae using chloroplast rbcL sequences. to this study, the maximum likelihood analysis shows That analysis showed a basal split of Lygodium at L. pal- L. microphyllum at the end of a long branch distant from matum followed by another for Lygodium microphyllum any of the other ‘‘species of interest”. Using genetic with the remainder of the genus examined forming a clade. distance as a criterion the closest ‘‘species of interest” to Our rooting with Anemia is followed by a basal split of Florida L. microphyllum is L. volubile at a sequence diver- L. articulatum and L. palmatum (Figs. 1 and 2). However, gence of 0.092 (9.2%). low bootstrap values suggest this could equally be a rooting at L. articulatum followed by L. palmatum. The large dis- 3.3. Rooting of Lygodium tance of the Anemias from the Lygodiums(Fig. 2B) sug- gests an ancient divergence where the exact placement of The choice of Anemia (Anemia phyllitidis, Anemia adian- the root cannot be resolved by this data set. While the tifolium) as the outgroup followed Hanks (1998) usage for higher evolution rates of the sequences used here give bet- her phylogenetic analysis using morphological characters. ter resolution within the Lygodium species the use of a Also, Hasebe et al. (1994) used rbcL gene sequences to more conserved sequence (such as rbcL) would probably show that Anemia and Schizaea are monophyletic with yield a more accurate root. Lygodium although they diverged earlier than most fern genera. Finally, Pryer et al. (2004) looked at early leptosp- 4.2. Relationships within Lygodium orangiate divergences using nuclear and chloroplast sequences. Their analysis places Anemia closer than Schiz- Following the L. articulatum/L. palmatum basal split(s) aea to Lygodium. the maximum likelihood analysis indicates another appar- The parsimony bootstrap analysis shows the root where ently ancient (long) branch with L. microphyllum and L. L. articulatum and L. palmatum are monophyletic occurred reticulatum (Fig. 2A, b). In the analysis of Wikstru¨m 50% of the time (Fig. 1). The maximum likelihood analysis et al. (2002) L. microphyllum also diverged following L. pal- also shows a monophyletic rooting for L. articulatum and matum. Hanks (1998) conducted a detailed cladistic analy- L. palmatum, however again the bootstrap analysis yields sis based on comparative morphology. This analysis placed support only 50% of the time (Fig. 2B). The equally prob- L. articulatum, L. palmatum, L. microphyllum,andL. retic- able alternative occurs with a rooting first with L. articula- ulatum as a derived lineage together in a group called Sec- tum followed by L. palmatum. When this alignment was tion Palmata (subgenus Gisopteris). Wikstru¨m et al. (2002) analyzed using Weighbor, a weighted neighbor joining pro- re-analyzed Hanks (1998) data and found that by con- cedure designed to reduce long branch attraction, it rooted straining L. palmatum to a basal split they added only a sin- first with both L. articulatum and L. microphyllum (they gle step to the tree length. Our analysis would consider were monophyletic). these species a paraphyletic group of ancient derivation. The remaining species that were analyzed appear mono- 4. Discussion phyletic and contain all the ‘‘species of interest” except L. palmatum. Within this clade (Fig. 2A, c) L. circinnatum 4.1. Phylogenetic origins of Lygodium and Lygodium flexuosum (L.) Sw. diverge first. Following this, two additional main branches appear, one containing Spores date the origins of the family Schizaeaceae back Lygodium lanceolatum Desv., L. volubile, L. smithianum, to at least the Triassic (Bolchovitina, 1971). The first L. oligostachyum and L. cubense and the other L. polystach- macrofossils identifiable as Lygodium (also possessing dis- yum, Lygodium venustum Sw. and L. japonicum. tinctly palmate leaves resembling extant L. palmatum) Cytologically, Lygodium represents an aneuploid series appear in the late Cretaceous as L. pumilum Brown with chromosomal base numbers n = 28, 29, 30. L. palma- (Brown, 1943). Gandolfo et al. (2000) examined charcoali- tum (Wagner, 1963), L. microphyllum (Mitui, 1968; Roy fied fossils of sorophores and spores of L. bierhorstiana and Manton, 1965), and L. reticulatum (Takamiya, 1995) Gandolfo, Nixon, Crepet, and Ratcliffe from the late Cre- all share a base ploidy of 30, with tetraploidy identified taceous. They interpreted constricted bases on the soro- in L. microphyllum. Among the species this study analyzed, phores as possible attachment sites to a non-laminar only L. flexuosum (a tetraploid) exhibited a base number of pinna suggesting greater similarity to L. palmatum and 28 (Roy and Manton, 1965) with two size classes of chro- Lygodium dimorphum Copel (where fertile pinna are non- mosomes indicating autopolyploidy. Other species with a laminar) than to other species in the genus. Lygodium spe- base number of 28 (Roy and Manton, 1965) include Lygo- cies with palmate leaves and dimorphic fertile segments dium longifolium (Willd.) Sw., Lygodium salicifolium Presl, become common in the fossil record from the Cenozoic and Lygodium trifurcatum Baker. Lygodium circinnatum P.T. Madeira et al. / Biological Control 45 (2008) 308–318 313
(Manton and Sledge, 1954), L. volubile (Walker, 1966, rently known from Japan, Okinawa and Hong Kong (Solis 1985), L. venustum (Walker, 1966), and L. japonicum (Man- et al., 2004). The development on L. palmatum is in line ton and Sledge, 1954; Mitui, 1968; Takamiya, 1995) all with the placement by Hanks (1998) but not reflective of have a base ploidy of 29. More than one ploidy level was its genetic distance from L. microphyllum. found in all but L. venustum for which only a tetraploid Host range testing results for the pyralid moth, Austro- was reported, however, with a bimodal distribution sug- musotima camptozonale Hampson, are taken from the tech- gesting allopolyploidy. We have arranged the base ploidy nical advisory group (TAG) petition (Buckingham et al., numbers onto the molecular taxonomy (Fig. 1). Note that, 2003) submitted to APHIS [submitted as Cataclysta camp- while incomplete, no homoplasy exists. Also, allopoly- tozonale] as well as the 2001 Annual Report of the Austra- ploidy in L. flexuosum, L. venustum, and possibly other spe- lian Biological Control Laboratory. Austromusotima cies indicates that relationships exist which are not reflected camptozonale was specific to the genus Lygodium. Adults in this study because we used maternally inherited chloro- developed on only two of the six ‘‘species of interest”, sig- phyll sequences. Identifying both parentals for allopolyp- nificantly on L. palmatum and to a lesser degree, on L. loids would require the use of nuclear sequences such as japonicum. In no-choice trials, an average of 20.3 adults/ the internal transcribed spacer (ITS) regions of rRNA. replicate developed on Australian L. microphyllum, 12.8 on Florida L. microphyllum, 13.8 on L. palmatum, and 4.3. Comparison of L. microphyllum with the ‘‘species of 1.9 on L. japonicum. In these trials no adults developed interest” on L. reticulatum whereas in the field L. reticulatum appears to be a host plant (Goolsby et al., 2003; Yen Clearly the closest relative to L. microphyllum among et al., 2004). This suggests that separate biotypes or sibling the species examined is L. reticulatum. Maximum likeli- species exist on the different plants. The host range devel- hood distances (in parentheses as sequence divergence) opment on L. japonicum does not particularly correlate between the United States L. microphyllum (L101A, with the molecular taxonomy; however, L. japonicum is L101B) and ‘‘species of interest” follow: L. volubile the host plant of related Asian musotimine moths. (0.092), L. japonicum (0.093), L. oligostachyum (0.095), L. Early host range testing of the eriophyid mite Floracarus cubense (0.099), and L. venustum (0.101). These distances perrepae from Queensland, Australia determined that it therefore differ by only about 9%, too small to be consid- would not rear on L. microphyllum sent from Florida. A ered predictive. L. palmatum, at a distance of 0.128, is search for a different mite population which would repro- somewhat further (if one assumes a constant molecular duce on the Florida fern commenced. Concurrently, Goo- clock across the phylogeny) than the other ‘‘species of lsby et al. (2006) sequenced the chloroplast trnL-F and interest”. rsp4-trnS introns of L. microphyllum from throughout the native range and discovered an exact match between the 4.4. Host range testing Florida type and specimens from the Iron Range, Queens- land, Australia and from Papua, New Guinea. Mites from Many methodologies and criteria may be studied during the Iron Range were also the most effective in creating rolls host range testing including oviposition, egg development, (mites can only reproduce on adequately rolled leaves) and larval development, and adult feeding. The primary crite- were chosen for further host range work. Goolsby et al. rion used in this discussion is ‘‘complete development” of (2005) report on the results of host range testing on plants the insect on the species under consideration, or as van of different ages of development. The youngest (sporeling Klinken (2000) described it, the ‘‘fundamental host range fern) appeared extremely sensitive to feeding and develop- described for the life cycle of the insect”. ment on non-target species and therefore was useful for Host range testing results for the pyralid moth, Neo- screening species for further testing. Leaf-rolling ability musotima conspurcatalis, are taken from the technical advi- was highest on L. microphyllum, followed by L. flexuosum sory group (TAG) petition submitted to APHIS and L. palmatum, with very low levels on L. japonicum, L. (Pemberton and Goolsby, 2005). Neomusotima conspurca- oligostachyum,andL. venustum. No rolling took place on L talis was tested in complete development trials, succeeding volubile. Modest numbers of mites developed to adulthood only on L. microphyllum, L. japonicum, and L. palmatum. on L. flexuosum and L. palmatum with low numbers devel- In one test, 549 adults were produced on L. microphyllum, oping on L. venustum and L. oligostachyum. However, 222 on L. palmatum, and 296 on L. japonicum. In another when presented with older plants, either in no choice tests test, 16 adults were produced on L. japonicum compared or in garden plots, the mites failed to produce rolls or with 48 on L. microphyllum. In no case did development develop adults on any of the ‘‘species of interest”. A low occur on the other ‘‘species of interest”. Complete develop- number of rolls were induced on older L. reticulatum ment on L. japonicum contrasts with the absence of com- fronds but no adults developed. This reflects L. reticula- plete development on L. oligistachyum and L. volubile, tum’s relationship to L. microphyllum in the molecular phy- which are at a similar genetic distance from L. microphyl- logeny. Floracarus perrepae collected from L. reticulatum at lum. Note that the closely related species Neomusotima Cape York, Queensland may represent a different biotype fuscolinealis Yoshiyasu occurs on L. japonicum and is cur- of the mite (Goolsby et al., 2005). 314 P.T. Madeira et al. / Biological Control 45 (2008) 308–318
cut material and whole plants, larvae placed on L. japoni- cum failed to feed and died. Separate host range trials are being conducted on ferns native to Florida and the Lygodi- um ‘‘species of interest” at the USDA-ARS, Invasive Plant Research Laboratory in Gainesville, Florida (Pemberton and Bennett, unpublished data). To date, in no-choice tri- als sawflies (collected on L. microphyllum from around Trat Province, east of Bangkok) have been able to complete their life cycles on L. volubile and L. japonicum.OnL. oli- gostachyum a few small, non-reproductive adults have been produced. Trials on other ‘‘species of interest” remain ongoing. Among the plants tested L. volubile (0.092), L. oli- gostachyum (0.095) and L. japonicum (0.093) are nearest in molecular distance to L. microphyllum.
4.5. Factors affecting host range
The primary purpose of this study was to construct a molecular phylogeny for the genus Lygodium, especially for ‘‘species of interest” to the biological control work on L. microphyllum. Secondarily, the study asked if the molec- ular phylogeny is predictive of the host ranges of herbivo- rous insects and mites associated with Lygodium species. The results do not appear particularly predictive, probably because of the large and similar relative distance of L. microphyllum from all the other species examined (except L. reticulatum). The absence of significant differences in genetic distance between L. microphyllum and the ‘‘species of interest” (except L. palmatum) presents an opportunity to examine the host range data for other influences. The pyralid moth, Fig. 1. Parsimony analysis of the trnL intron and trnL-F spacer N. conspurcatalis, which developed on L. japonicum and (chloroplast) sequence data for Lygodium species using the branch-and- produced large numbers of adults has a congeneric, Neo- bound criterion. The majority rule consensus tree (60 best equally musotima fuscolinealis, which uses L. japonicum as its host. parsimonious trees) generated by PAUP 4.0b10 is presented. The upper By comparison, host range tests showed a different pyralid numbers for each node represent consensus percentages for the 60 most moth, A. camptozonale, which has no close relative that parsimonious trees. The lower numbers at each node represent the bootstrap percentages (1000 replicates). Base chromosomal ploidy, if feeds on L. japonicum, produced only a few adults on L. available, is identified (n = 28, 29, 30) at the base of species branches. japonicum. At least one other Lygodium feeding member Samples are identified by species and sample identification (sample ID) of the Musotiminae, Lygomusotima stria Solis and Yen, number. The parsimony analysis is rooted using Anemia phyllitidis and is known and it appears to be a specialist on L. microphyl- Anemia adiantifolium as outgroup species. lum (Solis et al., 2004). Also of interest is the finding that Neostromboceras albicomus larvae collected from L. flexuo- Smith et al. (2002) reported on Neostromboceros albico- sum completed their life cycles on L. microphyllum while mus Konow, another potential biological control agent for those collected on L. microphyllum failed on L. flexuosum L. microphyllum. Field studies recovered N. albicomus from (Smith et al., 2002). Eggs and larvae of Neostromboceras both L. microphyllum and L. flexuosum. However, molecu- lutchti Malaise have been found on L. flexuosum and the lar investigation (D2 expansion domain of the 28S rRNA) larvae on L. microphyllum (Smith et al., 2002). While not indicated a difference of one base between the sawflies col- entirely predictive, the ability of the insect to develop on lected on the different plants (Smith et al., 2002). The saw- an additional host appears to be more frequent when the flies collected on L. flexuosum were able to complete their insect has a related biotype or species which feeds on the life cycles on L. flexuosum, L. microphyllum, and L. salicifo- additional host. Futuyma (2000), in his review of the evo- lium, although adults did not oviposit onto L. salicifolium lution of host range in herbivorous insects, expressed con- and were ‘reluctant’ to do so on L. microphyllum. The saw- cern that biological control should look ‘‘not only to plants flies found on L. microphyllum showed a preference for that are closely related to the insect’s normal host, but also feeding on that host plant. In oviposition tests most eggs plants that are hosts to the near relatives of the insect”. were laid on L. microphyllum with only a few eggs laid Futuyma and associates (Funk et al., 1995) tested this on L. flexuosum and none on L. japonicum. When provided hypothesis by comparing the molecular evolution of the P.T. Madeira et al. / Biological Control 45 (2008) 308–318 315
Fig. 2. Maximum likelihood analysis of the trnL intron and trnL-F spacer (chloroplast) sequence data for Lygodium species. A single best tree was generated by PAUP 4.0b10 using the best model (Akaike weighting) generated in MODELTEST 3.06. Numbers along the branches represent bootstrap percentages (1000 replicates). The lengths of branches represent distance as sequence divergence. Samples are identified by species and sample identification (sample ID) number. (A) Close-up view of the Lygodium species relationships. (B) View of Lygodium species along with outgroup Anemia species. leaf beetle genus Ophraella with the molecular phylogeny typically lead to an immediate host shift but instead to host of several tribes of Asteraceae (including the host plants, range expansion. There was a bias for establishment on a Ophraella spp.). The host plant usage displayed more of new host if it also served as the host of another species a host shifting paradigm than the classical co-speciation of Nymphalini. Finally, most of the larvae tested showed process (Ehrlich and Raven, 1964). In tests of genetically an ability to feed on Urtica dioica regardless of the species variable feeding responses by Ophraella species to plants of host plant used. Parsimony optimizations indicate an other than their normal host plants, hosts of related Ophra- ancestral association with the Urticaceae. ella species elicited a feeding response more readily than Models for genetic specialization include the classical hosts of more distantly related Ophraella species (Futuyma, model of allopatric fragmentation (Mayr, 1963) and sym- 2000). patric-speciation models where phytophagous insects speci- Janz et al. (2001) examined host range changes in nym- ate through host shifts (Berlocher and Feder, 2002). The phalid butterflies using a combination of parsimony opti- second process proposes mate choice coupled with habitat mizations of host plant data on the butterfly phylogeny selection resulting in assortative mating which restricts and larval survival tests. Evolution was dynamic with gene flow between conspecifics on divergent host plant spe- numerous expansions and reductions of the host plant cies and allows speciation (Hawthorne and Via, 2001). In range and with no particular support for specialization as either case, an early pruning of genetic diversity is the pre- the derived condition. Colonization of a new host did not dicted consequence (Berlocher and Feder, 2002; Diegisser 316 P.T. Madeira et al. / Biological Control 45 (2008) 308–318 et al., 2006), allopatric speciation due to the bottleneck of a spilomeline group polychotomy which is basal to the effect of isolation and sympatric speciation due to coloniza- pyraustine group. However, note that while the LEPTREE tion during the range expansion. However, Emelianov project reports on no Crambidae fossils there are two Pyral- et al. (2004) showed there is appreciable gene flow due to idae fossils, one in amber dated at 44 Ma, and the other at hybridization between allopatric host races of the larch 35 Ma, both with wing venation similar to extant species. budmoth in those linkage groups which are not under This infers that it is at least reasonable to speculate that active selection. That is, the pruning of genetic diversity Musotimine ancestors co-existed then. during allopatric speciation occurred primarily in those Another hypothesis would be that the development of linkage groups which are under selective pressures. these musotimine moths is simply a case of ‘‘ecological Janz et al. (2006) propose that the evolutionary process fitting” (Agosta, 2006) to a plant not directly involved in must also include a process which is inherently diversifying. their evolution. For a novel plant to be useable it must be They suggest the colonization of one or more novel hosts recognized by the insect for oviposition (i.e. oviposition (a polyphagous phase) allows the rapid expansion of geo- stimulants) and must contain nutrients which are physically graphic range and the penetration of new habitats (Janz (e.g. leaf toughness) and chemically (e.g. growth stage) et al., 2001). This process is followed by a more-gradual usable by the larvae, while not containing fatal secondary genetic diversification resulting in fragmentation and spe- defensive chemicals. Both musotimines showed significant cialization being restored. In examining the diversity of oviposition on L. palmatum in no-choice tests, second only host use within the butterfly family Nymphalidae using a to that for L. japonicum and L. microphyllum, indicating variant of independent contrast analysis they (Janz et al., shared secondary compounds. Oviposition on the other 2006) found diversity of host use drove insect species rich- ‘‘species of interest” was low. It certainly would not be sur- ness rather than the other way around. Although Lygodium prising for a congener, especially one basal in the Lygodium is a worldwide genus, 15 of the 26 species come from the phylogeny, to contain secondary chemicals recognized by Southeast Asian/Australian area. Further, L. japonicum, these insects, even without proposing direct ancestral host usage. L. flexuosum, and L. reticulatum are sympatric with L. microphyllum over significant portions of this range (Goo- 4.7. Implications for biological control lsby et al., 2003; Hanks, 1998). This diversity of host avail- ability appears to be driving speciation with allopatric Briese (2003) argued that formal taxonomic classification races of Neostromboceras albicomus on L. microphyllum is not always indicative of the true evolutionary history of and L. flexuosum (Smith et al., 2002) and Austromusotima a group, which might really be polyphyletic or paraphyletic, camptozonale on L. reticulatum and L. microphyllum (field a situation which proved true in this study. Earlier classifi- observations vs. host range results). Neomusotima cons- cations (Prantl, 1881; Reed, 1946) and morphological phy- purcatalis on L. microphyllum and N. fuscolinealis on L. logenies (Hanks, 1998)ofLygodium with their suggested japonicum are described as southern and northern allopat- sub-generic groupings did not align with our molecular tax- ric sister species (Solis et al., 2004). onomy or with that of Wikstru¨m et al. (2002). However, the use of molecular taxonomy to assemble plant lists for host 4.6. The curious case of L. palmatum range tests of insects to be used against L. microphyllum would have proved ineffective because of the large genetic Although L. microphyllum and L. palmatum share mor- distance between L. microphyllum and most of the other phological and cytological similarities, as well as a paraphy- Lygodium species. Instead, the alternate host usage found letic grouping of ancient derivation, they are separated by a during host range testing often reflects evolutionary usage large genetic distance and are geographically distantly iso- of that alternate sympatric host or its ancestor. lated. Host range trials showed that the musotimine moths, Therefore, while the molecular phylogeny of a host plant N. conspurcatalis and A. camptozonale, which normally and its congeners should indicate which plant species might develop on L. microphyllum will also complete development serve as alternate hosts for herbivorous arthropods, exam- on L. palmatum. The eriophyid mite, F. perrepae, completed ining the molecular phylogeny of the specialist insect and development on sporelings of L. palmatum but not on its congeners may also prove useful for this purpose. Host mature plants. So, in the context of this study, does ances- range trials should at least include plant species which occur tral host usage explain the success of the musotimines on within the potential geographic range of the target weed and L. palmatum? As noted earlier, L. palmatum-like fossils which are known to be exploited by closely-related insect are widespread in the fossil record (North America, Europe, species that are usually, but not necessarily, congeners. Northern Asia, Chile, Australia and New Zealand) during the Paleogene. Rozefelds et al. (1992) assigned an Eocene Acknowledgments dating to the Australian plants. It is unclear, lacking a fossil record or a molecular phylogeny in the literature for the The authors wish to recognize Christine Bennett who Crambidae, whether Musotiminae ancestors would have conducted host range studies on the 3 insects in the co-existed with Lygodium then. Solis and Maes (2002), USDA-ARS quarantine facility at the Florida Biological based on adult characters, place the Musotimines as part Control Laboratory. Thanks for the collaboration of John P.T. Madeira et al. / Biological Control 45 (2008) 308–318 317
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